Thai-Hoa Chung
email: chung@gustaveroussy.fr

Augusto Stancampiano
email: augusto.stancampiano@univ-orleans.fr

Kyriakos Sklias
email: kyriakos.sklias@u-psud.fr

Kristaq Gazeli
email: kristaq.gazeli@u-psud.fr

Franck M André
email: franck.andre@cnrs.fr

Sébastien Dozias
email: sebastien.dozias@univ-orleans.fr

Claire Douat
email: claire.douat@univ-orleans.fr

Jean-Michel Pouvesle
email: jean-michel.pouvesle@univ-orleans.fr

João Santos Sousa

Éric Robert
email: eric.robert@univ-orleans.fr

Lluis M Mir
email: luis.mir@cnrs.fr

Cell Electropermeabilisation Enhancement by Non-Thermal-Plasma-Treated PBS

Despite the panoply of anticancer therapies, many limitations remain to this day. The anticancer therapeutic strategies present specificity problems towards the tumour cells, thus causing unexpected and severe side effects. The precise targeting of tumours by local treatments is not always feasible. The development of multidrug resistance and the weak induction of immunogenic cell death (ICD) remain limiting factors. Thus, other therapeutic strategies based on new approaches are required to enhance the eradication of cancers, particularly the combination of different methods such as physical modalities that allow local treatment and are less subject to resistance in cancer cells. In the last thirty years, electrochemotherapy (ECT) -an established local eradication of tumour-based electroporation technology -has been widely employed in Europe in human and veterinary medicine with alleviated systemic side effects.

Meanwhile, non-thermal plasma (NTP) is gaining momentum for application in life science, specifically in oncology, thanks to their potential as rich sources of reactive oxygen and nitrogen species, amongst others. As a new specialised medical branch, NTP recently gave birth to plasma oncology and clinical plasma oncology as palliative care. Nevertheless, little is known about NTP anti-cancer effects, and there still are challenges to be overcome for both ECT and NTP oncology. This thesis was dedicated to enhancing electrochemotherapy through non-thermal plasma medicine. Indeed, although ECT is an established and standardised targeted anticancer treatment, given its nature, it still has some inherent drawbacks, e.g., causing some discomfort due to the application of intense pulsed electric fields (PEFs). This multidisciplinary approach, at the crossroads of physics, chemistry, biotechnologies and medicine, was

PREFACE

ii des Milieux Ionisés, UMR 7344, CNRS/Université d'Orléans, France) and the LPGP (Laboratoire de Physique des Gaz et des Plasmas, CNRS/Université Paris-Saclay, Orsay, France).

For the project, a novel helium NTP multijet device with a grounded and compensated circuit for stable uses has been designed and manufactured by the GREMI for in vitro and in vivo anticancer studies. By combining indirect NTP (i.e., plasma-treated liquids) with µsPEFs, we succeeded in reducing the electric pulsed amplitude in vitro and in vivo compared with that used in practical ECT for clinics or with NTP as mono-therapy while maintaining the anticancer efficiency. Moreover, the in vivo combined treatment with NTP and ECT improved the efficiency of ECT. The stability, the components and the pH over different storage conditions of the plasma-treated liquids have also been studied to establish the best production and storage conditions where reproducibly prepared plasma-treated liquids were stable and reproductive for various investigations and utilisations. All those studies within the novel approach combining NTP and µsPEFs contributed to complete anti-cancer therapies panoply.

During three years, my thesis led to the publication of two scientific articles, the submission of a third article, an article in preparation, and various scientific presentations at national and international conferences and workshops listed below. Pour ces trois années de doctorat, mes remerciements vont en premier à Lluis Mir, ancien directeur de notre UMR mais surtout mon directeur de thèse, sans qui cette thèse n'aurait pas vu lumière. Lluis, je vous remercie d'avoir été patient, pédagogue, compréhensible et par-dessus tout, disponible. J'ai apprécié nos conversations scientifiques (en admirant votre passion pour la science), politiques et sur la langue française (pendant lesquelles j'ai enrichi mon vocabulaire). Merci pour le Grand Maître que vous êtes.

Je remercie chaleureusement mon co-encadrant de thèse, Franck André, qui a su m'accompagner tout au long de ma thèse malgré son agenda chargé. Cet accompagnement allait de la paperasse pour mon recrutement bien avant notre rencontre, au travail quotidien au laboratoire où ses conseils m'étaient précieux. Un Franck (très) calme, un Franck gentil, un Franck positif, un Franck polyvalent, un Franck omniprésent ! Je ne pense pas pouvoir avoir mieux comme encadrants de thèse que Lluis et Franck. Qu'ils soient aussi remerciés de m'avoir fait partager leurs brillantes intuitions. C'est à leurs côtés que j'ai compris ce que rigueur et précision voulaient dire pour une thèse en sciences.

Mes sincères remerciements vont aux membres de mon jury de thèse qui, malgré les circonstances particulières causées par la pandémie de la Covid-19, ont accepté d'examiner mon travail et d'assister à ma soutenance de thèse :

• Au Professeur Emmanuel Odic -d'avoir accepté d'être Président du jury, d'avoir partagé ses avis très constructifs et de m'avoir fait part de ses compliments pour la qualité de mon travail. • Aux Docteurs Anne-Laure Bulteau et Thierry Dufour -de l'honneur que vous m'avez fait en acceptant d'être rapporteurs de ma thèse, d'y avoir consacré votre temps précieux pour partager des observations très constructives non seulement pour mon manuscrit de thèse mais aussi pour la suite du projet ANR dont ma thèse faisait partie. Je souhaite remercier particulièrement Thierry qui était également mon référent pour ces trois années de doctorat. Thierry, merci pour ta disponibilité, ton soutien et tes encouragements. Madame Bulteau, je suis touchée de la confiance que vous m'avez accordée, merci de m'avoir confié votre projet de recherche à travers duquel j'ai découvert le LVMH Recherche.

• Au Docteur Marie-Pierre Rols -d'avoir examiné ma thèse avec grande attention et d'y avoir apporté ses observations de qualité. Marie-Pierre, je garde une belle image de ta douceur en qualité de Directrice de recherche pionnier dans le domaine des impulsions électriques appliquées à la biologie.

• Aux Docteurs Muriel Golzio et Pierre-Marie Girard -d'avoir assisté à ma soutenance de thèse et partagé vos avis précieux.

Que tous les membres du jury ainsi que tous les collaborateurs et amis, famille sachent que leur présence à ma soutenance de thèse, ensemble avec l'estime qu'ils portaient pour mon travail, a fait de ce jour un des plus beaux jours de ma vie.

ACKNOWLEDGEMENTS x

As a matter of course, my profound gratitude to all my collaborators for their substantial contributions and professional cooperation:

• La dream team GREMI, toujours dynamique, sportive et joyeuse : Éric, un chef exceptionnel, discret mais qui ne manque pas d'humour ; Augusto, grazie mille per il tuo tempo e pazienza, la tua professionalità, per non parlare del tuo incoraggiamento al momento giusto; Claire, j'ai toujours admiré tes tresses et j'espère que tu m'en feras une quand mes cheveux seront suffisamment longs (en tout cas, tu me l'as promis !) ; Sébastien, merci pour ton soutien au niveau technique, sans toi nous n'aurions pas eu ces machines de plasmas, tout simplement ; Thomas, même si je n'ai pas eu l'opportunité de travailler avec toi, j'apprécie beaucoup tes présentations lors des congrès et qui reflètent la qualité de ton travail ; et en dernier pour un plus grand remerciement, Jean-Michel que j'estime tant, merci pour ta gentillesse et ta douceur envers nous -les thésards que tu as toujours su soutenir et encourager, merci pour les discussions très sympathiques sur le Viêt Nam, j'ai vu que mes observations t'ont fait rigoler. Tu fais partie des professeurs que j'ai pu tutoyer directement dès la première rencontre sans réfléchir (Amina m'a dit la même chose, tu sais que nous t'apprécions beaucoup !). Mieux qu'un collaborateur, tu étais un « mentor » pour moi pendant cette thèse. • L'équipe du LPGP : João, Kristaq, Kyriakos, Gérard, Thibault. Merci beaucoup pour ces belles années de thèse en collaboration avec vous. Toujours disponibles pour nous accueillir à Orsay et pour me préparer des échantillons quand la fameuse machine de plasma était capricieuse. I do not, unfortunately speak Greek but my sincere gratitude to Kristaq and especially to Kyriakos for being so kind. You know how to turn a long experimentation day interesting.

Je tiens à remercier tout particulièrement mes collègues à l'IGR avec qui j'ai partagé un « quotidien de thèse ». Ce n'est pas le plus simple des « quotidiens » et pourtant, grâce à leur compagnie, de proche ou de loin, ce quotidien était le plus rassurant. Dans l'ordre de connaissance, puisque 3 ans est mine de rien une longue période au cours de laquelle j'ai eu l'opportunité de rencontrer (ou croiser, comme disent les français) beaucoup de personnes :

• Isabelle, notre gestionnaire qui a su gérer toutes nos démarches administratives, « elle est un amour ». Isabelle, merci également de m'avoir confié ta plante d'araignée qui maintenant pousse en pleine vitesse chez moi. J'espère que mon orchidée se plaît dans ton bureau baigné de lumière. • Chère Amina, ma « coloc' de bureau » : qui peut croire, en nous voyant nous chamailler au bureau, que j'étais invitée à ton mariage ? Ce n'est pas pour rien que tout le monde a dit que l'on était comme un vieux couple ! Merci à toi pour ta bonne humeur de (presque) tous les jours, pour avoir été positive et optimiste, pour ta joie de vie (cela est contagieux), et particulièrement d'avoir pris le temps de me connaître et de me comprendre (pour autant). J'ai beaucoup de chance de t'avoir comme collègue et maintenant, une amie chère.

• Merci Adeline de m'avoir accompagnée au début de ma thèse pour les manips in vivo durant lesquelles j'ai beaucoup appris, en grande partie grâce à ta patience et gentillesse ; merci Tomás (ce qui m'a manqué depuis ton départ de l'équipe : « Holà, chica ! » puis « Allez ! Il faut travailler hein ! » ou encore « Réfléchis ! », puis pendant les pauses de café j'aurais pu sentir les arômes de mon café vietnamien préparé de façon très vietnamienne par un espagnol) ; un grand merci à l'équipe d'animalerie de s'être occupé de mes souris (surtout Mélanie de m'avoir accompagnée dans les grandes expérimentations) ; merci à Léna, Alain, Thierry, Félix, aux filles chez « les voisins » (Marie, Célia, Suzan, Linda), aux équipes de l'ex-UMR 8203 (David Castel, Jacques Grill et Birgit Geoerger) ainsi que les équipes formant notre nouvelle UMR 9018 (Catherine, notre directrice de l'Unité ; Nazanine, ma cheffe d'équipe ; Karim, Pierre, Yegor, Svetlana : merci à tous les chefs d'équipe de leur soutien ; un bonjour dans le couloir l'équipe. Heureuse d'avoir passé la moitié de ma thèse à tes côtés. Pas besoin d'être dans le même projet ni dans le même bureau pour avoir ton soutien en permanence. En plus, tu as un humour qui « colle » bien avec le mien donc si jamais je n'ai pas encore eu l'opportunité de te le faire savoir, saches que je me sens en pleine de sécurité quand je suis avec toi (j'en n'ai pas davantage d'explication). Merci à cette thèse de m'avoir offert toi et Amina comme collègues et amies.

• Céline, Giorgia, Jean-Rémi, Kenza, Camille, Alberto, Florian, Bảo Trâm, Valentin, Aurore : notre labo est formidable grâce à vous. Toujours souriants même quand je débarquais à l'imprévue pour vous emprunter du matériel ou vous poser mes questions naïves. Merci du fond du coeur d'avoir pris le temps de me connaître et « bienvenue » la personnalité que j'ai. Je tiens à remercier ma compatriote pour ses Bubble Tea qui m'ont réconfortée, pour son talent culinaire. Bé Trâm, toi qui es si calme, si douce, toi qui as su montrer à nos collègues la vraie « cliché » d'une vietnamienne (ce que je n'ai pas pu assurer), je suis heureuse de t'avoir rencontrée.

Il est peut-être out of scope, mais mes pensées vont aux différentes lignées de cellules et souris que j'ai pu étudier tout au long de ma thèse. Elles étaient, la plupart du temps, très collaboratives et « gentilles », si je peux le dire de cette manière pour leur remercier d'avoir contribué au succès de ce travail. Merci à la plus belle ville du monde, Paris mon amour, pour ces belles années ! A mes chers anciens collègues au CEA qui sont, depuis mon stage en 2012, devenus mes amis. Vous m'avez vue traverser certaines (grandes) épreuves et m'ont aussi vue rigoler très sérieusement. Je rédige ces remerciements avec une grande pensée à vous : Hamed, mon maître de stage, toi qui étais l'origine de ma passion pour les nanotechnologies, le stage dans ton équipe a été décisif pour mon parcours professionnel, je te remercie particulièrement pour ton soutien. Nous avons de la chance de t'avoir comme Directeur (j'en suis sûre, tu vas encore me dire « La chance, c'est aussi nous qui la provoquons ! ») ; Willem, mon co-encadrant de stage et surtout, un brillant chercheur très humble pour qui j'ai beaucoup d'estime ; Dominik -mon Domichou (si je pouvais, j'aurais déposé un brevet sur ce surnom), toi qui es toujours aussi doux, aussi gentil, toujours à mon écoute, je t'apprécie beaucoup et te souhaite le bonheur où que tu sois, à Paris ou à München ; David, merci de m'avoir fait découvrir Mestre, la sangria et le cap'b ; Aura, Joana, Rana, Shatha, puis enfin, Masha et son compagnon Joseph (the perfect couple dont l'un me prépare un superbe magret de canard bien rosé au BBQ en plein hiver et l'autre me sert de l'alcool, et qui m'ont confié deux charmantes demoiselleschats pendant une semaine sans rien me payer au retour à part des BBQ digne d'une étoile Michelin. A quand le prochain ?).

A Astrid ma coloc', je te remercie pour ta gentillesse, pour ton grand coeur. Nous n'avons pas vraiment le même caractère, n'empêche que j'étais heureuse d'avoir partagé avec toi notre charmant appartement parisien (ainsi que la passion pour le vintage) et je t'en remercie. C'est aussi en partie grâce à toi que Franck et moi sommes rencontrés, j'ai vu que Dieu a fait des merveilles en me guidant vers une coloc' avec toi.

A tous mes amies et amis grâce à qui je ne me suis jamais ennuyé en trois ans et trois mois : la thèse n'y était pas pour rien, mais vous vous êtes bien chargé du reste.

A Gia Thụy. Cảm ơn anh đã bên cạnh, yêu thương, chăm sóc em suốt 7 năm. La vie est faite de séparation, l'importance est de vivre des moments sincères et inoubliables. Je suis touchée de la dédicace me concernant dans ta thèse et t'en remercie. Sache que je te souhaite toujours le meilleur.

STATE OF THE ART

As a multidisciplinary thesis, this research work covered the field of biotechnologies, biomedicine, physics and chemistry for pre-clinical studies of novel medical physics approaches against cancer. Before presenting the achievements during those three years of research, state of the art will be introduced to give insight into the medical physics era with a particular interest in electroporation-based technology and the non-thermal plasma domain.

The main applications of those two technologies, both general and medical, focusing on oncology, will also be presented.

STATE OF THE ART From iatrophysics to medical physics: then and now -11 -

From iatrophysics to medical physics: then and now

Modern medical practice and research depend significantly on technology, and hospitals are equipped with physics-based devices for clinical measurement, diagnosis, and treatment.

Today, the exertion of physicists in medical institutions generally refers to the term medical physics, which originated from iatrophysics. It is essentially related to medical applications of radiation, diagnostic imaging, and clinical measurement. Although the implication of physics in clinics was only instigated this last century, the relation between physics, biology and medicine has a much longer, more prosperous and more profound heritage, dating back to the earliest recorded period of medical history. Even if historical origins are often arguable, thus difficult to determine, this chapter will trace a brief history of the contribution that physics has made to medical sciences, from ancient Greece to the study of the human body mechanics by da Vinci in the late 15 th century to the emergence of radiology, phototherapy and electrotherapy at the end of the 19 th century, with particular focus on two physical-based technologies of the 20 th century, the electroporation and non-thermal plasma technologies, being the centre of this thesis for their applications in oncology.

A historical perspective

In Classical Greece, Hippocrates (c. 460 -c. 370 BC) -the Father of Medicine, prescribed sunbathing (heliotherapy) for medical and psychological purposes, establishing the therapeutic use of ultraviolet (UV) radiation. Throughout the centuries, the discovery and application of physics have been fundamentals for scientists to explore the living's functions.

Electrophysiology, biomechanics, cardiology, ophthalmology, neurology, audiology, physiotherapy, orthopaedics, and radiology directly apply physics to the medicine field [START_REF] Keevil | Physics and medicine: A historical perspective[END_REF]. Other specialities such as environmental health, dermatology, and even general surgery have medical physics sources. Leonardo da Vinci (1452-1519), one of the most famous polymaths of human history, could be considered the first medical physicist with his detailed studies of human body mechanics. Da Vinci described mechanical science as "the noblest and above all others the most useful", bringing physics closer to clinical engineering and medicine. However, science had to wait for more than a hundred years later to demonstrate by William Harvey (1578Harvey ( -1657) that blood flows rapidly around the human body under the action of a single mechanical pumping system STATE OF THE ART From iatrophysics to medical physics: then and now -12 -of arteries and veins [START_REF] Harvey | An Anatomical Disquisition on the Motion of the Heart and Blood in Animals[END_REF]. At the same time, the microscope of Anton van Leeuwenhoek (1632Leeuwenhoek ( -1723) ) opened up new vistas of biological structure, and the pioneering work of Santorio Sanctorius (1561-1636) on clinical measurement of temperature, pulse rate, and body mass are early examples of modern clinical practice [START_REF] Porter | The Greatest Benefit to Mankind: A Medical History of Humanity[END_REF]. René Descartes and later materialist philosophers of the 17 th century stated that creatures, either human beings or animals as mechanisms without a soul. This early school of thought merely interested in fundamental issues about the body's function and the nature of life, leading to the development of the biomechanics field [START_REF] Keevil | Physics and medicine: A historical perspective[END_REF]. Indeed, following Harvey's work, Giovanni Alfonso Borelli (1608-79) examined the human body in its static as well as dynamic positions, from the structure to the motion, balance, and forces vis-à-vis almost all the principal joints, with the recognition that the muscles balance the body-weight (Figure 1a). Borelli's famous De motu animalium (published posthumously in 1679) is the first comprehensive treatise on biomechanics. Borelli was probably the first to determine man's centre of gravity experimentally [START_REF] Marquet | Iatrophysics to biomechanics. From Borelli (1608-1679) to Pauwels (1885-1980[END_REF]. The following century saw the first introduction of the term Medical Physics (or, more accurately, Physique médicale) in Paris in 1778 by Félix Vicq d'Azir , the general secretary of the Société royale de médecine [START_REF] Parent | Félix Vicq d'Azyr: Anatomy, medicine and revolution[END_REF] which set Paris on the road to becoming the leading centre for medical training and research in Europe for the first half of the 19 th century (Société royale de médecine, 1790). We find Mauduyt de la Varenne (1732-92) making a critical study into the medical uses of electricity [START_REF] Mauduyt | Mémoire sur les différentes manières d'administrer l'électricité, et observations sur les effets qu'elles ont produits[END_REF] following the demonstration of charge storage in a Leyden Jar in 1746 by Pieter van Musschenbroek (1692Musschenbroek ( -1761)), and the observations of Abbé Jean-Antoine Nollet (1700-70) on the biological effects of electricity in his experimental animals and plants after electrification (Figure 1b) [START_REF] Nollet | Recherches sur les causes particulieres des phénoménes électriques et sur les effets nuibles ou avantageux qu'on peut en attendre[END_REF]. Physicists had rapidly engaged in exploring its possible medical applications. In Geneva, Jean Jallabert (1712-68) was amongst several who reported successful treatment of paralysis using electric shocks [START_REF] Jallabert | Experiences sur l'électricité, avec quelques conjectures sur la cause de ses effets[END_REF]. In 1786, Italian physician, physicist, biologist and philosopher Luigi Galvani (1737-98) discovered "animal electricity" in his famous experiments with the muscles of dead frogs' legs twitched when struck by an electrical spark. This one of the first forays into the study of bioelectricity has made Galvani the pioneer of bioelectromagnetics. Together with Alessandro Volta (1745[START_REF] Arnott | Elements of physics, or natural philosophy, general and medical: explained independently of technical mathematics[END_REF], who demonstrated his electric pile in 1800, they founded the electrophysiology science [START_REF] Porter | The Greatest Benefit to Mankind: A Medical History of Humanity[END_REF]. If the spine of a stevedore is bent and supports a load of 120 pounds carried on the neck, the force exerted by Nature in the intervertebral disks and the extensor muscles of the spine is equal to 25,585 pounds. The force exerted by the muscles alone is not less than 6404 pounds". [START_REF] Marquet | Iatrophysics to biomechanics. From Borelli (1608-1679) to Pauwels (1885-1980[END_REF]. (b) Abbé Nollet's experiment on animals and plants [START_REF] Nollet | Recherches sur les causes particulieres des phénoménes électriques et sur les effets nuibles ou avantageux qu'on peut en attendre[END_REF]. Nollet measured the change in weight of cats and small birds after placing them either near an electrified body or within an electrified cage. Electrified animals lost more weight than the controls [START_REF] Duck | The origins of medical physics[END_REF]. (c) Hand mit Ringen (Hand with Rings): a print of one of the first X-rays by Wilhelm Röntgen of his wife Anna Bertha Ludwig's left hand on November 8 th , 1895. This first-ever X-ray image was presented to Professor Ludwig Zehnder of the Physik Institut, University of Freiburg, on January 1 st , 1896. From Wikimedia Commons, the free media repository.

The early 19 th century was marked by critical investigation of physics in biomedicine and physiology, from the famous wave theory of light by Thomas Young (1773-1829) to the invention of the ophthalmoscope by Hermann von Helmholtz (1821-94) and the invention of the stethoscope in 1816, the most iconic of medical instruments, by René Laennec . A complete, accurate and concise definition of medical physics in French arose in the 1814 revised edition of Nysten's medical dictionary [START_REF] Nysten | Dictionnaire de médecine et des sciences accessoires à la médecine: avec l'étymologie de chaque terme[END_REF]:

Physics applied to the knowledge of the human body, to its preservation and to the cure of its illnesses ("Physique appliquée à la connaissance du corps humain, à sa conservation et à la guérison de ses maladies").

Nevertheless, the term "medical physics" took more than a decade to first appear in English in one of the most influential textbooks ever written on general science Elements of Physics [START_REF] Arnott | Elements of physics, or natural philosophy, general and medical: explained independently of technical mathematics[END_REF]. This has created a shared understanding of the connection between physics and medicine, which marked the beginning of medical physics as a coherent discipline. Neil Arnott (1788Arnott ( -1874) ) was also the inventor of the water bed -labelled the "Hydrostatic Bed" -first used STATE OF THE ART From iatrophysics to medical physics: then and now -14 -professionally in 1873 at St Bartholomew's Hospital (London, UK). From the late 19 th century onwards, fundamental physics was a compulsory element in medical education in the UK, recognising the growing importance of physics in medical practice.

The late 19 th century saw a historical revolution in medical physics since, in the early evening of November 8 th , 1895, an illuminating accident led to the discovery of "a new kind of ray" by Wilhelm Röntgen which changed everything [START_REF] Stanton | On a new kind of rays. By Wilhelm Conrad Röntgen[END_REF]. The first X-ray image Röntgen took of his wife Bertha's hand with her wedding ring visible (Figure 1c) is one of the most famous scientific images in history. Röntgen's discovery of a new form of energy would be named after him. Still, he always preferred the term X-radiation or X-rays -from the mathematical designation "X" denoting something unknown -as no one understood these great rays. Researchers worldwide could experiment on X-rays to develop body imaging, amongst others, as Röntgen refused to patent his findings, convinced that his "inventions and discoveries belong to the world at large" (from Nobelprize.org). Within four years since the discovery that earned Röntgen the first Nobel Prize in 1901, and of which the medical implications were immediately realised, human history witnessed some of the most significant discoveries in life science. A few months after Henri Becquerel (1852Becquerel ( -1908) ) discovered radioactivity (uranic rays) in 1896, Pierre and Marie Curie (1859-1906 and 1867-1934, respectively) (Figure 2a) discovered radium and isolated radioactive isotopes. They shared a Nobel prize in Physics in 1903 (and later a second Nobel prize for Madame Curie in 1911, this time in Chemistry). None of these investigations was uplifted by the prospect of medicine. Still, their medical potential was recognised without delay, with the first use of radiography on the battlefield in March 1896 and by the next month, the creation of the first scientific journal dedicated to medical imaging, Archives of Clinical Skiagraphy (from Greek σκiα for a shadowan early term for radiography) [START_REF] Keevil | Physics and medicine: A historical perspective[END_REF]. From then on, the use of ionising radiation in medicine was widely recognised, in particular via the rapid progress of radiotherapy and X-ray imaging. The first cancer treatment by X radiation was performed in Savoie, France, by Dr Victor Despeignes for a patient with an epigastric tumour. That cancer had shrunk by about 50% [START_REF] Sgantzos | The physician who first applied radiotherapy, Victor Despeignes, on 1896[END_REF]. Inspired by this, radiologist Jean Bergonié (1857Bergonié ( -1925) ) and histologist Louis Tribondeau (1872-1918) have laid the first biological basis for the use of Xrays, demonstrating that cancer cells are more sensitive to X-rays than healthy cells (formulating the following law: "X-rays are more effective on cells which have a greater reproductive activity; the effect is greater on those cells which have a long lineage, on those STATE OF THE ART From iatrophysics to medical physics: then and now -15 -cells the morphology and the function of which are least definitively fixed") [START_REF] Bergonié | Interpretation of Some Results from Radiotherapy and an Attempt to Determine a Rational Treatment Technique[END_REF]. With the right hand, she lifts progressively the weight applied to the quartz; in the left hand, she holds the chronometer [START_REF] Adloff | THE LABORATORY NOTEBOOKS OF PIERRE AND MARIE CURIE AND THE DISCOVERY OF POLONIUM AND RADIUM[END_REF]. (b) One of the world's first megavoltage radiotherapy units at St Bartholomew's Hospital, London, UK, in 1937 [START_REF] Keevil | Physics and medicine: A historical perspective[END_REF].

Occurring after, and mainly because of World War II (1939)(1940)(1941)(1942)(1943)(1944)(1945), nuclear medicine was one of the greatest innovations in radiotherapy that extended the applications of medical physics, especially in the treatment of cancer. ¹³¹I produced at the Berkeley cyclotron (Berkeley CA, USA) was the first radioisotope used in the clinic for thyroid investigations in 1939, then for treating hyperthyroidism and thyroid cancer [START_REF] Hamilton | STUDIES IN IODINE METABOLISM BY THE USE OF A NEW RADIOACTIVE ISOTOPE OF IODINE[END_REF]. Figure 2b depicted one of the world's first megavoltage radiotherapy units implemented at St Bartholomew's Hospital in London in 1937 [START_REF] Keevil | Physics and medicine: A historical perspective[END_REF]. Imaging techniques and standardisation operating procedures have also benefited from tremendous technological development: ultrasound imaging in the 1930s for non-destructive examination followed by medical applications in the 1950s [START_REF] Wells | Physics and engineering: Milestones in medicine[END_REF]; positron emission tomography (PET), invented in the 1950s; electron linear accelerators (linacs) for delivery of megavoltage x-rays, a spin-off from war-time research on the radar, used at Hammersmith Hospital (London, UK) by 1953; nuclear tomographic imaging, also known as single-photon emission computed tomography, developed in the 1960s, etc. One of the most significant advances in medicine in the last millennium is the invention of the X-ray computed tomography (CT) scanner in 1973 by Godfrey Hounsfield (1919Hounsfield ( -2004) ) and Allan Cormack (1924-98). The revolution that CT brought about in medical imaging has been surpassed only by the invention of magnetic resonance imaging or MRI in 1973 [START_REF] Lauterbur | Image formation by induced local interactions: Examples employing nuclear magnetic resonance[END_REF][START_REF] Mansfield | NMR 'diffraction' in solids?[END_REF] STATE OF THE ART From iatrophysics to medical physics: then and now -16 -1973). Based on nuclear magnetic resonance (NMR) that applies magnetic fields rather than ionising radiation, MRI is a powerful and flexible technique for imaging physiological and mechanical function and structure, such as the brain with its consciousness, a notion which Descartes and fellows of the past centuries ignored. Following those imaging techniques, multimodality imaging -true imaging fusion -garnered momentum for clinical oncology with the complementary capabilities of PET and CT [START_REF] Beyer | A combined PET/CT scanner for clinical oncology[END_REF] or, more recently, PET and MRI [START_REF] Marsden | Simultaneous PET and NMR[END_REF] in one imaging system. Another essential invention in physics and medicine history was the laser (an acronym for "light amplification by stimulated emission of radiation"). The medical applications of laser (laser medicine) were settled within a year of constructing the first functioning laser in 1960 [START_REF] Müller | The medical laser[END_REF], starting in ophthalmology and dermatology, followed by surgery and in photodynamic therapy.

Many physics-based medical innovations, such as CT and PET, have resulted from focused research with clinical purposes in mind. Nonetheless, generally significant developments have been unpredictable spin-offs from basic science research. Their translation into clinical practice, if this takes place, has not always been as rapid or straightforward. On the other hand, by focusing on these historical events, we can easily forget that other medical applications of physics have their origins at this same time. The therapeutic use of infrared (IR) and ultraviolet (UV) radiation was pioneered by the Nobel prize-winning Danish doctor Niels Ryberg Finsen , who introduced the "red room" treatment for smallpox lesions in 1893, and who, in 1895 showed that Lupus vulgaris, a disfiguring form of tuberculosis, responded to treatment with concentrated light radiation. Thus, phototherapy was born. Another investigation on the biological responses of electrical currents and the problem of industrial deaths from electrocution was performed by Arsène d'Arsonval (1851-1940), professor of biophysics at the Collège de France (Paris, FR), who introduced the first clinical high-frequency heat therapy unit, in the Hôtel Dieu, in 1891. Thus, modern electrotherapy was born. At the same time and following the path of Abbé Nollet, who reported the first systematic observations of the appearance of red spots on animal and human skin exposed to electric sparks [START_REF] Nollet | Recherches sur les causes particulieres des phénoménes électriques et sur les effets nuibles ou avantageux qu'on peut en attendre[END_REF], Ritter in 1802 [START_REF] Noah | Lectures on Electricity: Comprising Glavanism[END_REF], Frankenhaeuser & Widén in 1955[START_REF] Frankenhaeuser | Anode break excitation in desheathed frog nerve[END_REF], [START_REF] Stämpfli | Membrane potential of a ranvier node measured after electrical destruction of its membrane[END_REF][START_REF] Stämpfli | Membrane potential of a ranvier node measured after electrical destruction of its membrane[END_REF] reported that nerve membrane "electrical breakdown" might explain the electrical conductivity changes in nerves which electric fields have damaged. The bio-medical application of pulsed electric fields began quickly in 1982 with the seminal work of Neumann and colleagues to temporarily permeabilise cell membranes to STATE OF THE ART From iatrophysics to medical physics: then and now -17 -deliver foreign DNA into cells [START_REF] Neumann | Gene transfer into mouse lyoma cells by electroporation in high electric fields[END_REF]. They termed the phenomenon "electroporation". Thus, electroporation-based technology was born.

Another modern science, plasma science, finds its fundamentals established in 1879 by Sir William Crookes (1832Crookes ( -1919) ) when he was experimentally ionising gas in an electrical discharge tube thanks to the application of high voltage through a voltage coil [START_REF] Crookes | On Radiant Matter[END_REF]. Crooks designated the ionised gas "radiant matter". Almost fifty years later, in 1927, when Irvin Langmuir (1881Langmuir ( -1957) ) observed that the flow of certain highly ionised gases in a closed atmosphere resembled that of blood flowing through the vascular system, he proposed, "We shall use the name plasma to describe this region containing balanced charges of ions and electrons" [START_REF] Langmuir | Oscillations in Ionized Gases[END_REF]. This observation gave birth to the current plasma term, whose technology has since been applied in many domains over the past few decades, including medicine. Thus, non-thermal plasma technology was born.

A broad and necessarily selective history of physics's contribution to medicine has been presented. Even briefly introduced, this contribution is undoubtedly essential and diverse. With specific respect to the clinic, most contemporary medical procedures involving those physical inventions have been refined over decades and now are considered safe and beneficial. We now stand on the brink of a new revolution in post-genomic personalised medicine, with physics-based technologies again at the forefront. The following sections of this chapter will offer a general view of electroporation-based and non-thermal plasma technologies that have garnered the rising interest of physicists, biologists, and physicians for their potent applications in various life science fields, including for medical research against cancer -the centre of this thesis.

Electroporation-based technology

History of cell membrane impermeability rupture by electric pulses

The appearance of red spots on animal and human skin exposed to electric sparks observed by Abbé Nollet [START_REF] Nollet | Recherches sur les causes particulieres des phénoménes électriques et sur les effets nuibles ou avantageux qu'on peut en attendre[END_REF] (see history in Chapter 1.1) could be considered the first systemic observations of the thermal damage tissues by pulsed electric fields. This marks the beginning of the history of the electropulsation phenomenon. Over the next two centuries, as mentioned above in that history, this effect was reported in a series of investigations: from Frankenhaeuser and Widén [START_REF] Frankenhaeuser | Anode break excitation in desheathed frog nerve[END_REF] to [START_REF] Stämpfli | Membrane potential of a ranvier node measured after electrical destruction of its membrane[END_REF] for the excitable STATE OF THE ART From iatrophysics to medical physics: then and now -18 -membrane at the Ranvier nodes, followed by a study by Stämpfli where he compared the phenomenon to the breakdown of the dielectric field of a capacitor and reported that under certain conditions, membrane breakdown is irreversible whereas in others it is reversible [START_REF] Stämpfli | Reversible electrical breakdown of the excitable membrane of a Ranvier node[END_REF]. In 1967, a pivotal study by Sale and Hamilton on non-excitable cells of microorganisms under electric fields [START_REF] Hamilton | Effects of high electric fields on microorganisms. II. Mechanism of action of the lethal effect[END_REF][START_REF] Sale | Effects of high electric fields on microorganisms. I. Killing of bacteria and yeasts[END_REF] set the basis for the field of irreversible electroporation. The observations reported by Sale and Hamilton laid the foundation for many future studies in electroporation in general. Kinosita and Tsong, in the 1970s showing high-amplitude pulsed electric fields (PEFs) could be tuned to generate defects of different sizes in erythrocyte membranes ("pores") and that those pores eventually resealed, allowing for selective internalisation of naturally impermeant molecules (Kinosita et al., 1977a[START_REF] Kinosita | Survival of sucrose-loaded erythrocytes in the circulation[END_REF]. These researchers also demonstrated the increase in conductivity that follows the application of high voltage (HV) pulses and the ability of the electric fields to directly cause haemolysis (Kinosita et al., 1977b., Kinosita et al., 1977c). These early contributions supported the theory of aqueous pore formation, published the following year [START_REF] Sugár | A theory of the electric field-induced phase transition of phospholipid bilayers[END_REF].

Those studies were followed by similar ones for planar lipid bilayers in 1979 [START_REF] Benz | Reversible electrical breakdown of lipid bilayer membranes: A charge-pulse relaxation study[END_REF] and lipid vesicles in 1981 [START_REF] Teissié | Electric Field Induced Transient Pores in Phospholipid Bilayer Vesicles[END_REF].

Less than ten years later, as presented in the previous section, Neumann and colleagues managed to use short and intense electric pulses to introduce DNA into a mouse lyoma cell line in suspension to induce the expression of the exogenous gene carried by the DNA (Neumann, 1982;[START_REF] Wong | Electric field mediated gene transfer[END_REF], coining the term "electroporation" and opening the gate for what was called electrogenetherapy (EGT) henceforth. The same year witnessed the use of reversible electroporation to produce a fusion between cells [START_REF] Zimmermann | Electric field-mediated fusion and related electrical phenomena[END_REF]. More than two years were necessary before the second publication on the successful electrotransfer of DNA to eukaryotic cells in vitro by Potter and colleagues [START_REF] Potter | -specific gene expression/B-lymphocyte development/gpt selection)[END_REF], achieved using the ISCO 494 generator -a piece of classical laboratory equipment for proteins and DNA gels electrophoresis. Ever since, microbiologists have employed electrophoresis power supplies as accessible equipment to perform gene transfection in bacteria by electroporation. Another remarkable discovery of PEFs in biology, which has revolutionised the medical field, was the introduction of cytotoxic agents into malignant cells after electroporation, an investigation pioneered by our group in 1987in -1988in (Mir et al., 1988;;Orlowski et al., 1988). Those studies paved the way for the potential of PEFs in cancer treatment since the studied hydrophilic molecules (anticancer drugs netropsin and bleomycin) could not cross the plasma membrane STATE OF THE ART From iatrophysics to medical physics: then and now -19 -in ordinary conditions but diffuse directly into the cytosol under the delivery of PEFs that transiently permeabilised the cell membrane. Indeed, only a few years later, in 1991, our group published two breakthrough papers on the use of reversible electroporation in oncology, an in vivo study (Mir et al., 1991a) and the first clinical trial in the field (Mir et al., 1991b). We termed that application "electrochemotherapy" or ECT. Note that there were also important series of studies: the demonstration of irreversible electroporation in the case of tissue electrical trauma [START_REF] Gaylor | Significance of cell size and tissue structure in electrical trauma[END_REF][START_REF] Lee | Electrical injury mechanisms: Electrical breakdown of cell membranes[END_REF]., Lee et al., 1988), the first skin electroporation used for transdermal drug delivery [START_REF] Prausnitz | Electroporation of mammalian skin: A mechanism to enhance transdermal drug delivery[END_REF], the first in vitro application of HV pulses of sub-microsecond duration (60 ns) [START_REF] Schoenbach | The effect of pulsed electric fields on biological cells: Experiments and applications[END_REF], the first report of the blood flow blockage in the area where applied PEFs during tissue reversible electroporation [START_REF] Ramirez | Electrochemotherapy on liver tumours in rabbits[END_REF], a phenomenon that our group later referred to as the vascular lock [START_REF] Gehl | Vascular reactions to in vivo electroporation: Characterization and consequences for drug and gene delivery[END_REF].

The fundamental principles introduced by those investigations still underlie our current understanding of the electropulsation phenomenon, whose mechanisms will be briefly discussed in the next section. This cell membrane rupture of impermeability by the electric pulses has resulted in the electroporation-based technology, whose versatility has found its applications in daily life and biomedicine. Those applications will be further described in chapters 2 and 3, focusing on electrochemotherapy as this thesis's primary field of study.

A mechanistic perspective

Electropermeabilisation at the cell level

Let us look back at our cellular membrane, whose detailed organisation remains somewhat elusive. Typical mammalian plasma membranes (PM) contain hundreds of different lipids with a highly non-uniform lateral mixing [START_REF] Ingólfsson | Lipid Organization of the Plasma Membrane[END_REF]. Of only 5 nm of thickness with a eukaryotic cell of about 15 to 50 µm in diameter, the PM regulates all the exchanges between the outside and the inside of the cell in an exact manner. In particular, the PM acts as a barrier that prevents the free diffusion of not only vitally essential molecules, such as all the substrates or products of the cell metabolism, ions, sugars, and amino acids, but also other large hydrophilic molecules. Life would not be possible without controlling the transport of substances across the cell membrane. Together with the small hydrophilic molecules that are STATE OF THE ART From iatrophysics to medical physics: then and now -20 -not transported through channels or pumps of the PM, these molecules (termed "nonpermeant" molecules) can only reach specific cell compartments of the cell (but neither the cytosol nor the nucleosol) by endocytosis/exocytosis.

To allow the uptake of those non-permeant molecules, perturbations must be initiated at the PM level, i.e., concerns that transiently break up the PM impermeability. One of the most popular and practical methods to reversibly permeabilise the PM consists of applying adequate PEFs -a theory firstly elaborated by Neumann and colleagues, as mentioned above, and which they termed "electroporation". Their electroporation theory states that the electric pulsesinduced transmembrane voltage difference can enlarge the normal fluctuations of the PM, resulting in their large hydrophobic pores. These pores become hydrophilic by rotating the lipids at the limits between the lipid bilayer and the aqueous medium that tries to fill the hydrophobic "pore" (conduit).

To explain this theory as early as it was reported, some 1D approximations of the PM were analytically modelled [START_REF] Bernhardt | On the generation of potential differences across the membranes of ellipsoidal cells in an alternating electrical field[END_REF][START_REF] Litster | Stability of lipid bilayers and red blood cell membranes[END_REF]. If considering the cell as a 1D model (Figure 3a) at a physiological state, the cell PM has a native transmembrane potential difference or ΔψO, whose value depends on the species and the cell type [START_REF] Escoffre | Electrochemotherapy: Progress and Prospects[END_REF].

ΔψO is found generally around -70 mV for excitable cells. The exposure of living cells to shortduration and intense electric HV pulses induces electrophoretic movement of ionic charges (mostly small ions) on both sides of the plasma membrane, i.e., an induced transmembrane potential (Δψi). This generates a transient increase in the permeability level of the PM.

Δψi is defined by the Schwan equation (a derivation of Laplace's equation published by H.

Schwan [START_REF] Schwan | Electrical properties of tissue and cell suspensions[END_REF]):

Δψi = 3 /2 × Eext × r × cos (Θ)
where [START_REF] Gimsa | Analytical description of the transmembrane voltage induced on arbitrarily oriented ellipsoidal and cylindrical cells[END_REF][START_REF] Kotnik | Analytical description of transmembrane voltage induced by electric fields on spheroidal cells[END_REF][START_REF] Schwan | Electrical properties of tissue and cell suspensions[END_REF]:

Eext: the external electric field strength r: the radius of the cell Θ: the polar angle between the field line and a normal from the centre of the cell (approximated by a sphere) to the point of interest on the cell membrane When the transmembrane potential difference net value (the sum of the vectorial values of the induced and resting potential differences or ΔψO + Δψi) is more significant than 200 to 300 mV, there will be the formation of short-lasting pores in the PM with chemical modification of the lipids. These transient permeation structures are generated at the cell membrane level STATE OF THE ART From iatrophysics to medical physics: then and now -21 -because the membrane structure has to endure the electro-compressive forces caused by this potential difference. Under the electric field, water dipoles orient and form almost single molecule-thick columns that penetrate the lipid tails' layer. Phospholipid heads also move towards the interior of the PM, and columns rapidly enlarge to form hydrophilic conductive large pores ("pre-pore"), resulting in its permeabilisation.

With time, there are many more studies investigated not only in in silico using molecular dynamics (MD) simulations (some will be discussed in the next section) but also in vitro to better under the electroporation phenomenon [START_REF] Azan | Cell membrane electropulsation: Chemical analysis of cell membrane modifications and associated transport mechanisms[END_REF]Breton et al., 2012a;[START_REF] Gabriel | Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane[END_REF]Kinosita, 1977a;[START_REF] Kotnik | Membrane Electroporation and Electropermeabilization: Mechanisms and Models[END_REF]Rols et al., 1998a;[START_REF] Teissié | An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization[END_REF] The localisation and asymmetry of electropermeabilisation can be detected by propidium iodide uptake in a CHO cell submitted to a train of 10 electric pulses, 5 ms, 1 Hz at 700 V/cm. Adapted from [START_REF] Bernhardt | On the generation of potential differences across the membranes of ellipsoidal cells in an alternating electrical field[END_REF][START_REF] Escoffre | Electrochemotherapy: Progress and Prospects[END_REF]Kinosita, 1977a;[START_REF] Teissié | An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization[END_REF].

The resting transmembrane potential is essential for the permeabilisation threshold and the succession of events. Permeabilisation will initially happen at the pole of the cell facing the anode (the positive electrode), where the capacitance of the membrane is first exceeded when an external electric field is applied. The second event is the permeabilisation of the pole of the STATE OF THE ART From iatrophysics to medical physics: then and now -22 -cell facing the cathode (the negative electrode). The extent of permeabilisation (area of the permeabilised membrane) on the pole facing the anode can be controlled by pulse amplitude, i.e., the higher the pulse amplitude, the greater the area through which diffusion can take place [START_REF] Gabriel | Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane[END_REF]. The degree of permeabilisation can be controlled by the pulse duration (and pulse number), i.e., the longer the pulse, the greater the perturbation of the membrane in a given area [START_REF] Gabriel | Direct observation in the millisecond time range of fluorescent molecule asymmetrical interaction with the electropermeabilized cell membrane[END_REF]. Depending on the electric field strength and the duration of the exposure of cells, the membrane electroporation can be either reversible or irreversible, with or without thermal damage (Figure 4).

Figure 4| Schematic of different effects on cells exposed to electric fields, including the consequent thermal effects

Reversible electroporation, irreversible electroporation and thermal damage zones are displayed as functions of electric field strength and pulse length (exposure duration). Note that the field scale is logarithmic and approximative. Figure adapted from [START_REF] Bower | Irreversible electroporation of the pancreas: Definitive local therapy without systemic effects[END_REF][START_REF] Yarmush | Electroporation-Based Technologies for Medicine: Principles, Applications, and Challenges[END_REF]).

• Reversible electroporation is defined as the temporary permeabilisation state of a cell exposed to an electric field. The presence of the electric field disturbs the phospholipid bilayer, allowing molecules to reach the cell interior through the cell plasma membrane. The cell membrane will recover its initial state afterwards (Figure 4, lower panel).

• Irreversible electroporation also referred to as non-thermal irreversible electroporation or IRE, is defined as the permanent permeabilisation state of a cell exposed to HV electric pulses. It induces the loss of homeostasis (lethal biochemical imbalance) and apoptotic-like cell death by irrecoverable disruption of the cell membrane (Figure 4, upper panel). Consequently, sooner or later, all molecules will STATE OF THE ART From iatrophysics to medical physics: then and now -23 -be able to freely cross the cell membrane when it cannot fully recover its initial impermeability.

Electroporation of the lipid bilayer: molecular mechanisms

Since electroporation, as it is called, is a succession of events, our group also proposed the term electropulsation (comprises cell electroporation followed by cell electropermeabilisation) to better describe the action of PEFs on the cell membrane. The kinetic of electropulsation phenomenon can be described in five major steps, with timescale [START_REF] Kotnik | Membrane Electroporation and Electropermeabilization: Mechanisms and Models[END_REF][START_REF] Rols | Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon[END_REF][START_REF] Teissié | Mechanisms of cell membrane electropermeabilization: A minireview of our present (lack of ?) knowledge[END_REF]:

• Initiation or creation of pores (ns for the conductivity, and µs for the permeability):

the applied PEF induces a membrane potential difference and reaches the critical value (E = EPoration). The plasma membrane is destabilised (mechanical stress with a magnitude that depends on the buffer composition), some membrane defects are generated and permeabilisation begins.

• Expansion (up to µs): these defects expand on the cell surface as long as the PEF strength is larger than a critical value (E > EPoration) and the density of the defects increases as long as the field is present (cumulated duration);

• Stabilisation and partial recovery (µs for the conductivity, and ms for the permeability): points of cell permeabilisation for small molecules remains but the membrane retrieves its organisation as soon as the PEF is subcritical (E < EPoration);

• Resealing (seconds to minutes at about 20-37°C, and hours at about 4°C): once the PEF is turned off (E < EPoration until E = 0), the membrane gradually loses its permeability (the annihilation of leaks is slow), returning to its initial impermeable state (unless damages are irreversible, leading to the loss of cell viability after this step);

• Memory (hours): some structural changes and physiological properties recover on a much longer time scale while the cell viability is preserved.

MD simulation was employed to simulate and understand the effect of PEFs at the cell membrane level (Figure 5), offering a better visualisation of the electroporation (Delemotte et al., 2012;[START_REF] Tarek | Membrane electroporation: A molecular dynamics simulation[END_REF]. This computational methodology simulates the evolution of the atoms composing the cell membrane and its surroundings for tens of nanoseconds. Pore generation can occur during the computational time if the force corresponding to the simulated electric STATE OF THE ART From iatrophysics to medical physics: then and now -24 -field is high enough. This process is stochastic, and simulations often require a high field amplitude. Since, in the calculations of MD, each molecule is considered, the region of interest is limited by the number of elements that the computer can handle simultaneously; thus, the constraints in both sample size and the duration of the simulation (especially that of ns timescale) [START_REF] Azan | Cell membrane electropulsation: Chemical analysis of cell membrane modifications and associated transport mechanisms[END_REF]. Otherwise, too heavy MD simulations have to be run, with a computational cost challenging to afford. Using MD simulation, Vernier and colleagues have demonstrated that electroporating fields target oxidatively damaged areas in the cell membrane (Vernier et al., 2009); Yusupov et al. also revealed the synergistic effect of electric field and lipid oxidation on the permeability of cell membranes (Yusupov et al., 2017), elucidating the dynamics of the permeation process under the influence of combined lipid oxidation and electroporation. [START_REF] Kotnik | Membrane Electroporation and Electropermeabilization: Mechanisms and Models[END_REF][START_REF] Levine | Life cycle of an electropore: Field-dependent and fieldindependent steps in pore creation and annihilation[END_REF]. (b) Electrotransfer of a double-stranded siRNA (yellow and green spheres) through an aqueous pore within a model DOPC membrane (headgroups showed as blue and grey spheres and hydrophobic tails as cyan lines) driven by nanosecond electric pulses. When being submitted to a pulse of sufficient field strength, the siRNA is dragged by electrophoresis towards the membrane and several of the siRNA phosphate groups strongly associate with the cholines of the phospholipid headgroups. If the siRNA is close to an electropore (i.e., a field-induced hydrophilic pore), the siRNA can slide electrophoretically along this pore, against the field direction, being finally detached from the membrane phospholipids by electrophoresis. In the simulation, the translocation of a 23 bp siRNA can be completed within less than 10 ns under a transmembrane potential difference of 1.6 V. For clarity, water molecules are omitted, and the lipid headgroups initially in interaction with the siRNA strand are depicted in red to highlight their lateral diffusion. (Breton et al., 2012b;[START_REF] Mir | Electroporation-based gene therapy: Recent evolution in the mechanism description and technology developments[END_REF] Figure 5a illustrates an electropore in a lipid bilayer [START_REF] Kotnik | Membrane Electroporation and Electropermeabilization: Mechanisms and Models[END_REF][START_REF] Levine | Life cycle of an electropore: Field-dependent and fieldindependent steps in pore creation and annihilation[END_REF]. Pore creation in an electric field begins with introducing water into the bilayer, followed by the reorganisation of the phospholipid head groups in each leaflet around the defect (pore construction). Migration of additional water and head groups into the created pore continues until an arbitrarily defined mature pore structure is formed (pore maturation). Pore annihilation begins with the removal of the "porating" electric field. The pore structure is quasistable at this time (pore destabilisation). Still, soon after that, there is a decrease in pore size as head groups and water begin to migrate out of the membrane interior (pore degradation).

The head groups separate again into two groups (pore deconstruction). Water quickly follows (pore dissolution), and the intact structure of the bilayer is restored. Figure 5b illustrates the mechanism of electrotransfer of a double-stranded siRNA through an aqueous pore within an STATE OF THE ART From iatrophysics to medical physics: then and now -26 -artificial lipid membrane of a giant unilamellar vesicle (GUV) synthesised with 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), under nanosecond pulses (Breton, 2012b;[START_REF] Mir | Electroporation-based gene therapy: Recent evolution in the mechanism description and technology developments[END_REF].

Despite the constraints of heavy MD simulations, our group succeed to predict the electropulsation phenomenon with ns pulses (Breton, 2012b).

For the in vitro study of the electroporation phenomenon, there are several techniques developed to detect plasma membrane electroporation. Those physical and chemical methods are highlighted in the graphic shown in Figure 6 [START_REF] Napotnik | In vitro electroporation detection methods -An overview[END_REF].

Electric pulse generator and electrodes

The delivery of EPs to cells or tissues can be performed using both home-made [START_REF] Zerbib | Electric field mediated transformation: Isolation and characterization of a TK+ subclone[END_REF] or commercially available electric pulse generators. Some CE approved generators authorised for application in humans are presented in Figure 7. parameters for immediate management and later retrieval and analysis. Furthermore, this square-wave pulses Cliniporator™ can deliver a high pulse frequency, enabling the use of more complex electrodes (Marty et al., 2006). Many preclinical [START_REF] André | Efficiency of high and low voltage pulse combinations for gene electrotransfer in muscle, liver, tumor and skin[END_REF][START_REF] Čemažar | Control by pulse parameters of DNA electrotransfer into solid tumors in mice[END_REF] and clinical [START_REF] Gehl | Results of the ESOPE (European Standard Operating Procedures on Electrochemotherapy) study: Efficient, highly tolerable and simple palliative treatment of cutaneous and subcutaneous metastases from cancers of any histology[END_REF][START_REF] Snoj | Limb sparing treatment of bleeding melanoma recurrence by electrochemotherapy[END_REF][START_REF] Spanggaard | Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma: Safety and efficacy results of a phase i firstin-man study[END_REF] studies using electroporation involved the Cliniporator™. This computer-controlled medical device provides two types of EPs:

• short (microsecond time scale), high voltage (HV, 100-1000 V) pulses

• long (millisecond time scale), low voltage (LV, 20-200 V) pulses

Considerable advances have been made in the fabrication of electroporation equipment for pre-clinical studies and clinical uses, among which has found the electrodes -one essential aspect of the electroporation [START_REF] Gehl | In vivo electroporation of skeletal muscle: Threshold, efficacy and relation to -147 -electric field distribution[END_REF][START_REF] Gilbert | Novel electrode designs for electrochemotherapy[END_REF]. Four types of fixed geometry electrodes are available (Figure 8):

• Non-invasive plate electrodes For successful therapy, the entire target tissue must be exposed to the local electric field strength above the reversible threshold. There are thus electrodes developed recently for the treatment of deep-seated tumours. Those will be presented in chapter 3. The choice of the EP type and the choice of the electrodes will also be addressed in sections dedicated to electrochemotherapy (ECT) and electrogenetransfer (EGT). The efficiency of ECT strongly depends on the local electric field distribution inside the target tissue.

Non-thermal plasma technology

Plasma and the introduction of non-thermal plasma in biology and medicine

There are four fundamental states of matter: solid, liquid, gas, and plasma (Figure 9a). As the energy exerted on atoms increases, the thermal motion of atoms in the solid aggravates and finally overcomes the restrictive interaction between particles in the solid such as ionic bonds and forms liquid. Similarly, when the atoms in liquid obtain adequately significant energy to overcome the restrictive Van der Waals force from surrounding atoms, these liquid atoms will transfer into gas atoms. The translational energy of atoms in gas is much larger than that in liquid and solid. When the enthalpy is large enough for the electron to overcome the electrostatic potential barrier, the electron will be stripped away, creating a free electron and a positively charged ion (an ionisation process). The field energy causes the free electrons to accelerate and ionise the gas atoms and molecules, which release more free electrons that provoke new ionisations. At the same time, excited electrons produce molecular dissociations, forming new atoms and free radicals, which can excite atoms and molecules to higher energy STATE OF THE ART From iatrophysics to medical physics: then and now -29 -levels. When returning to a more stable state, excited atoms and molecules emit excess energy in broad-spectrum electromagnetic radiation, such as ultraviolet (UV) radiation. As a result, the plasma is constituted essentially by molecules and atoms in an excited state, positive and negative ions, free radicals, electrons, UV radiation and reactive oxygen and nitrogen species (RONS), such as atomic oxygen (including ozone (O3)), superoxide (O2 -), hydroxyl radicals ( • OH), singlet oxygen ( 1 O2), nitric oxide (NO • ) or nitrogen dioxide (NO2). Plasma is thus defined as an ionised gas in which many atoms have lost one or several electrons and exist in a mixture of free electrons and positive ions [START_REF] Eliezer | The Fourth State of Matter: An Introduction to Plasma Science[END_REF]. In simple terms, plasma is an electrified gas with a chemically reactive media mixing a large number of different species such as electrons, positive and negative ions, free radicals, gas atoms and molecules, in the ground or any higher state, of any form of exciting species. Plasma is formed when a high voltage or temperature knocks off the outermost electrons from an atom, which in turn knock off electrons in the neighbouring atoms.

Temperature (enthalpy) increases when matters transform from solid to liquid to gas and plasma. The plasma temperature is determined by the thermal motions of electrons and heavy particles (atoms and ions); hence plasma is generally distinguished into two main groups: the STATE OF THE ART From iatrophysics to medical physics: then and now -30 -high temperature or fusion plasmas and the low temperatures or gas discharges. The lowtemperature plasma is further divided into two groups, resulting in a classification of three distinctive kinds of plasma:

• High temperature (equilibrium) plasma: intensive collisions between all particles (electrons, ions and neutral species) increase their density. All particles have the same temperature and thus are in thermal equilibrium.

• Thermal (quasi-equilibrium) plasma: plasma in a local thermal equilibrium state.

• Non-thermal (non-equilibrium or cold) plasma: generated at reduced pressures, the heavy particle temperature of NTP ranges between +25°C and +50°C (usually less than 40°C at the point of the application). All particles are not in thermal equilibrium.

The major components of a non-thermal plasma are electrons, ions, and radicals such as reactive oxygen and nitrogen species (ROS, RNS or also called RONS), mild thermal radiation, UV photons and visible light (Figure 9b).

Typical classification and parameters of plasma with some common applications are listed in Adapted from [START_REF] Sakudo | Disinfection and sterilization using plasma technology: Fundamentals and future perspectives for biological applications[END_REF] In nature, we find plasma in lightning as temporary electrical discharges, the solar wind or the northern light (aurora borealis). More than 99% of the Universe is composed of plasma. Our

Sun is in a gigantic plasma ball whose core reaches 108 K, and since almost all of the stars that we observe in the sky are suns, they are in the high-temperature plasma state. Thunderstorm lightning is an example of thermal plasma that can find a temperature of approximately 2 × 10 4 K. For daily life applications, we see the plasma technology applied for microplasma welding (a method used to join paper-thin sheets of metals, e.g., for the manufacture of stainless-steel water storage tanks or kitchen implements), plasma spray (the only process to coat any material onto any other one), printers (plasma makes plastics attract or repel liquids), neon lights, and plasma TV. Examples of plasma physics applications for applied sciences are international communications, energy power generators, and the nuclear fusion bomb (the hydrogen bomb).

Plasma Science and technology have offered a valuable contribution to human health for more than 50 years. Since thermal plasma can reach up to several thousand Celsius degrees, it is used in applications where high temperatures are required, such as casting processes in the metallurgical industry or chemical synthesis processes (e.g., production of acetylene from natural gas). Non-thermal plasma (NTP), with temperatures close to ambient temperature, is, on the contrary, suitable for the treatment of heat-sensitive materials even though the mean electron energies of NTP is about 20 eV, considerably higher than those of the components of the ambient gas. NTP thus finds its utilisation in biotechnology and medicine, starting with experiments conducted in the 1960s and 1970s for biological applications. For example, NASA (The National Aeronautics and Space Administration) investigated oxygen plasma to destroy natural matter and inactivate microorganisms for space applications [START_REF] Bol'shakov | Radio-Frequency Oxygen Plasma as a Sterilization Source[END_REF][START_REF] Mogul | Impact of low-temperature plasmas on Deinococcus radiodurans and biomolecules[END_REF]. After decades of experience in biomaterials, in 1971, scientists began working with a device that took advantage of "the plasma state" properties to produce simultaneous division of tissue and coagulation of blood vessels. Thermal plasma, e.g., plasma knife (plasma scalpel), was first demonstrated to heal the wound of mouse skin (Link et al., STATE OF THE ART From iatrophysics to medical physics: then and now -32 -1973) and a few years later, was used for surgery -the work pioneered by Glover and colleagues in 1978[START_REF] Glover | The use of thermal knives in surgery: Electrosurgery, lasers, plasma scalpel[END_REF][START_REF] Glover | The plasma scalpel: A new thermal knife[END_REF]. Despite this historical connection, thermal plasma is typically associated with high energy physics or low-pressure processes employed in the semiconductor industry while being rarely used in medical applications directly. The few known uses of thermal plasma in medicine are based mainly on the high temperature generated by conventional thermal discharges. One good example of this is the Argon Beam or Argon Plasma Coagulator (APC), developed primarily to cauterise wounds [START_REF] Ginsberg | The argon plasma coagulator: February 2002[END_REF]. This device generates thermal plasma in flowing argon through HF (≥ 350 kHz) electrical discharge ionisation. The argon flow takes thermal plasma outside the ionisation tube, creating a jet that impinges onto the tissue. The high temperature of the thermal plasma (about 10,000 K) leads to rapid cauterisation and tissue desiccation. This APC technique has been employed successfully in open surgery and endoscopy for haemostasis and thermal devitalised pathological tissues, e.g., in the otorhinolaryngology [START_REF] Bergler | Argon plasma coagulation (APC) surgery in otorhinolaryngology[END_REF][START_REF] Grund | Endoscopic argon plasma coagulation (APC) first clinical experiences in flexible endoscopy[END_REF][START_REF] Peng | Efficacy and Safety of Argon Plasma Coagulation for Hemorrhagic Chronic Radiation Proctopathy: A Systematic Review[END_REF]. The procedure is yet painful, but significant thermal tissue damage (up to 7 mm deep) results in prolonged healing [START_REF] Ginsberg | The argon plasma coagulator: February 2002[END_REF][START_REF] Vargo | Clinical applications of the argon plasma coagulator[END_REF]). Today's medical community is shifting the preference toward non-equilibrium room temperature (RT) discharges where thermal damage is minimised or eliminated. Fridman and colleagues' study has introduced direct NTP treatment of living tissue where sterilisation occurs without any visible or microscopic damage, and blood is coagulated in an open wound without damaging the surrounding tissue [START_REF] Fridman | Blood coagulation and living tissue sterilization by floatingelectrode dielectric barrier discharge in air[END_REF] (see Figure 12 in chapter 2.2). Initial steps in eliminating thermal damage by NTP have been made earlier, e.g., pouring saline over the arc to cool it off, preventing any significant tissue desiccation. A more sophisticated NTP-based surgical tool that has recently been reported is the Pulsed Electron Avalanche Knife (PEAK), where thermal damage to the tissue was reduced by keeping the current pulses short (microseconds) and the electrode thin (microns) [START_REF] Miller | Precision and Safety of the Pulsed Electron Avalanche Knife in Vitreoretinal Surgery[END_REF]. The resulting streamers (micro-sparks) of NTP, forming under the micro-wire, deposit significant energy into the tissue, rupturing it quickly without destroying surrounding areas, thus performing the precision cutting. Further advances in the non-thermal direction have been made by further size reduction of the needle tip and introduction of noble gases, where discharge power can be significantly lower. A discharge capacity of a much gentler, non-thermal interaction with tissue, "plasma needle", has been proposed to treat dental cavities and skin disorders demonstrated to destroy cells and bacteria in a highly localised manner without disturbing the nearby tissues STATE OF THE ART From iatrophysics to medical physics: then and now -33 -(Stoffels et al., 2002). It involves a "glow" discharge igniting at the end of a sharp pin in flowing helium upon applying an RF electromagnetic excitation (about 13 MHz). This discharge operates near RT, dissipating milliWatts in several cubic millimetres. Physicists, chemists, engineers and even medical doctors have actively engaged in the race for the research and development of NTP devices suitable for applications in biotechnology and medicine. Details of those innovative devices and their implications in different fields will be discussed later in chapters 2 and 3.

Plasma sources and diagnostics

Primary sources of NTP for biological applications

NTP in biomedical applications is mainly generated by electrical discharge methods whose type of discharge depends on the waveform and the frequency of the power source, such as direct current (DC) and alternating current (AC) discharge, as well as ambient gas pressure, such as low-pressure and atmospheric pressure plasma, and the precise shape and configuration of the electrodes. Electrical discharge methods are generally categorised into glow discharge, corona discharge, atmospheric pressure plasma jet (APPJ), dielectric barrier discharge (DBD), micro-hollow cathode discharge (MHCD), DC discharge, and pulse discharge or high/lowfrequency discharge. Table 2 lists some of those working at atmospheric pressure NTP and used for biomedical applications [START_REF] Sakudo | Disinfection and sterilization using plasma technology: Fundamentals and future perspectives for biological applications[END_REF]. Adapted from [START_REF] Sakudo | Disinfection and sterilization using plasma technology: Fundamentals and future perspectives for biological applications[END_REF].

Discharge type (at

STATE OF THE ART

From iatrophysics to medical physics: then and now -34 -There is no universal prerequisite for the composition of plasmas. Most NTP is produced with noble gases, commonly pure helium or argon but can also be made from the air or other mixtures. There are, however, limits to the production of plasmas concerning electric current, UV radiation (maximum dose allowed) and the output of RONS (limits for O3, NO2, NO etc.) for the safety of their utilisation [START_REF] Heinlin | Plasma medicine: possible applications in dermatology[END_REF].

Two predominant sources of NTP discharge devices used in biomedicine can be distinguished [START_REF] Heinlin | Plasma medicine: possible applications in dermatology[END_REF][START_REF] Hoffmann | Cold Atmospheric Plasma: Methods of production and application in dentistry and oncology[END_REF][START_REF] Pouvesle | Applications thérapeutiques des plasmas froids atmosphériques[END_REF]:

• Direct plasma discharge sources, typically dielectric barrier discharge (DBD), use the target area as a counter electrode (Figure 10a), creating relatively homogenous plasmas that contain high concentrations of plasma-generated species with relatively weak UV radiation. The electrical discharge between two electrodes of a DBD (an HV electrode and a grounded one) is isolated by an insulating layer (barrier).

DBD devices must also be charged only with bipolar or HV pulses to ensure capacitive coupling. Since the target itself (tissue/skin/biological sample) serves as an electrode, the electrical current flow through the target (body). The distance between the DBD and the biological target is typically 1 mm. DBD is one of the most, if not the most, popular devices to generate NTP. Originally called silent (inaudible) discharge, DBD was also known as partial discharge or ozone production discharge, as it was firstly reported in 1857 by Ernst Werner von Siemens (1816-92). He generated ozone by subjecting a flow of oxygen or air to the influence of a DBD maintained in a narrow annular gap between two coaxial glass tubes. This was achieved by an alternating electric field of sufficient amplitude [START_REF] Kogelschatz | Dielectric-barrier Discharges: Their History, Discharge Physics, and Industrial Applications[END_REF].

• Indirect discharge sources, typically plasma jets (Figure 10b andc), refer to various discharge systems (hence numerous configurations) used in plasma science. The plasma jet commonly refers to a system where the carrier gas discharge (noble gas/air) is operated in a non-sealed electrode arrangement, generating a relatively more robust UV radiation and lower density of reactive species on the target in comparison to the DBD. The gas temperature at the production site of the jets is around 40-60°C. The plasma jets can be categorised according to discharge geometry, electrode arrangement, excitation frequency or pattern [START_REF] Winter | Atmospheric pressure plasma jets: An overview of devices and new directions[END_REF]. Thanks to the configuration of the plasma jet, the distance between the STATE OF THE ART From iatrophysics to medical physics: then and now -35 -production site of the jet and the biological target can easily be greater (up to a few centimetres) than that using the DBD.

• Moreover, larger surfaces can also be created by joining single jets or multielectrode systems. Recently, the team of Robert at the GREMI (Orléans, France) has developed NTP multi-jet systems using two different setups based on a single plasma source, offering a large treatment surface covered by multiple single jets (Figure 10c) [START_REF] Robert | New insights on the propagation of pulsed atmospheric plasma streams: From single jet to multi jet arrays[END_REF]. This thesis used one of the NTP multi-jet systems from the

GREMI.

Another NTP discharge source, the hybrid plasma devices or coronal barrier discharges, combines both principles described above but are currently applied only at the practical level.

Other sources for the plasma generation are corona pin electrode, HF discharge, gliding arc, pen-type Tsukuba mild plasma, and Nagoya non-equilibrium atmospheric pressure plasma (NEAPP). Different NTP sources are recapitulated in the review of [START_REF] Braný | Cold Atmospheric Plasma: A Powerful Tool for Modern Medicine[END_REF][START_REF] Tanaka | State of the art in medical applications using non-thermal atmospheric pressure plasma[END_REF]. The four specific types of NTP devices having CE approval for clinical practice will be [START_REF] Maho | Atmospheric Pressure Multijet Plasma Sources For Cancer Treatments[END_REF][START_REF] Robert | New insights on the propagation of pulsed atmospheric plasma streams: From single jet to multi jet arrays[END_REF].

Although the control of the plasma composition remains an important challenge, the direct discharge sources can control the plasma composition more easily as compared with other discharge devices. Nonetheless, their major disadvantage is the application distance (between the electrodes) which must remain within a close range, generally less than 3 mm, thus limiting its use for small areas of the human body. To that, even if the concentration in RONS generated by the indirect discharge sources is lower than that of direct discharge sources and the plasma approach allows for novel treatment of biological and medical surfaces where no tissue damage is observed while some biological processes are initiated and/or catalyzed. Additionally we offer a model describing the influence of this e-plasma on concentrations of various ions in blood plasma. 4 The principle of operation of the proposed e-plasma is similar to Dielectric Barrier Discharges (DBD) introduced by Siemens in the middle of 19th century [7,8]. It occurs at atmospheric pressure in air when sufficiently high voltage of continuous waveform or pulses of short duration are applied between two insulated electrodes. The presence of the insulator between the electrodes prevents the build-up of high current. As a result, the discharge creates e-plasma without substantial heating of the gas, thus offering no medium limitations (i.e. need for lower pressure or noble gases). We found that it is possible to replace one of the electrodes by an object with high capacity for charge storage-a "floating electrode"(FE). Living tissue of animal or human body with its high water content and a relatively high dielectric constant has the required high capacity for charge storage [9,10] and, therefore, can easily be employed as the FE of the DBD e-plasma. In this case the FE-DBD e-plasma is created in the gap between the living tissue and the other insulated electrode. While the current in the gaseous discharge gap is mainly due to motion of charge carriers (electrons and ions), it continues mostly in the form of displacement current through the tissue. Moreover, most of the energy due to the e-plasma current is dissipated in the gap, just like in the case of a conventional DBD. Thus, in the FE-DBD we obtain non-thermal e-plasma that remains at room temperature throughout the operation of the system while active species, radicals, ultraviolet radiation, and sub-millimeter scale temperature fluctuations offer "synergetic" effect in tissue treatment. Moreover, the e-plasma generated in this way can be applied directly to a living human tissue without thermal or chemical damage [11][12][13]. In addition to sterilization of tissue, we demonstrate that the FE-DBD e-plasma rapidly coagulates blood. Rather than a physical influence, we observe that e-plasma catalyses the natural blood coagulation processes as was confirmed by experimental data and a model based on e-plasma influence on ion concentrations in blood plasma.

STATE OF THE ART From iatrophysics to medical physics: then and now -36 -generated is less controllable, the use of different specific carrier gases, the variation of the distance to the target, the configuration of the electrodes, amongst several other characteristics, make plasma jet possible to obtain changes in plasma composition using classical plasma jets (Isbary et al., 2013b).

Plasma experimental observations and diagnostics

As discussed above, one of the essential properties of NTP is its high chemical reactivity at low gas temperatures and even at RT. These properties are necessary for interaction with biological targets. For essential applications such as plasma medicine, the knowledge of the fluxes of the discharge species and the relevant elementary processes, i.e., plasma observations and diagnostics, are essential. Having usually small dimensions (typical from micrometre to the millimetre) and non-uniform distributions of the species densities, amongst other parameters [START_REF] Schulz-Von Der Gathen | Visible tumor surface response to physical plasma and apoptotic cell kill in head and neck cancer[END_REF], NTP is not easy to characterise. Non-invasive optical diagnostic techniques with the high special resolution are, to date, the methods of choice for the diagnostics of NTP and for the slightest disturbance of the plasma properties. Those techniques are selectively described in the review of [START_REF] Lu | Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects[END_REF]. To cite some:

Plasma experimental observations by:

• Fast imaging: can be obtained with phase-resolved imaging or optical emission spectroscopy (to image dynamic processes in plasma with picosecond time resolution at MHz acquisition rates) or with gated and triggered intensified chargecoupled device (ICCD) cameras (to detect and monitor plasma bullets with pico-to nanosecond precision) or with streak cameras (high time resolution (but 2D) for the monitoring of highly-dynamic breakdown processes)

• Laser-induced fluorescence (LIF) spectroscopy: single-and two-photon absorption laser-induced fluorescence, scattering techniques (Rayleigh scattering, Thomson and Raman scattering), optical emission and optical absorption spectroscopies, selected non-optical methods (molecular beam mass spectrometry, flow visualisation, electron paramagnetic resonance spectroscopy)

Plasma electrical characterisation:

• Voltage-current characteristics: the temporal behaviour of the voltage is measured using an HV probe and a voltage divider (with specific characteristics) for voltages above 500 V; the temporal behaviour of an electric current is calculated using STATE OF THE ART From iatrophysics to medical physics: then and now -37 -voltage measurements across known impedances (another method is to measure pulsed and alternative currents using current transformers and Rogowski coils). The DIN SPEC 91315:2014-06 (Deutsches Institut für Normung e.V. Berlin, DE) is implemented to characterise plasma sources with their biological performance (e.g., risk and safety potential).

• Electric field: investigated by an electro-optic sensor equipped with an isotropic crystal probed by a laser beam. One example is the Kapteos eoProbe™ from the Kaptéos Society (France). This Ultra-Wide Band sensor allows accurate electric field measurements in almost any location and environment (air, liquids, gases, vacuum) under harsh conditions.

Besides, electron paramagnetic spectroscopy can detect radical species generated in the plasma by measuring unpaired electrons (dangling bonds, radicals, transition metal complexes, etc.).

Principles of non-thermal plasma treatment in biomedicine

NTP has a myriad of potential not only in the physics and material industry (ozone generation, gas purification, electrostatic precipitation, combustion and surface functionalisation of materials, etc.) but also in biotechnology and medical applications.

Alongside well-known applications such as using low energy plasmas for decontamination, sterilisation or even wound healing or using higher energy plasmas to cut bones during an operation or for blood coagulation, etc., therapeutic applications of NTP are under research.

They have recently garnered significant interest, especially in the cancer treatment [START_REF] Fridman | Applied plasma medicine[END_REF].

Despite that wide range of applications, the interaction and thus the effects of NTP on microorganisms or living tissues and cells are not fully understood. Whether there are or not distinctive effects of NTP treatment on bacteria, microorganisms or cells, the biological impact of NTP could be explained by the generation of a large number of different species within the plasma region and the afterglow (electrons, ions, gas atoms and molecules of any form of exciting species, UV and heat radiation, electromagnetic fields, and especially free radicals), as shown in Figure 9b. If the plasma is in contact with a biological target (e.g., microorganisms, skin, tissue, cells) or is exposed to a liquid (e.g., water, saline solutions, cell culture media, etc.), STATE OF THE ART From iatrophysics to medical physics: then and now -38 -those species that are delivered from the plasma to the targets will interact across different regions in the gaseous and liquid states, generating a "cocktail" of reactive species of varying life span and characteristics. And yet radicals are essential to induce physiological outputs in living cells and tissues [START_REF] Trachootham | Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?[END_REF].

For example, depending on the plasma dose and voltage value producing the plasma discharges, NTP may trigger either physical destruction of the bacteria or a programmed cell death [START_REF] Lunov | The interplay between biological and physical scenarios of bacterial death induced by non-thermal plasma[END_REF][START_REF] Sakudo | Disinfection and sterilization using plasma technology: Fundamentals and future perspectives for biological applications[END_REF]. In general, the effects of NTP treatment were mainly attributed to [START_REF] Bourke | Microbiological interactions with cold plasma[END_REF][START_REF] De Geyter | Nonthermal Plasma Sterilization of Living and Nonliving Surfaces[END_REF]: UV-based DNA damage in combination with the erosion of microorganism structures by UV-based photo-desorption and etching processes by reactive species; the deleterious impact of ionised particles on the membranes (membrane damage, membrane perforation by etching due to highly reactive gas radicals); ion accumulation increases the eternal electrostatic pressure that destroys mechanically rigid bacterial wall structures; or interactions with RONS leading to oxidative damage and modification of cytoplasmatic membrane, proteins, and DNA, etc. Indeed, according to the current state of knowledge, the biomedical effects of NTP were mainly based on the action of reactive oxygen and nitrogen species (RONS) and attributed to redox-controlled cellular processes [START_REF] Bauer | Mechanisms of Selective Antitumor Action of Cold Atmospheric Plasma-Derived Reactive Oxygen and Nitrogen Species[END_REF]Graves, 2012a[START_REF] Graves | Mechanisms of Plasma Medicine: Coupling Plasma Physics, Biochemistry, and Biology[END_REF][START_REF] Privat-Maldonado | ROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy[END_REF][START_REF] Von Woedtke | Plasma medicine: A field of applied redox biology[END_REF]. That applied redox biology will be further discussed in chapter 3.3, focusing on cancer cells.

The three most essential effects offered by NTP treatment for biomedical applications are consistently reported in various studies using different plasma sources and devices under varying plasma parameters and treatment conditions (Bekeschus et al., 2018b;[START_REF] Braný | Cold Atmospheric Plasma: A Powerful Tool for Modern Medicine[END_REF][START_REF] Dubuc | Use of cold-atmospheric plasma in oncology: a concise systematic review[END_REF][START_REF] Liao | Inactivation mechanisms of non-thermal plasma on microbes: A review[END_REF]Von Woedtke, 2019;[START_REF] Weltmann | Low temperature plasma applications in medicine[END_REF]:

• Inactivation of a broad spectrum of microorganisms, including multidrug-resistant pathogens • Stimulation of cell proliferation and angiogenesis via redox-regulated pathways where pro-angiogenetic factors are promoted (effects occur with a low NTP treatment intensity and time)

• The initialisation of (programmed) cell death, even triggering ICD with higher NTP treatment intensity and time, primarily in cancer cells.

The cellular effects of RONS can be summed up as follows: STATE OF THE ART From iatrophysics to medical physics: then and now -39 -

• Reactive Oxygen Species (ROS) such as O, O2-, O3, OH, H2O2… have several effects on cell membrane: Peroxidation of lipids, oxidation of proteins (chain of amino acids) that play the role of gateways between the exterior and interior of the cells, DNA oxidation, ...

• Reactive Nitrogen Species (RNS) such as N, NO, NO2, NO2 -, NO3 -, N2O5 ... affect the cell signalling.

Nevertheless, it is worth to be pointed out that almost all those observed effects were from nearly all studies whose plasma devices that are in-lab-made designed and developed, i.e., their plasma devices have different forms of plasmas electrode source designs, types of energy used (RF, DC and microwave), the frequency used (ns or µs, at Hz range or kHz range), the gas used (argon, helium or nitrogen, etc. versus air), not speaking of the time of treatment (contact time between the plasma and the target). Those different parameters are the primary sources of the physical and chemical properties of the plasma generated and, in fine, their reported therapeutic effects.

In need of communal efforts to tackle major obstacles essential to move forward in medical-oriental plasma research, five major fields were identified in the Future in Plasma Science II (FIPS) workshop in February 2016 in Greifswald (Germany): (1) plasma-based biomedical materials, (2) plasma decontamination, (3) plasma biology, (4) plasma wound healing, and (5) plasma oncotherapy (Bekeschus et al., 2018a). At least the most important, those applications will be discussed over the following two chapters.

STATE OF THE ART Biophysical-based treatments, except for cancer treatment -40 -

Biophysical-based treatments, except for cancer treatment

After a long-winded road of discovery from physicists laying down the theoretical fundamental of pulsed electric fields (PEFs) and non-thermal plasmas (NTP), through uncountable pre-clinical investigations with various models, from artificial ones to 2D and 3D cells in vitro and finally to animals in vivo, PEFs and NTP technologies came to be applied extensively in different fields, including medicine. This chapter introduces the leading and most known applications of those technologies in the biomedical field except oncology. Their applications in oncology -the focus of my thesiswill be addressed in detail in the next chapter.

Electroporation-based treatments

General applications of electroporation

The use of the electric field in agriculture research has a long history [START_REF] Bertholon | De l'électricité des végétaux[END_REF].

Ever since, positive effects of PEF have been described in fungi, soy, microalgae, and other cells [START_REF] Wohlgemuth | Perspective on Pulsed Electric Field Treatment in the Bio-based Industry[END_REF]. Controllable and reliable growth and compound stimulation have been achieved under nsPEF conditions (and only) while maintaining techno-functional properties of the remaining compounds, as demonstrated for foaming, emulsification, and colour compounds [START_REF] Buchmann | Effect of nanosecond pulsed electric field treatment on cell proliferation of microalgae[END_REF], with a yet unknown specific mechanism. Operated at relatively high field amplitude PEFs (up to 80 kV/cm), electroporation is an alternative to conventional non-thermal technology in food processing and biorefinery. It improves food quality and preservation by reducing the cost of energy by lowering treatment temperature and shortening processing time, and inactivating microorganisms such as vegetative bacteria and yeast cells selectively [START_REF] Sitzmann | Pulsed electric fields for food industry: Historical overview[END_REF]. A typical example of electroporation in food manufacturing is the production of French fries with pre-treatment through a gauntlet of PEFs -a machine known as the E-FLO™ (Heat and Control, 2019). This PEF process allows sugars and amino acids to be released from the potato before cooking, which lessens acrylamide -a hazardous substance categorised as "probably carcinogenic to humans" by the WHO. Besides creating a healthier product, PEF treatment also increases the amount of starch in the outer layers of the potato, resulting in a crunchier chip, and also reduces the time and need to blanch the potatoes before cooking. Moreover, cooking and kitchen operations assisted by PEF have found their places in daily life with commercialised products, such as the Nutri-Pulse® E-cooker STATE OF THE ART Biophysical-based treatments, except for cancer treatment -41 -utilised for fast preparation of fish, meat, and vegetables at a moderate PEF strength V/cm) upon heating above only 40-50 °C (Blahovec et al., 2017;[START_REF] Ixl Netherlands | -PULSE® E-COOKER® FOR PULSED ELECTRIC FIELDS FOOD RESEARCH[END_REF].

In recent years, besides utilising PEF for pasteurising liquid food products (such as juices, milk or liquid egg products), other applications have been increasingly investigated, particularly regarding its potential for improving mass transfer, offering higher extraction yields and valuable compounds recovery. Electroporation is thus applied in juice and oil production processes (biorefinery), for fermentation by raising yeast metabolism, for vinification as elimination of pathogenic microorganisms, reduction of maceration time, the release of polyphenolic compounds into grape juice, acceleration of wine ageing and inactivation of oxidative enzymes [START_REF] Ozturk | Pulsed electric fields (PEF) applications on wine production: A review[END_REF], for accelerating the drying process in vegetables or meat via the acceleration of water transfer, for cryopreservation (efficient permeabilisation of cryoprotectant into the cell for freezing tolerance of spinach leaves) or for meat curing (infusion of curing agent such as salt) [START_REF] Gómez | Application of pulsed electric fields in meat and fish processing industries: An overview[END_REF][START_REF] Mahnič-Kalamiza | Electroporation in Food Processing and Biorefinery[END_REF][START_REF] Toepfl | Overview of pulsed electric field processing for food[END_REF].

Medical applications of electroporation, except for cancer applications

Electrogenetherapy (gene transfer mediated by electroporation)

Historical aspects of electrogenetherapy

The concept of gene therapy arose in the mid-60s when the benefit of therapeutic genes to genetically modify viruses with the replacement of deleterious viral genes was evoked [START_REF] Tatum | Molecular biology, nucleic acids, and the future of medicine[END_REF]. Five years later (i.e., forty years ago), the potential of the delivery of genetic material to the nuclei of cells to generate a therapeutic effect has been recognised [START_REF] Rogers | Gene therapy: a potentially invaluable aid to medicine and mankind[END_REF], either for the correction of a defective gene by silencing it or by providing a functional replacement or for the introduction of a gene that encodes a foreign therapeutic protein or whose expression provokes cell death. Gene therapy has evolved to encompass not only the delivery of therapeutic DNA (plasmid DNA or pDNA) but also messenger-(mRNA), micro-(miRNA), short hairpin-(shRNA) and small interfering RNAs (siRNA) and oligodeoxynucleotides (ODNs). On its journey to target cell nuclei, nucleic acids encounter several biological barriers, such as the extracellular matrix, the cell membrane, the cytoplasm, and the nuclear envelope whose nuclear pores are impermeable to molecules more significant than 70 kDa (ca. 10 nm Ø) STATE OF THE ART Biophysical-based treatments, except for cancer treatment -42 -(McCrudden et al., 2013;[START_REF] Melchior | Mechanisms of nuclear protein import[END_REF]. These are the main reasons gene therapy has yet to be successfully translated. Significant effort has been allocated to developing viral and non-viral techniques for the vectorisation of nucleic acids. Non-viral strategies are making meaningful progress in the quest for a prominent gene delivery vehicle in the clinical medicine [START_REF] Nakayama | Comparison of Current Regulatory Status for Gene-Based Vaccines in the U.S., Europe and Japan[END_REF], amongst which we find electrogenetransfer (EGT), also known as gene electrotransfer (GET), proven to be one of the most efficient and safest techniques [START_REF] Spanggaard | Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma: Safety and efficacy results of a phase i firstin-man study[END_REF][START_REF] Villemejane | Physical methods of nucleic acid transfer: General concepts and applications[END_REF].

Based on electroporation technology, pioneer works regarding EGT were performed in 1982 by Neumann and colleagues for in vitro induction of exogenous gene expression, as presented in previous sections, followed by numerous investigations up to date to develop and improve this physics-based gene therapy. The development of a square-wave pulse generator with modulable intensities by Teissié [START_REF] Zerbib | Electric field mediated transformation: Isolation and characterization of a TK+ subclone[END_REF] found its application not only in the transformation of bacteria with plasmid DNA [START_REF] Fiedler | Transformation of bacteria with plasmid DNA by electroporation[END_REF] but also in the transfection of animal and plant cells, as well as the delivery of genes into animal and human tissues [START_REF] André | DNA electrotransfer: Its principles and an updated review of its therapeutic applications[END_REF][START_REF] André | Efficiency of high and low voltage pulse combinations for gene electrotransfer in muscle, liver, tumor and skin[END_REF][START_REF] Daud | Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma[END_REF][START_REF] Gothelf | Gene Electrotransfer to Skin; Review of Existing Literature and Clinical Perspectives[END_REF][START_REF] Mir | High-efficiency gene transfer into skeletal muscle mediated by electric pulses[END_REF]., Mir et al., 2005;Rols et al., 1998b;[START_REF] Rosazza | Gene Electrotransfer: A Mechanistic Perspective[END_REF][START_REF] Spanggaard | Gene electrotransfer of plasmid antiangiogenic metargidin peptide (AMEP) in disseminated melanoma: Safety and efficacy results of a phase i firstin-man study[END_REF].

Mechanisms and applications concerning other gene delivery strategies

Mechanisms of EGT

Electroporation is a crucial component of EGT, where the electric pulses are used to electropermeabilise the cell membranes and also to favour the electrophoretic transfer of extracellular genetic materials into the cells while securing the cell viability afterwards (Rols, 1998b;[START_REF] Satkauskas | Mechanisms of in vivo DNA electrotransfer: Respective contribution of cell electropermeabilization and DNA electrophoresis[END_REF][START_REF] Titomirov | In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA[END_REF]. The seven major steps of EGT are: (1) Plasmid DNA is delivered to the tumour environment; (2) EPs permeabilise the cell membrane;

(3) pDNA interacts with cell plasma membrane on cathode facing site [START_REF] Faurie | Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation[END_REF]; (4) pDNA is inserted into cell membrane via actin cytoskeleton [START_REF] Rosazza | The actin cytoskeleton has an active role in the electrotransfer of plasmid DNA in mammalian cells[END_REF]; (5) Plasmids are internalised by endocytosis, and microtubules actively transport DNA molecules through the cytoplasm; (6) DNA leaves endosomes and crosses the nuclear envelope [START_REF] Lechardeur | Nucleocytoplasmic transport of plasmid DNA: A perilous journey from the cytoplasm to the nucleus[END_REF]; (7) After DNA expression, the proteins are released into the cytoplasm.

EGT has a different electrotransfer mechanism than small molecules (smaller than the usual size of plasmid) electrotransfer, such as drugs. Simple diffusion happens through the cell STATE OF THE ART Biophysical-based treatments, except for cancer treatment -43 -membrane after permeabilisation for drugs and other small molecules. Diffusion will occur as long as the cell membrane is permeabilised, i.e., after the pulses have been delivered. On the contrary, DNA (and other nucleotides too large to pass through the hydrophilic pores by diffusion) are polyanions with an overabundance of negative charges, enabling the molecules to move in an electric field. In in vivo, the membrane of muscle cells or tumour cells remains permeabilised for several minutes after the delivery of a train of eight square wave pulses of 100 μs duration each, as discussed above in chapter 1. However, no transfection (or at least no more transfection than the one obtained by the injection of naked DNA in the absence of PEFs) is found in DNA is injected into the tissue after the delivery of the electric pulses (Gehl, 1999;Mir, 1999;[START_REF] Satkauskas | Mechanisms of in vivo DNA electrotransfer: Respective contribution of cell electropermeabilization and DNA electrophoresis[END_REF]. Thus, DNA must be present at the time of the delivery of PEFs.

However, moving DNA is insufficient; the cell membrane must be permeabilised to allow the DNA molecule's passage. Hence, optimisation of pulses is necessary to achieve either a greater degree of permeability of the cell membrane (for passive diffusion) or a greater degree of electrophoretic effect. Our group has demonstrated that generally, a series of short high voltage pulses (e.g., eight pulses of 0.1 ms duration each, at 1000 V/cm voltage to electrode distance) is optimally used for drug delivery [START_REF] Gothelf | Electrochemotherapy: Results of cancer treatment using enhanced delivery of bleomycin by electroporation[END_REF], while a combination involving permeabilising short HV pulses and electrophoretic long LV pulses (e.g., one short pulse of 0.1 ms duration, at 800 V/cm and one long pulse of 400 ms duration, at 80 V/cm) are used for very effective DNA transfer [START_REF] André | Efficiency of high and low voltage pulse combinations for gene electrotransfer in muscle, liver, tumor and skin[END_REF]. There are also studies displaying efficient electrotransfer in several tissues (tumours, liver, skeletal muscle) using long pulses only, ranging from 5 ms to 50 ms (see review [START_REF] Mir | Electroporation-based gene therapy: Recent evolution in the mechanism description and technology developments[END_REF]).

Applications of EGT

The EGT is used in research and medicine for many applications such as transfection of reporter genes, gene therapy, DNA vaccination and generation of iPS. DNA vaccination consists of injecting a DNA sequence coding for a pathogenic cell epitope (e.g., cancerous cell), leading to the activation of the immune system against the cells exposing that epitope. With the possibility of multi-potent vaccines, the high level of safety due to the absence of viral elements and the stability of the DNA molecule in lack of need for cold storage, DNA vaccines offer considerable advantages and the possibility to produce good manufacturing practices (GMP)grade plasmid DNA (GMP-DNA) for direct gene transfer into humans [START_REF] Gothelf | What you always needed to know about electroporation based DNA vaccines[END_REF]. For example, a recent study done in our laboratory by Calvet et al. used a plasmid that encodes a STATE OF THE ART Biophysical-based treatments, except for cancer treatment -44 -modified form of the human telomerase reverse transcriptase gene (hTERT) and its intradermal electrotransfection in mice. The transgene expression was 100-fold higher when combined with EGT than when the DNA was injected alone. After optimising the electrical parameters (choice of suitable electrodes, HV pulse intensity and the best combination of HV-LV), the telomerase antigen will be used as a cancer DNA vaccine called INVAC-1 (Calvet et al., 2014a).

Since LV pulses application is favourable for the delivery of DNA vaccines [START_REF] André | Efficiency of high and low voltage pulse combinations for gene electrotransfer in muscle, liver, tumor and skin[END_REF][START_REF] Gothelf | What you always needed to know about electroporation based DNA vaccines[END_REF][START_REF] Mir | Nucleic acids electrotransfer-based gene therapy (electrogenetherapy): past, current, and future[END_REF], it is worth highlighting the most recent news related to EGT and the ongoing COVID-19 pandemic. On November 24 th , 2020 -a week before the end of this thesis writing, when the world is facing the SARS-CoV-2 virus that has already infected ca. 64.5 M people and taken more than 1.5 M lives, for less than a year since the first confirmed case

Irreversible electroporation

First reported with persuasive results in 1987-88, irreversible electroporation (or IRE), as presented above in section 1.2, is permanent electroporation with promising clinical achievements. Research conducted by Davalos and co-investigators tested this phenomenon using a mathematical model and in vivo conditions, showing that IRE could indeed induce cell death without using a thermal energy [START_REF] Davalos | Tissue ablation with irreversible electroporation[END_REF], leading to the first successful in vivo IRE procedure on mice tumours [START_REF] Al-Sakere | Tumor Ablation with Irreversible Electroporation[END_REF]. Clinical applications of IRE were followed (were following?), wherein micro-to millisecond HV pulses were delivered to induce cellular necrosis or apoptosis while mitigating deleterious thermal effects of ablated tissues and neighbouring [START_REF] Rubinsky | Irreversible electroporation: A new ablation modality -Clinical implications[END_REF]. IRE finds its applications primarily for cancer treatment, but those will be presented later in chapter 3.1. Other applications in medical treatment (except STATE OF THE ART Biophysical-based treatments, except for cancer treatment -45 -oncology) of IRE are: cardiology (prevent arterial restenosis following angioplasty) or hypertension treatment through the renal sympathetic nerve denervation [START_REF] Rolong | History of electroporation[END_REF]Rubinsky, 2007). Nevertheless, this treatment modality has to proceed with caution since adverse effects have been reported on patients, such as cardiac arrhythmias, complete heart block, increasing pacing thresholds or atrial and ventricular electrical and mechanical dysfunction (directly related to the PEF strength) [START_REF] Aras | Irreversible electroporation: Proceed with caution[END_REF], whence the adoption of electrocardiogram (ECG)-synchronised delivery of pulses to gate IRE with R-wave of ECG to deliver the PEFs during the absolute refractory period of the ventricles [START_REF] Deodhar | Irreversible Electroporation Near the Heart: Ventricular Arrhythmias Can Be Prevented With ECG Synchronization[END_REF].

A systemic review reported an improvement in cardiac safety of ECG-gated IRE Field [START_REF] Scheffer | Irreversible electroporation for nonthermal tumor ablation in the clinical setting: A systematic review of safety and efficacy[END_REF], but no systemic evaluation of short and long-term cardiac injury associated with this combined technique.

IRE is a technology of ongoing development to enhance existing treatments or for novel applications, such as tissue engineering or dermatology (regenerative medicine, scarless skin regeneration, skin rejuvenation, wound healing, and wound burn disinfection), etc. [START_REF] Rolong | History of electroporation[END_REF].

The application of PEFs in cancer research and treatments will be expressly detailed later in section 3.2.

Non-thermal plasma technology for biological applications

General applications of NTP

NTP is a cutting-edge technology that has gained considerable attention during the last decade for different applications in various fields of research and development and innovative industry. Similar to electroporation technology discussed above, NTP is a promising technology for surface modification [START_REF] Foest | Non-thermal atmospheric pressure discharges for surface modification[END_REF] or in agriculture and food industry sectors thanks to not only its effectiveness in microbial pathogens inactivation such as spoilage fungi, bacterial in addition to degrading toxins but also its simple design, ease of use (especially at room temperature), cost-effective operation with short treatment times, lack of toxic effects, and significant reduction of water consumption [START_REF] López | A review on non-thermal atmospheric plasma for food preservation: Mode of action, determinants of effectiveness, and applications[END_REF][START_REF] Sakudo | Disinfection and sterilization using plasma technology: Fundamentals and future perspectives for biological applications[END_REF]. Plasma agriculture possesses great potential in stimulating seeds germination and plants growth, tailoring plants' dormancy and improving plants' resistance to common diseases (Adhikari et al., 2020a., Adhikari et al., 2020b). The research team led by Dr T. Dufour has demonstrated the STATE OF THE ART Biophysical-based treatments, except for cancer treatment -46 -ability of NTP treatment of tap water or demineralised water and liquid fertiliser (used to irrigate seeds) to increase plant growth and promote seed germination [START_REF] Judée | Plasma-activation of tap water using DBD for agronomy applications: Identification and quantification of long lifetime chemical species and production/consumption mechanisms[END_REF][START_REF] Zhang | Promoting lentil germination and stem growth by plasma activated tap water, demineralized water and liquid fertilizer[END_REF]. The advantage of the concept of a contact-free application of NTP to sterilise, for example, Candida biofilm-contaminated surfaces and devices in health care settings [START_REF] Maisch | Contact-free inactivation of Candida albicans biofilms by cold -154 -atmospheric air plasma[END_REF] might have a positive impact on preventing community-acquired and nosocomial infections in health care facilities. NTP also acquires strong microbicidal properties, including those against bacterial spores. Yost and Joshi used FE-DBD plasma to treat PBS and the resulting plasma-treated PBS induced severe oxidative stress in Escherichia coli as a consequence of a direct and non-specific attack of • OH (generated from H2O2 and O2 •-) on various microbial structures and components, including rapid loss of membrane potential and membrane integrity, lipid peroxidation and DNA damage [START_REF] Yost | Atmospheric nonthermal plasma-treated PBS inactivates Escherichia coli by oxidative DNA damage[END_REF]. Guo et al. demonstrated that plasma pre-treatment enhances the sensitivity of methicillin-resistant Staphylococcus aureus to antibiotics. The short-lived RONS generated by NTP played a primary role in inducing the increase of many species of RONS in the bacteria cells (Guo et al., 2018a).

The same group is also assumed, together with some other studies, that NTP is effective in the virus inactivation [START_REF] Ahlfeld | Inactivation of a foodborne norovirus outbreak strain with nonthermal atmospheric pressure plasma[END_REF]Guo et al., 2018b;[START_REF] Volotskova | Cold atmospheric plasma inhibits HIV-1 replication in macrophages by targeting both the virus and the cells[END_REF]. Similar modes of action of NTP have determined its application in the food industry. Treatment with NTP is used for food preservation or to decontaminate various kinds of foods with minimal impact on the food quality, even if it has been demonstrated that its effectiveness as a food decontamination technique varies amongst different foods [START_REF] López | A review on non-thermal atmospheric plasma for food preservation: Mode of action, determinants of effectiveness, and applications[END_REF]. NTP can also sterilise contaminated matter [START_REF] Laroussi | Sterilization of contaminated matter with an atmospheric pressure plasma[END_REF] or decontaminate abiotic surfaces such as stainless steel, various packaging materials, paper, glass and plastics of diverse nature. Notably, the NTP jet is also used medically for disinfection and sterilisation of medical instruments [START_REF] Sakudo | Disinfection and sterilization using plasma technology: Fundamentals and future perspectives for biological applications[END_REF].

Nevertheless, the rapid industrial implementation of NTP still faces some issues and unanswered questions, such as its impact on the nutritional and sensory quality of treated products; our knowledge of the NTP components and reactive species responsible for the antimicrobial activity; possible toxicity of (or some of) the chemical species generated, its scaleup by designing fit-for-purpose apparatus, etc. STATE OF THE ART Biophysical-based treatments, except for cancer treatment -47 -

Medical applications of NTP except for cancer applications

Plasma in dentistry: from sterilisation to whitening

Conventional treatments such as unique teeth brushing products, fluoride uptake, antibiotics, and vaccines for oral disease or infections (dental caries, periodontal and intraoral illness caused by bacteria) still have limitations. Therefore, with antibacterial capacity, NTP is an inexpensive approach for treating dental diseases. It has been demonstrated that NTP sterilised biofilms efficiently on root canals, dental implants or oral mucosa and eradicated bacteria and microbes. Given its characteristic, the NTP jet can access small irregular structures and narrow channels within the diseased tooth to be cleaned, e.g., for the treatment of dental caries or for root canal disinfection (a property that is proven as more advantageous than lasers, by some studies) [START_REF] Arora | Cold Atmospheric Plasma (CAP) in Dentistry[END_REF]. NTP is also used to assist in cleaning and optimising tooth and implant surfaces to improve bone integration or restore the adhesive of dental composite. It was observed that NTP treatment could modify the dentine surface and increase the dentine/adhesive interfacial bonding [START_REF] Ritts | Dentin surface treatment using a non-thermal argon plasma brush for interfacial bonding improvement in composite restoration[END_REF]Stancampiano et al., 2019).

Numerous methods are available in dentistry to restore natural tooth colour, amongst which found NTP -is considered "the next-generation technology" [START_REF] Arora | Cold Atmospheric Plasma (CAP) in Dentistry[END_REF]. NTP has been tested for its tooth bleaching effect and has produced successful results, shedding light on the chemical effect of NTP exposure. Indeed, hydrogen peroxide (H2O2), one of the oldest bleaching techniques, has long been used for tooth whitening by producing free radicals, which have also been found in NTP treatment.

Skin decontamination

Similar to its capacity to disinfect or sterilise a wide range of targets, ranging from material to biological targets, NTP showed antiseptic effects on bacteria, yeast, and fungus, as described above in section 2.2.1. Thus, NTP is also used to decontaminate skin thanks to the abundant production of free radicals, which are toxic to organisms at a critical concentration [START_REF] Klebes | Effects of tissue-tolerable plasma on psoriasis vulgaris treatment compared to conventional local treatment: A pilot study[END_REF].

Skin wound healing

Since NTP demonstrated its antiseptic effects in the decontamination of skin, it can support wound healing by stimulating of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect STATE OF THE ART Biophysical-based treatments, except for cancer treatment -48 - [START_REF] Haertel | Non-thermal atmospheric-pressure plasma possible application in wound healing[END_REF] (Figure 11). The first prospective randomised controlled phase II trial to decrease bacterial load with 5 min daily argon NTP and standard wound care on chronic wounds in 36 patients resulted in a significantly less bacterial load (Figure 11d) [START_REF] Isbary | A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients[END_REF].

Another randomised controlled trial engaging 50 patients demonstrated the healing effect of NTP in pressure ulcers with a significantly better pressure ulcer scale for healing scores and exudate [START_REF] Chuangsuwanich | The Healing Effect of Low-Temperature Atmospheric-Pressure Plasma in Pressure Ulcer: A Randomized Controlled Trial[END_REF].

NTP has been demonstrated in vitro and in vivo to be able to change the gene expression of key molecules of the wound healing machinery (activation of angiogenesis-related molecules in skin keratinocytes, fibroblasts and endothelial cells), thus improving the wound healing (Arndt et al., 2013b[START_REF] Arndt | Cold atmospheric plasma (CAP) activates angiogenesis-related molecules in skin keratinocytes, fibroblasts and endothelial cells and improves wound angiogenesis in an autocrine and paracrine mode[END_REF][START_REF] Kubinova | Nonthermal air plasma promotes the healing of acute skin wounds in rats[END_REF]. [START_REF] Heinlin | Plasma medicine: possible applications in dermatology[END_REF] and (d) the MicroPlaSter® plasma torch (Microwave 2.46 GHz, 86 W; Ar 2.2 slm; distance to torch 2 cm) used in the treatment of a chronic infected wound [START_REF] Isbary | A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients[END_REF].

Besides the effectiveness in skin decontamination and the healing of skin wounds, mild NTP treatments have shown to enhance the expression of collagen I, fibronectin, and vascular endothelial growth factor (VEGF) in fibroblasts significantly to boost angiogenesis and repair of connective tissues, hence application in dermatology as rejuvenation [START_REF] Choi | Low-temperature atmospheric plasma increases the expression of anti-aging genes of skin cells without causing cellular damages[END_REF][START_REF] Foster | Advances in plasma skin regeneration[END_REF].

Blood coagulation

Haemorrhage or blood loss is considered a significant issue during surgical intervention, especially in patients under anticoagulant therapy [START_REF] Van Der Meer | Bleeding Complications in Oral Anticoagulant Therapy: An Analysis of Risk Factors[END_REF]. To treat and prevent blood clots, new oral anticoagulants (blood thinner) such as rivaroxaban (Xatelro®) are increasingly prescribed instead of former anticoagulative therapy using vitamin K antagonists STATE OF THE ART Biophysical-based treatments in cancer therapy -49 -(e.g., warfarin) [START_REF] Carter | Rivaroxaban: A review of its use in the prevention of stroke REFERENCES -143 -and systemic embolism in patients with atrial fibrillation[END_REF]. However, there still is a need for pro-coagulant therapy [START_REF] Weiner | Hemostasis in the era of the chronic anticoagulated patient[END_REF] due to the lack of proper antidotes [START_REF] Riley | Role of agents for reversing the effects of target-specific oral anticoagulants[END_REF].

Besides the capacity to sterilise and disinfect living tissues, NTP has been suggested as an alternative haemostatic direct treatment to meditate haemorrhage during surgery, an application similar to the one fulfilled by the extensively used electrical knives. An ex vivo investigation pioneered by Fridman and colleagues fifteen years ago showed blood coagulated from oozing haemorrhage of human spleen using FE-DBD [START_REF] Fridman | Blood coagulation and living tissue sterilization by floatingelectrode dielectric barrier discharge in air[END_REF] (Figure 12). The tissue integrity was preserved in that study. The group pursued their investigation of the mechanisms of blood coagulation by NTP. It attributed a central role to a plasma-induced fibrin clot [START_REF] Kalghatgi | Mechanism of blood coagulation by nonthermal atmospheric pressure dielectric barrier discharge plasma[END_REF]. This mechanism was also speculated in another study on blood plasma sample [START_REF] Ke | Haem-assisted dityrosine-cross-linking of fibrinogen under nonthermal plasma exposure: one important mechanism of facilitated blood coagulation[END_REF]. However, a recent in vivo study rejected that proposed mechanism and emphasised the critical regulator role of platelets in the haemostasis facilitated by the kINPen® MED via the increased activation of platelets by the RONS oxidation [START_REF] Bekeschus | Platelets are key in cold physical plasma-facilitated blood coagulation in mice[END_REF]. Although the mechanisms of NTP blood coagulation are yet unrevealed, and NTP treatment could only offer a limited treatment zone, practical results of NTP already open the gate for clinical benefits of tissue-tolerable NTP as a coagulant agent in surgery. Top cut: blood continues to ooze from an untreated area; bottom cut: blood coagulates while the wound remains wet [START_REF] Fridman | Blood coagulation and living tissue sterilization by floatingelectrode dielectric barrier discharge in air[END_REF].

The application of NTP in cancer research and treatments will be expressly detailed later in section 3.3.

Biophysical-based treatments in cancer therapy

This chapter will get a glimpse of cancer with its updated basic facts. It will then focus on the applications of the electroporation-based approaches and the non-thermal plasma technologies against cancer.

STATE OF THE ART Biophysical-based treatments in cancer therapy -50 -

Cancer at a glance

The landscape and epidemiology of cancer

Characterised by uncontrolled growth and spread of abnormal cells, cancer -a generic term for malignant tumours and leukaemias -is a group of diseases with the highest impact on global health. According to the World Health Organization (WHO), cancer is the second leading cause of death globally (behind ischaemic heart disease and stroke). Cancer occurs principally in low-and middle-income countries (approximately 70% of the world's cancers). Note that all the statistics mentioned in this section are from, if not otherwise stated, the WHO (World Health Organization, 2018., World Health Organization, 2020), the WHO Cancer Fact Sheet, the (American Cancer Society®, 2009), and from [START_REF] Siegel | Cancer Statistics[END_REF] In 2018, the WHO estimated 18.1 million new cases and 9.6 million deaths from cancer (Figure 13a), in which the ten most common cancers for both sexes are responsible for 60-70% of cancer cases and deaths (Figure 13b). By 2040, those figures will nearly double, with the most significant increase in low-and middle-income countries. Around one-third of deaths from cancer are due to the five leading behavioural and dietary risks: high body mass index, low fruit and vegetable intake, lack of physical activity, tobacco use, and alcohol use. Tobacco use is the most critical risk factor, responsible for approximately 22% of cancer deaths. Female breast cancer (11.6%), lung cancer in men (11.6%) and colorectal cancers (10.2%) are the most frequently diagnosed cancers worldwide. Due to increasing life expectancy, epidemiological and demographic transitions, and frequency of diagnosis, the global numbers of new incidence and mortality continue to rise [START_REF] Van Hoeve | Long-term effects of a regional care pathway for patients with rectal cancer[END_REF]. Despite all the recent technological improvements, recurrence and metastasis remain the principal cause of death from cancer.

The causal factors triggering cancer, either external (tobacco, chemicals, infectious organisms, radiation, etc.) or internal (inherited mutations, mutations that occur from metabolism, hormones or immune conditions, etc.), may act together or in sequence to initiate or promote carcinogenesis, with ten or more years passed between the exposure to those factors (especially the external ones) and the detection of cancer.

The World Cancer Research Fund and the American Institute for Cancer Research have determined additional cancer causes, including beta carotene, red meat, processed meats, low fibre diets, not breastfeeding, obesity, increased adult height and sedentary lifestyles [START_REF] Blackadar | Historical review of the causes of cancer[END_REF]. In that excellent review article tracking back the history of the cause of STATE OF THE ART Biophysical-based treatments in cancer therapy -51 -cancer, Blackadar was surprised himself when he discovered that natural factors (hormones, UV radiation, parasites, fungus, bacteria, wood dust, alcohol, food preparation technique, herb, etc.) are an additional and relatively underappreciated and woefully ignored cause of cancer. Rather than responding appropriately to the signals that control normal cell behaviour, cancer cells grow, divide, and proliferate uncontrollably, invading normal tissues and organs and eventually spreading throughout the body (metastasis). That is to say, all cancers involve the malfunction of genes that control cell growth and division. However, only about 5% of all cancers are strongly hereditary, in that an inherited genetic alteration confers a very high risk of developing one or more specific types of cancer. Most cancers do not result from inherited 6.9%

6.65% 6.55% 6.46% 6.36% 6.27% 6.17% 6.07% 5.98% 5.88% 5.79% 5.69% 5.59%

5.50% Cancerologists and oncologists distinguish the two following risks for an individual to develop cancer:

• Lifetime risk is the probability that an individual, throughout a lifetime, will develop or die from cancer (e.g., in the US, men have slightly less than a 1 in 2-lifetime risk of developing cancer compared to women, whose risk is a little more than 1 in 3);

• Relative risk refers to measuring the strength of the liaison between risk factors and particular cancer by comparing the risk of developing cancer in persons with a specific exposure or characteristic to the risk in persons who do not have this trait.

For the two most common cancers, the American Cancer Society estimated that male smokers are about 23 times more likely to develop lung cancer than nonsmokers, i.e., their relative risk is 23; and women who have a first-degree relative (mother, sister or daughter) with a history of breast cancer have about twice the risk of developing breast cancer as compared with women who do not have this family history.

There is no better way against disease than to prevent it after studying its causation or origination (aetiology). Humans have won the battle against deadly diseases throughout history, including smallpox and polio, by vanquishing them through prevention. The actual cancer control continuum defined by WHO, shown in Figure 14 Even if the WHO indicates that at least 30-50% of all cancer deaths are preventable with current knowledge and interventions [START_REF] Danaei | Causes of cancer in the world: Comparative risk assessment of nine behavioural and environmental risk factors[END_REF], the journey towards achieving cancer prevention remains challenging and full of obstacles. Effective screening methods are available for only a few cancers, and advances in cancer treatment have not been as practical as those for other chronic diseases, not speaking of the cost of treatment (the total annual economic cost of cancer in 2010 was estimated at approximately US$ 1.16 trillion). Moreover, as stated earlier, ten or more years pass between the exposure to cancerous factors (especially STATE OF THE ART Biophysical-based treatments in cancer therapy -54 -the external ones) and the detection of cancer; late-stage presentation and inaccessible diagnosis and treatment are common. Humankind has thus likely to treat and live with cancers rather than prevent them.

Recognising the rapidly increasing global burden, governments have increased the importance of comprehensive cancer control. Firm commitments have been made in United Nations and World Health assemblies during the past decade. The set priorities, which have to be feasible, evidence-based, comprehensive and inclusive, are: strengthen tobacco control to reduce cancer death by 25%; vaccinate against HPV and hepatitis B, reaching >90% coverage; screen for cervical cancer with >70% participation; focus on early diagnosis and treatment for curable cancers, e.g., some childhood cancer to save 1 million lives by 2030; scale-up capacity to manage 200 million cancer cases in next decades; provide palliative care for all. There is also a global initiative for childhood cancer to ensure the survival of at least 60% of children with cancer by 2030, thereby saving an additional one million lives and reducing suffering for all.

The development of cancer

Types of cancer

Cancer can result from uncontrolled proliferation of any of the different kinds of cells in the body, resulting in numerous distinct types of cancer, varying substantially in their behaviour and response to treatment. The International Classification of Diseases (revision 11) lists more than 600 types of cancer, most of which require unique diagnostic and management approaches (World Health Organization, 2020). The most critical issue in cancer pathology is the distinction between benign and malignant tumours. A tumour is an abnormal proliferation of cells, which may be either benign or malignant. A benign tumour, such as a common skin wart, remains confined to its original location, neither invading surrounding normal tissue nor spreading to distant body sites. However, a malignant tumour can invade surrounding normal tissue and spread throughout the body via the circulatory or lymphatic systems (metastasis).

Only malignant tumours are appropriately referred to as cancers, with their dangerous ability to invade and metastasise specific organs, including lung, liver, brain, and bone (Cooper, 2000). Metastasis proceeds through the progressive acquisition of characters that allow malignant cells originating in one organ to disseminate and colonise a secondary site. Although these traits are depicted as part of a continuous biological sequence, their acquisition during metastatic progression need not follow this particular order. Although, in some cases, several factors may be crucial to implementing a single step in this cascade, other mediators of metastasis may facilitate the execution of multiple stages simultaneously. Likewise, the specific steps of this sequence that are rate-limiting for metastatic progression may also vary from one tumour to another [START_REF] Gupta | Cancer Metastasis: Building a Framework[END_REF].

Both benign and malignant tumours are classified according to the type of cell from which they arise, mostly three main groups: carcinomas, sarcomas, and leukaemia or lymphomas (their definition are given in the Glossary & Key Terms found at the end of this thesis manuscript). Note that carcinomas, which are malignancies of epithelial cells, comprise approximately 90% of human cancers, as opposed to sarcomas (solid tumours of connective tissues), rare in humans. Tumours are further classified according to the tissue of origin and the type of cell involved. For example, fibrosarcoma arises from fibroblasts and erythroid leukaemia from precursors of erythrocytes (red blood cells). The most common cancer type varies amongst countries. Certain cancers are much more common in countries at the lower end of the human development index (HDI) than in high-HDI countries, e.g., cervical cancer and Kaposi sarcoma. (Cooper, 2000) Tumour clonality has been demonstrated as one of the fundamental features of cancer, i.e., the development of tumours from single cells that begin to proliferate abnormally. STATE OF THE ART Biophysical-based treatments in cancer therapy -56 -Cancer development is a multistep process involving mutation and selection for cells with progressively increasing capacity for proliferation, survival, invasion, and metastasis at a cellular level, as described above in Figure 16. This progress could be resumed in two significant steps: tumour initiation, probably the result of a genetic alteration leading to abnormal proliferation of a single cell (Cell proliferation then leads to the outgrowth of clonally derived tumour cells), and tumour progression that continues as additional mutations occur within cells of the tumours. Some of these mutations present a selective advantage to the cell (e.g., more rapid growth). The descendants of a cell bearing such a mutation will become dominant within the tumours -a clonal selection process. The clonal selection continues throughout tumour development, so tumours continuously become more rapidly growing and increasingly malignant.

The development of cancer

Ninety per cent of cancer deaths are not caused by the primary tumour but because metastasis [START_REF] Torre | Global Cancer Statistics[END_REF]. The primary tumour selectively and actively modifies organs of future metastasis before the occurrence of metastatic spread. Tumours induce the formation of microenvironments (pre-metastatic niches) in distant organs that are favourable to the survival and outgrowth of tumour cells before they arrive at these sites [START_REF] Peinado | Pre-metastatic niches: Organ-specific homes for metastases[END_REF].

Metastases are usually multiple and resistant to conventional therapies, jeopardising successful surgical resection, chemotherapy and radiation treatment.

The hallmarks of cancer

In human oncology, one of the most pervasive characteristics of tumours is genomic instability [START_REF] Hanahan | Hallmarks of cancer: The next generation[END_REF], leading to the generalised loss of growth control of malignant cells -the net result of accumulated abnormalities in multiple cell regulatory systems, reflecting in several characteristics of cell behaviour that distinguish cancer cells from their normal counterparts (expression of neoantigens, differentiation antigens or cancer-testis antigens, which can lead to the presentation of peptides bound to major histocompatibility class I (MHCI) molecules on the surface of cancer cells) [START_REF] Chen | OncologyMeetsImmunology: TheCancer-ImmunityCycle[END_REF]. • Resisting cell death: When an event irreversibly damages noncancerous cells, these latter will trigger apoptosis (programmed cell death). Contrariwise, cancer cells often present mutations on genes responsible for sensing such damages or triggering cell death;

• Enabling replicative immortality: Noncancerous cells cannot divide more than a defined number of times due to telomere shortening (Hayflick limit). Contrariwise, cancer cells can reactivate the telomerase to synthesise telomeres continuously and thus escape senescence;

• Inducing angiogenesis: Cancer cells require sufficient nutrients and energy to sustain their growth. By releasing factors that promote the creation of new blood vessels, they ensure their blood supply;

• Activating invasion and metastasis: by downregulating the expression of adhesion molecules and secreting proteases, cancer cells can invade surrounding tissues or even reach the blood flow through which they may colonise distant tissues;

• Deregulating cellular energetics and metabolism: cancer cells can alter their utilisation of energy sources-notably glucose-to support their proliferation;

• Avoiding immune destruction: incipient neoplasia could find ways to circumvent active surveillance by the immune system that would otherwise eliminate the proliferation of premalignant cells aberrantly. [START_REF] Dejeans | Addicted to secrete -novel concepts and targets in cancer therapy[END_REF][START_REF] Hanahan | Hallmarks of cancer: The next generation[END_REF].

Those capabilities are familiar to many, if not most, forms of human cancer. Each one serves a distinct functional role in supporting tumours' development, progression, persistence and constituent cells. Genome instability facilitates their acquisition with subsequent gene mutation and tumour-promoting inflammation. Integrating these hallmark capabilities encompasses heterotypic interactions between multiple cell types populating the "tumour microenvironment". The tumour microenvironment comprises cancer cells and a tumourassociated stroma, including three major classes of recruited support cells: angiogenic vascular cells, various subtypes of fibroblasts, and infiltrating immune cells. Their functions are hijacked to participate in tumour initiation, progression, and metastasis [START_REF] Hanahan | Hallmarks of cancer: The next generation[END_REF][START_REF] Valkenburg | Targeting the tumour stroma to improve cancer therapy[END_REF]. Moreover, the neoplastic cells populating individual tumours are typically heterogeneous, in that cancer cells can undertake a variety of distinctive phenotypic states and undergo genetic diversification during tumour progression. Hence, the hallmarks of cancer constitute a valuable heuristic tool for elucidating mechanistic bases and commonalities underlying the pathogenesis of diverse forms of human cancer, with potential applications to cancer therapy that will be discussed after that. STATE OF THE ART Biophysical-based treatments in cancer therapy -59 -As cancer remains a worldwide health problem with yet unsolved puzzle, the WHO underlines that cancer cannot be prevented or controlled by the health sector alone but requires a coordinated, multisectoral, multidisciplinary approach (World Health Organization, 2020). Hence, efforts are put not only into daily life prevention and intervention but also into the research and development of cancer therapies to diagnose better, prevent and treat cancer.

Concerning traditional cancer treatment modalities, the following section will highlight advances in current anticancer therapies as multidisciplinary approaches.

Anticancer therapies

The development of anticancer therapies was initially based on empirical observations due to the lack of knowledge on the characteristics of cancer cells and their microenvironment, as well as their role in tumour progression. Historically cancer has been treated based on broad pathologic criteria involving various regimens of cytotoxic therapy -a ''one size fits all'' approach. Over time, better knowledge in oncology and cancerology has been acquired, thus allowing the development of novel and more appropriate therapeutic strategies that consider the tumour size, stage, and other characteristics and patient preferences. The most commonly utilised anticancer therapies to date are surgery, chemotherapy and radiotherapy.

Tumour ablation

By far, surgery is the first and most considered cancer treatment modality. William Halsted's approach to mastectomy took the medical world by storm at the turn of the last century [START_REF] Halsted | The Results of Operations for the Cure of Cancer of the Breast Performed -149 -at the Johns Hopkins Hospital from June, 1889[END_REF]. Ever since ablative therapy has diverse approaches with a very similar concept but distinguished technology [START_REF] Seror | Ablative therapies: Advantages and disadvantages of radiofrequency, cryotherapy, microwave and electroporation methods, or how to choose the right method for an individual patient?[END_REF].

Tumour ablation can be performed with:

• Surgery: the surgical act consists of operating on the patient to remove the tumour or the affected organ and a part of the healthy tissue surrounding the tumour with the neighbouring lymph nodes and vessels to avoid any risk of cancer extension.

Tumour resection remains an invasive treatment that can be unmanageable, depending on the size of the tumour mass. Nevertheless, surgery is responsible for a large part of cancer cures.

• Cryoablation [START_REF] Erinjeri | Cryoablation: Mechanism of action and devices[END_REF]: Referred to all methods of destroying tissue by freezing, cryoablation instigates cellular damage, death, and necrosis of tissues by STATE OF THE ART Biophysical-based treatments in cancer therapy -60 -cold-induced injury to cells (direct mechanisms) and by change of the cellular microenvironment and impair tissue viability (indirect mechanisms). By both mechanisms, cell injury can be influenced by cooling rate, target temperature, time at the target temperature, and thawing rate. Indeed, this invasive cancer treatment targets the lesions, under imaging guidance, by percutaneous or laparoscopic insertion of cryoprobes (one or several) cooled with liquid nitrogen. Almost all tissues exhibit subsequent cell death via a rapid cooling below -40°C (some techniques also use high-pressure room temperature argon gas to reach -80°C of cryoprobes). Since the cooling of tissues and nerves provide an anaesthetic effect, cryoablation tends to be less painful than the heat-based thermal ablation techniques like radiofrequency or microwave ablation that will be next discussed.

• Hyperthermia: As introduced in chapter 1.1., the application of radiofrequency (RF) energy and its thermal effects on tissue were described in 1891 by d'Arsonval.

Employing RF or micro-wave, these techniques are used for tumours whose localisation does not allow surgical intervention. Based on thermal destruction, hyperthermia is governed by three phenomena: thermal production, i.e., the energy delivered as well as its interaction with the tissues; thermal conduction, i.e., how the heat is dissipated by transmission due to the physical contact between the target tissue and the surrounding tissues (it differs from one tissue to another); thermal convection, i.e., the dissipation of heat via its transport by the fluids which pass through the tissues (thus, blood vessels more significant than 2-3 nm act as heat sinks [START_REF] Ringe | Experimental Evaluation of the Heat Sink Effect in Hepatic Microwave Ablation[END_REF]). RF ablation takes advantage of the heat produced by applying a high-frequency electric current, c.a. 460-480 kHz [START_REF] Hong | Radiofrequency ablation: Mechanism of action and devices[END_REF], reaching around 60 to 100°C at the treated site. Depending on the tumour location, the RF is delivered percutaneously or by laparoscopy employing multiple curved uninsulated electrodes deployed from the needle tip central cannula (an umbrella shape). RF ablation is the primary technique used today to treat hepatic tumours [START_REF] Pepple | Laparoscopic-Assisted Ablation of Hepatic Tumors: A Review[END_REF]. Nevertheless, certain parts of the tissues, such as blood vessels and the hepatic bile ducts, strongly dissipate the deposited heat, interfering with the treated areas' proper heating effects. On the other hand, microwave ablation radiates into the tissue through an interstitial antenna. For medical purposes, the frequency of microwave ablation varies between 915 and 2450 MHz. It causes the STATE OF THE ART Biophysical-based treatments in cancer therapy -61 -very rapid oscillations of the dipoles, thus causing friction of the water molecules in the tissues, leading to a temperature rise of up to 180 °C in the target tissues (without current passage). That temperature rise is more rapid than in RF ablation.

Besides, not like RF ablation, microwave ablation seems less prone to induce a heat sink effect when the used microwave are capable of propagating through and effectively heating many types of tissues, even those with low electrical or thermal conductivity and high baseline impedance [START_REF] Lubner | Microwave tumor ablation: Mechanism of action, clinical results, and devices[END_REF].

• Irreversible electroporation (IRE) and high-frequency irreversible electroporation (H-FIRE): these recent approaches are nowadays employed when thermal ablation turns out to be too risky. IRE and HFIRE target tissues with an electroporation-based strategy (described above in chapter 1.2.) using a series of brief electric pulses, whose parameters cause the cell electroporation but may also result in secondary Joule heating of the tissue [START_REF] Aycock | Irreversible Electroporation: Background, Theory, and Review of Recent Developments in Clinical Oncology[END_REF]. Davalos and colleagues were the first to propose that IRE could be an efficient soft tissue ablation approach [START_REF] Davalos | Tissue ablation with irreversible electroporation[END_REF]. Those results were followed by the first successful in vivo application of IRE for the minimally invasive treatment of aggressive cutaneous tumours implanted, achieved by our group, using plate electrodes to deliver 80 monopolar pulses of 100 µs at 0.3 Hz with a PEF magnitude of 2500 V/cm, with minimal heating of the tissue thanks to mathematical models of the electrical and thermal fields [START_REF] Al-Sakere | Tumor Ablation with Irreversible Electroporation[END_REF], and the first clinical trial to treat renal tumours [START_REF] Thomson | Investigation of the safety of irreversible electroporation in humans[END_REF]. Recently, H-FIRE has been introduced to outperform IRE by using HF voltage waveforms with integrated bursts of 0.25-10 µs bipolar pulses to more uniformly penetrate the heterogeneous system [START_REF] Arena | Theoretical Considerations of Tissue Electroporation With High-Frequency Bipolar Pulses[END_REF]. H-FIRE has since been used to treat intracranial malignancies in the canines [START_REF] Garcia | Predictive therapeutic planning for irreversible electroporation treatment of spontaneous malignant glioma[END_REF], and low-amplitude H-FIRE waveforms have been used to transiently disrupt the blood-brain barrier [START_REF] Arena | Focal blood-brain-barrier disruption with high-frequency pulsed electric fields[END_REF].

• Radiotherapy: ionised radiations (X-rays, gamma rays, radiations using protons, neutrons, electrons, carbon ions) used to locally target the treated tumour (see history in chapter 1.1.). To date, more than half of cancer patients resort to radiotherapy in their approved health care pathway, primarily in combination with other treatments such as chemotherapy or surgery. The main challenge remains to target cancer cells without specifically harming surrounding normal cells.

STATE OF THE ART Biophysical-based treatments in cancer therapy -62 -As for unresectable and disseminated cancerous tumours, and this is, unfortunately, the case of numerous cancer cases, there are developments and applications of other therapies introduced below.

Photodynamic therapy

Since the Ancient Indian and Chinese civilisations more than 3000 years ago, light has been employed to treat various diseases, mainly in combination with reactive chemicals, e.g., vitiligo, psoriasis or skin issues [START_REF] Ackroyd | The History of Photodetection and Photodynamic Therapy[END_REF]. However, it was only after discovering phototherapy in 1895 (see history in section 1.1.) that the use of light and chemicals emerged.

Currently being used as an alternative treatment for the control of malignant diseases, photodynamic therapy (PDT) is based on the uptake of a photosensitiser molecule that reacts with oxygen after being excited by light in a determined wavelength and generates oxidant species in target tissues (such as radicals, singlet oxygen, triplet species), leading to cell death.

The photosensitised oxidations of nucleic acids, lipids, and proteins within the cell that leads to severe alteration in cell signalling cascades or gene expression regulation have been identified as the main cytotoxic properties of PDT. The key step in terms of the performance of the photosensitiser is the formation of triplet exciting species [START_REF] Dos Santos | Photodynamic therapy in cancer treatment -an update review[END_REF].

With well-established safety profiles and selectivity towards tumour cells for cellular and vascular-targeted PDT, verteporfin (Visudyne®) and porfimer sodium (Photofrin®) are amongst the most commonly photosensitising agents approved or in clinical trials.

Chemotherapy

Chemotherapy was first developed at the beginning of the 20 th century, though it was not originally intended as a cancer treatment. Throughout World War II, it was noticed that individuals exposed to nitrogen mustard developed significantly reduced white blood cell counts. This discovery led researchers to scrutinise whether mustard agents could be used to halt the growth of rapidly dividing cells such as cancer cells. In the 1940s, two distinguished Yale pharmacologists, Alfred Gilman and Louis Goodman examined the therapeutic effects of mustard agents in treating lymphoma. First, they established lymphomas in mice and showed that the tumours could be treated with mustard agents. Then, together with Gustav Linskog, a thoracic surgeon, they injected a less volatile form of mustard gas termed mustine (nitrogen mustard) into a patient having non-Hodgkin's lymphoma. Since then, several other molecules have shown to be effective antineoplastic agents (Figure 18). • Palliative treatment, i.e., to relieve symptoms caused by cancer when the latter is at an advanced stage, probably cannot be controlled, and has metastasised. This palliation (also called palliative chemotherapy or treatment with palliative intent) is in the hope to improve the quality of life or help the patient feel better by shrinking a tumour that's causing pain or pressure.

Chemotherapeutic agents not only damage actively dividing cells but can also affect tissues

with a low mitotic potential (e.g., neurons). Common adverse effects of chemotherapy occur in: gastrointestinal tract (e.g., chemotherapy-induced nausea and vomiting, diarrhoea, constipation, intestinal perforation, etc), blood (e.g., myelosuppression, often dosedependent), skin (alopecia), central nervous system (e.g., vomiting, chemotherapy-induced peripheral neuropathy), sexual organs (gonadal damage).

Chemotherapy is thus a non-targeted therapy or at least not a precise strategy treatment, as opposed to targeted therapies which will be discussed here below.

STATE OF THE ART Biophysical-based treatments in cancer therapy -64 -

Targeted anticancer therapies

Despite numerous improvements in many anticancer approaches (improvement of surgical techniques, radiotherapy procedures, introduction and extension of treatments by immunotherapy, alone or in combination...), there are nevertheless many situations for which cancerous disease cannot be controlled, and side effects are unavoidable. More efficient techniques are thus needed, amongst which found targeted anticancer therapies. With cuttingedge translational research discoveries and based on either the unique characteristics of molecules, peptides, proteins, antibodies or the structures, metabolisms and other phenotypic properties of cancer, targeted anticancer therapies engage in destroying cancer cells in a more precise manner, therefore, may significantly improve the treatability of cancer.

Targeted anticancer therapies encompass a variety of direct and indirect approaches.

Direct approaches target tumour cells directly to alter their molecular pathways by either mAbs

or small molecular inhibitors. Indirect approaches target tumour stroma, a critical component of the tumour microenvironment, by mAbs, peptide ligands or radiolabelled chemical agents that inhibit angiogenesis or disrupt the tumour vasculature fibroblasts, macrophages, hence affecting tumoural micro-environment. In other words, there are many different types of targeted therapies for cancer, and some may fit into more than one category, but those can be distinguished into at least two main strategies [START_REF] Bashraheel | Update on targeted cancer therapies, single or in combination, and their fine tuning for precision medicine[END_REF]: The main current approaches and treatments deemed promising and even used for clinical applications in the fine-tuning of precision medicine to target cancer with maximal tumour destruction, and minimal side effects are described here. 3.1.4.4.1 Hormone-replacement therapy Tumours require certain hormones to grow. Thus, hormone therapy is proposed to slow or impede the growth of hormone-sensitive tumours. This systemic treatment prevents the body from producing the hormones or interfering with the hormones' action. Hormone therapies have been approved for breast and prostate cancer which depend on sex hormones to grow [START_REF] Lobo | Hormone-replacement therapy: Current thinking[END_REF].

•
The introduction of hormonal therapy took place in 1939 by Charles Huggins, based on an early observation on the effect of oestrogens on breast cancer made by Beatson in 1896 [START_REF] Beatson | On the Treatment of Inoperable Cases of Carcinoma of the Mamma: Suggestions for a New Method of Treatment, with Illustrative Cases[END_REF] treated men with prostate cancer with hormones and was able to show responses through the decreases in acid phosphatase levels [START_REF] Huggins | Studies on Prostatic Cancer. I. The Effect of Castration, of Estrogen and of Androgen Injection on Serum Phosphatases in Metastatic Carcinoma of the Prostate[END_REF]. This exciting discovery was an essential addition to the systemic treatment of cancer and earned Huggins a Nobel Prize. Since then, many medications have been discovered to prevent cancer cells from getting the hormones needed for growth and proliferation and have been used in combination

or not with other cancer treatments, such as chemotherapy or radiation therapy.

Some frequently used hormone therapy drugs in clinics to treat common cancer types, mainly breast cancer and prostate cancer, are anastrozole (Arimidex®), exemestane (Aromasin®), fulvestrant (Faslodex®), letrozole (Femara ® ), leuprolide (Eligard®, Lupron Depot®), tamoxifen (Nolvadex®), abiraterone (Zytiga®). 3.1.4.4.3 Apoptosis inducers

Apoptosis is a programmed cell death process mediated by several intrinsic and extrinsic signalling pathways, which are triggered by multiple factors, including cellular stress, DNA damage and immune surveillance. Healthy cells of an organism may undergo apoptosis to get rid of unneeded or abnormal cells, but cancer cells have strategies to avoid this process. Antiapoptotic oncogenes such as BCL2 (B-cell lymphoma 2) and BCL2L1 (BCL2-like 1) that prevent cell death [START_REF] Hockenbery | Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death[END_REF]) are a powerful mechanism for tumour growth and drug resistance [START_REF] Rathmell | Pathways of apoptosis in lymphocyte development, homeostasis, and disease[END_REF]. Apoptosis inducers can get around these strategies to cause (or restore) the normal cell death of cancer cells. To date, there is a limited number of FDAapproved apoptotic inducers, which are small molecules designed to inhibit anti-apoptotic BCl-2 family members. For example, velcade (Bortezomib®) induces apoptosis in lymphoma and multiple myeloma cells. Other promising therapeutic strategies to activate cancer cell apoptosis include agents that trigger the extrinsic apoptosis pathway, target tumour suppressor pathways or the tumour microenvironment, and combination drug therapies [START_REF] Carneiro | Targeting apoptosis in cancer therapy[END_REF].

Scientists are also investigating plant compounds like resveratrol (found in red wine) to see if they might trigger cancer cell death.

Angiogenesis inhibitors

To get abundant nutrients and oxygen necessary for tumours to grow, progress and metastasise beyond a specific size, cancer cells form new blood vessels from pre-existing ones by releasing pro-angiogenic chemical signals -a process named tumour angiogenesis [START_REF] Teleanu | Tumor Angiogenesis and Anti-Angiogenic Strategies for Cancer Treatment[END_REF]. Treatments that interfere with angiogenesis (angiogenesis inhibitors) may block tumour growth by preventing new blood supply formation. For example, the treatment of glioblastoma or hepatocellular carcinoma based on some targeted therapies such as Pazopanib (Votrient®), Sorafenib (Nexavar®), or Sunitinib (Sutent®) inhibits angiogenesis interfere with the action of vascular endothelial growth factors (VEGF), a substance that stimulates new blood vessel formation (angiogenic activator) or platelet-derived growth factors (PDGF), that interact with cell surface tyrosine kinase receptors important for regulating cell proliferation, cellular STATE OF THE ART Biophysical-based treatments in cancer therapy -67 -differentiation, cell growth and development. Other angiogenesis inhibitors support the removal of existing vessels, i.e., at the end of an inflammatory response. Those chemical signals, such as angiostatin, endostatin, platelet factor 4 (PF4), thrombospondin-1 and 2, IL-12, etc., facilitate the termination of neovascularisation through anti-apoptosis gene suppression [START_REF] Mousa | Angiogenesis and Anti-Angiogenesis Strategies in Cancer[END_REF].

The FDA has approved several angiogenesis inhibitors to treat cancers, with some being used in conjunction with other anticancer chemotherapeutics [START_REF] Rajabi | The role of angiogenesis in cancer treatment[END_REF]. The most applied amongst the currently available anti-angiogenic drugs of enzymatic or mAb origin are bevacizumab, sunitinib, pazopanib, endostar, regorafenib, axitinib, sorafenib, ranibizumab, and aflibercept (Teleanu, 2019). 3.1.4.4.5 Immune cytokine-based therapies

As discussed above, in the hallmarks of cancer, tumours can develop mechanisms to escape the immunologic detection and elimination [START_REF] Palucka | The Basis of Oncoimmunology[END_REF]. Therefore, anticancer immunotherapy seeks to stimulate the immune system to favour anticancer immunity and decrease the control of cancer cells over immunity. Some immunotherapies use mAbs to recognise specific molecules on the surface of cancer cells and bind to those target molecules, resulting in the immune destruction of cancer cells. Other mAbs bind to specific immune cells to help these cells better kill cancer cells.

Since the discovery of anti-tumour activity of interferon α (IFN-α) against several cancer cell lines in 1970 [START_REF] Gresser | Antitumor effects of interferon preparations in mice[END_REF], scientists have investigated many cytokines such as (in conjunction with electroporation approaches) interleukin 2 (IL-2) or into IL-12, in preclinical studies [START_REF] Cha | Human Vaccines & Immunotherapeutics Plasmid IL-12 electroporation in melanoma[END_REF][START_REF] Mir | Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses[END_REF]., Mir et al., 1997) and clinical trials in both human [START_REF] Daud | Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma[END_REF][START_REF] Greaney | Intratumoral Plasmid IL12 Electroporation Therapy in Patients with Advanced Melanoma Induces Systemic and Intratumoral T-cell Responses A C[END_REF] and veterinary (Mir, 1997;[START_REF] Pavlin | IL-12 based gene therapy in veterinary medicine[END_REF], exploiting their ability to stimulate immune responses, either innate or adaptative, against cancer. Indeed, whereas systemic IL-12 is associated with potentially life-threatening toxicity [START_REF] Atkins | Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies[END_REF][START_REF] Leonard | Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production[END_REF] as part of the incapability of cytokines (which are peptides) to cross the cell lipid bilayer to enter the cytoplasm, the intratumoural plasmid IL-12 electroporation therapy is safe, well-tolerated and can induce tumour regression at distant sites, especially in a patient with metastatic melanoma. Those strategies using electroporation technology to overcome the plasma membrane impermeability were described in chapters 1 and 2 and are part of gene therapy in cancer treatment which will be mentioned in the next section.

STATE OF THE ART Biophysical-based treatments in cancer therapy -68 -Despite some drawbacks of cytokines as monotherapies, the (mild) anti-tumour activities shown by the IL-2 and IFN-α led to their FDA approval to treat several types of cancer. For example, IL-2 has been approved for the treatment of advanced renal cell carcinoma and metastatic melanoma, and IFN-α for the treatment of hairy cell leukaemia, follicular non-Hodgkin lymphoma, melanoma and AIDS-related Kaposi's sarcoma [START_REF] Bashraheel | Update on targeted cancer therapies, single or in combination, and their fine tuning for precision medicine[END_REF] As one of the fastest-growing categories of protein therapeutics with an increasingly improved safety profile, monoclonal antibodies (mAbs) technologies can cause a specific cancer cell death via the delivery of toxic molecules. Once the mAb has bound to its target cell, the toxic molecule linked to the Ab (such as a radioactive substance or a cytotoxic chemical) is taken up by the cell, ultimately killing that cell. Cells that lack the target for the antibody will not be affected by the toxin -i.e., the vast majority of cells in the body.

Nevertheless, mAb-based medicines also have limitations that halt their clinical utilisation.

To date, the most prominent challenges consist of the short pharmacokinetic properties of mAbs and stability issues during their production (manufacturing, transport and storage) that can lead to aggregation and protein denaturation [START_REF] Awwad | Overview of Antibody Drug Delivery[END_REF]. Cytokines have other disadvantages for several types of cancer treatment (low response rate, modest efficacy and high toxicity due to the need for high doses of IL-2 and IFN-α). Therefore, besides the use of excipients (e.g., surfactants and amino acids) or the production of more stable constructs (e.g., protein scaffolds and bispecific molecules), many strategies are being pursued to improve the pharmacokinetic and stability profiles of mAbs, such as Fc fusions, PEGylation, and combination with other therapies (Bashraheel, 2020). 3.1.4.4.6 Cellular adoptive immunotherapy Cellular adoptive immunotherapy (also called adoptive T cell therapy, adoptive cell transfer or T-cell transfer therapy) is immunotherapy in which genetically modified autologous T cells could be intravenously re-infused into patients to produce substantial clinical benefit, at least in specific B cell malignancies [START_REF] Grupp | Chimeric Antigen Receptor-Modified T Cells for Acute Lymphoid Leukemia[END_REF], bypassing vaccination. The most welldeveloped therapy is the chimeric antigen receptor T-cell (CAR T-cell or "CARs") therapy in which patient's T cells are transfected with a construct encoding an antibody against a tumour surface antigen (typically CD19) fused to T cell signalling domains, resulting in a potent antitumour activity in, e.g., lymphoid malignancies [START_REF] Kochenderfer | Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors[END_REF]. With a similar principle, there is tumour-infiltrating lymphocyte (TIL) therapy that employs these lymphocytes, STATE OF THE ART Biophysical-based treatments in cancer therapy -69 -already primed to recognise the tumour cells and destroy the tumour itself. TILs are collected from the tumour during a biopsy or surgical resection and grown in considerable numbers in a laboratory with IL-2 that promotes rapid TIL growth before infusing into the patient [START_REF] Geukes Foppen | Tumor-infiltrating lymphocytes for the treatment of metastatic cancer[END_REF].

Multiple approaches to cancer therapy exist, but few are as complicated as immune-based therapy. Figure 19 depicts some therapies currently under preclinical or clinical evaluation to target the cancer-immunity cycle [START_REF] Chen | OncologyMeetsImmunology: TheCancer-ImmunityCycle[END_REF].

Vaccination

Vaccination allows immune cells to better irradicate cancerous cells by efficiently recognising antigens presenting on those cell surfaces. Here vaccination is not a preventive approach but one more treatment of established, slow-growing (or otherwise already treated) tumours. The anticancer vaccines could be either from the patient's tumour cells (antigenpresenting cells or APCs) or from DCs that stimulate the immune system to respond to an antigen present on cancer cells. More recently, a novel approach called oncolytic virotherapy was proposed and which uses oncolytic virus (natural or engineered) to enhance systemic antitumour immune responses [START_REF] Andtbacka | Talimogene laherparepvec improves durable response rate in patients with advanced melanoma[END_REF]. These oncolytic viruses have tumour selectivity and, through genetic engineering methods, the capacity of being transformed to express tumour-killing factors only in the cancer cells while being non-pathogenic for the noncancerous counterparts. They have been ever since used in combination with other anticancer therapies [START_REF] Cao | The Oncolytic Virus in Cancer Diagnosis and Treatment[END_REF].

STATE OF THE ART Biophysical-based treatments in cancer therapy -70 -

Gene therapies

Gene therapies, as their name indicate, are treatments dealing with nucleic acids rather than chemical agents for therapeutic ends. Gene therapy is sometimes considered a targeted therapy because, like cancer vaccines, this therapy, for anticancer aims, interferes with the growth of specific cancer cells.

It has been nearly half a century since the introduction of the gene therapy concept for human genetic disease [START_REF] Friedmann | Gene therapy for human genetic disease?[END_REF] and almost twenty years since the beginning of the successful clinical translation of gene therapies in the 2000s. In between those historical moments, the early development of gene delivery vectors such as an adeno-associated virus (AAV) and replication-defective retroviruses, coupled with encouraging results in preclinical disease models, was hindered by, unfortunately, therapy-related severe toxicities in the initial clinical trials in the early 1990s, including inflammatory responses to the virus vectors and malignancies caused by vector-mediated insertional activation of proto-oncogenes [START_REF] Dunbar | Gene therapy comes of age[END_REF].

Building on decades of pre-clinical research and clinical assays, with manufacturing advances, gene therapies have begun to be improved using plasmid or non-viral vectors as an alternative to classical viral gene delivery. The delivery of nucleic acids is enhanced by various approaches, including a polymer or lipid conjugation, particle-mediated delivery, hydrodynamic delivery, ultrasound or electroporation [START_REF] Dunbar | Gene therapy comes of age[END_REF][START_REF] Heller | Electrically mediated delivery of plasmid DNA to the skin, using a multielectrode array[END_REF][START_REF] Heller | Gene Electrotransfer Clinical Trials[END_REF]. These safer and more efficient plasmid vectors were used to transduce autologous hematopoietic stem cells in patients with immunodeficiencies, hemoglobinopathies, and metabolic and storage disorders. The US FDA approved the first gene therapy products in 2017, with CAR-T cells (described above in section "cellular adoptive immunotherapy") to treat B cell malignancies and AAV vectors for in vivo treatment of congenital blindness [START_REF] Dunbar | Gene therapy comes of age[END_REF].

Up to 2019, twenty approved human gene and cell-based gene therapy products have already been approved in clinics and markets of mainly North America, Europe and Asia [START_REF] Shahryari | Development and clinical translation of approved gene therapy products for genetic disorders[END_REF], primarily based on AAV and lentiviral vectors, and over two thousand human gene therapy clinical trials have been reported worldwide (http://clinicaltrials.gov).

Even if all those abovementioned targeted therapies have been proven efficient in various cancers, targeted therapies can instigate serious side effects. Common ones are diarrhoea, hepatitis (amongst other liver problems), and changes to the patient's skin, hair, and nails. The STATE OF THE ART Biophysical-based treatments in cancer therapy -71 -toughest for most cancer patients is skin problems because targeted cancer therapies attack the same growth factors and blood vessels needed for healthy skin.

Personalised medicine

After decades of research, scientists now understand how genetic changes in patients' tumours can cause cancer to grow and spread. They have also discovered that the changes that drive cancer in one person may not occur in others who have the same type of cancer, i.e., individuals with the same disease respond differently to the same therapy. Moreover, the same cancer-causing changes may be found in different categories of cancer. Until recently, cancer patients with the same type and stage of cancer were receiving the same treatment. Thus, the inefficiency of the therapies has been observed in numerous cases.

Personalised medicine, also known as precision medicine, allows the selection of a treatment based on the patient's genetic makeup. For cancer, the treatment is tailored to consider the genetic changes that may occur in the individual's tumour, i.e., the approach takes advantage of knowing the genetic changes in a patient's tumour to determine its selective treatment. Hence, personalised medicine could be defined as the targets involved in targeted therapy, with similar strategies to target cancer as described above.

Despite the richness of therapies, challenges remain to this day in oncology. The employed anticancer therapeutic strategies present indeed problems of specificity for tumour cells or a lack of validated biomarkers and also insufficient characterisation of the patient populations appropriate for treatment, amongst others, thus causing considerable side effects. The precise targeting of tumours by local treatment is not always practicable. Furthermore, cancer recurrence or the development of multidrug resistance and the weak induction of immunogenic cell death, amongst other issues, remain to limit factors. Therefore, different therapeutic strategies based on new approaches are required, particularly physical modalities that allow local treatment and are less prone to developing resistance in cancer cells. Several strategies potentiate anticancer drugs' distribution in tumours, improving their therapeutic index.

Recent years have witnessed a considerable increase in research, development and application of anti-cancer therapies based on physical principles, amongst which electrochemotherapy and non-thermal plasma technology have raised great excitement. These approaches offer eradication of cancerous tumours, directly or indirectly, and in the case of electroporation, a safe and efficient vectorisation technique for delivering molecules of therapeutic interest (e.g., STATE OF THE ART Biophysical-based treatments in cancer therapy -72 -nucleic acids, drugs, ions). The following two sections are interested in electrochemotherapy, an anti-cancer treatment based on electroporation, and non-thermal plasma technology applied in oncology.

Electrochemotherapy

Basic knowledge

Electrochemotherapy (ECT) enhances the delivery to the tumour site of very low-or nonpermeant chemotherapeutic agents, at a low dose in the tumour site, by the local exposure to electric pulses transiently permeabilise the tumour cell membrane. ECT selectively kills the tumour cells and accordingly reduces associated systemic side effects. Being the first electroporation-based application in the targeted drug administration (Mir, 1991b), ECT is a non-thermal, safe and efficient established local eradication of solid tumours. The effectiveness of ECT has made it nowadays widely utilised in more than 150 clinics in the European Union and abroad in the human (Campana et al., 2019a;[START_REF] Probst | Electrochemotherapy as a new modality in interventional oncology: A review[END_REF] and veterinary oncology (Čemažar et al., 2008). ECT indeed has an excellent therapeutic index over other chemical-based anti-cancer treatments, i.e., very low concentrations of the chemotherapeutic agents are needed to achieve antitumour effectiveness; thus, eventual systemic toxicity is spared.

The theory of ECT is based on the properties mentioned above of reversible electroporation (see chapter 1.2 and Figure 20) combined with the therapeutic efficiency of chemotherapeutic molecules [START_REF] Escoffre | Electrochemotherapy: Progress and Prospects[END_REF]Mir, 1991a).

Figure 20| Principle of electrochemotherapy

Low-or non-permeant cytotoxic drugs (BLM or cis-Pt) are i.v. or i.t. administered to a patient, then short intense electric pulses are applied to the tumour via electrodes connected to an electric pulse generator, resulting in tumour cell electropermeabilisation and, therefore, in an increased internalisation of chemotherapeutic drugs in the cytosol, leading to necrotic/apoptotic cell death by drug cytotoxicity, systemic anti-tumour reaction (Calvet et al., 2014b); vasoconstriction of the tumour neovasculature to the ischemia of the tumour cells [START_REF] Jarm | Antivascular effects of electrochemotherapy: Implications in treatment of bleeding metastases[END_REF]. Scheme adapted from [START_REF] Escoffre | Electrochemotherapy: Progress and Prospects[END_REF].

In general, a patient receiving ECT will first get an intravenous or intratumoural administration of an anti-cancer drug, commonly used bleomycin (BLM) or cis-platin (cis-Pt), resulting in an even distribution over the vascular system and extracellular space of the tissue.

Electrodes located around (see Figure 8) or inside the tumour (see Figure 24) will deliver defined electric pulses, which enable diffusion through the plasma membrane of the otherwise very low or non-permeant anti-cancer drug into the target cells. After a short time ranging from a few seconds to several minutes after exposure to the PEF, the plasma membrane permeability will return to its initial state. The specific anticancer chemotherapeutics will elicit DNA singleand double-strand breaks (bleomycin) or intra-and inter-strand DNA bonds (cis-platin). Those cytotoxic effects are much more pronounced in fast-dividing tumour cells than in quiescent (non-malignant) cells because unrepaired strand breaks mainly affect dividing cells [START_REF] Mir | Bases and rationale of the electrochemotherapy[END_REF][START_REF] Tounekti | The ratio of single-to double-strand DNA breaks and their absolute values determine cell death pathway[END_REF]. Therefore, this mechanism of action only requires two main conditions to achieve an efficient ECT: first, although low, a necessary concentration of cytotoxic drug has to reach the tumour, and second, the whole tumour nodule has to be covered by a permeabilising pulsed electric field (PEF). Similar to EGT, the electroporation parameters should be carefully chosen in ECT to avoid "excessive electroporation" because cells should survive the electroporation process.

Benefits arising from this combination therapy of pulsed electric fields (PEFs) and chemotherapeutic agents include a considerable reduction of the dose of a cytostatic drug due to locally increased cytotoxic effect (BLM, 1000-fold and cis-platin, 80-fold increase of toxicity) STATE OF THE ART Biophysical-based treatments in cancer therapy -74 - [START_REF] Gehl | Enhancement of cytotoxicity by electropermeabilization: An improved method for screening drugs[END_REF]Orlowski, 1988;Poddevin et al., 1991), as, in the case of the bleomycin, a few hundreds of molecules per cell only are sufficient to induce cell death in a single treatment session. ECT is used in clinics to treat almost all types of tumour nodules, as all cells are permeabilised by electric pulses of appropriate electric field amplitude and bleomycin is effective on all dividing cells, whatever their origin. Beyond its effectiveness and benefits mentioned above, ECT is a repeatable and minimally invasive intervention that is easy and quick to perform (about 25-30 minutes), thus offering a short hospitalisation and mitigating the symptom burden.

Our group published the first clinical study on ECT in 1991, demonstrating this target anticancer modality's feasibility, safety, and effectiveness (Mir, 1991b). This pioneered study enthused groups in the United States (Tampa), Slovenia (Ljubljana), and France (Toulouse and Reims) to accomplish further clinical studies [START_REF] Domenge | Antitumor Electrochernotherapy New Advances in the Clinical Protocol[END_REF][START_REF] Heller | Treatment of cutaneous nodules using electrochemotherapy[END_REF][START_REF] Rudolf | Electrochemotherapy with bleomycin. The first clinical experience in malignant melanoma patients[END_REF], which were then compiled in a joint paper published seven years later [START_REF] Mir | Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy[END_REF].

Figure 21|

Photos demonstrating a complete response to ECT in a patient with cutaneous metastasis from malignant melanoma Photos of the course of treatment for a patient treated with i.v. bleomycin and type III (hexagonal) electrodes under general anaesthesia. Before treatment (left panel), the metastasis was ulcerated with consequently haemorrhage, pain and discomfort. One month after treatment (middle panel), the lesion was covered by a crust. Around the crust, needle marks in normal tissue are visible, because the tumour was covered including a margin of normal tissue. Whereas the tumour area became necrotic, the normal tissue was very little affected, indicating a therapeutic window. Six months after treatment (right panel), complete regression of the treated nodule has been observed. The crust fell off after 10 weeks, revealing normal skin that had healed underneath the nodule (from Gehl J., Ugeskrift for Laeger, 2005).

The current and potential applications of ECT are focused on: (i) primary tumours (breast, ovary, colon-rectum, pancreas); (ii) cutaneous metastasis (melanoma and non-melanoma skin cancer, breast, head and neck) and (iii) non-cutaneous metastasis (bone, liver, brain, soft tissue sarcomas) [START_REF] Esmaeili | Electrochemotherapy: A Review of Current Status, Alternative IGP Approaches, and Future Perspectives[END_REF].

STATE OF THE ART Biophysical-based treatments in cancer therapy -75 -

Chemical aspects of ECT

Bleomycin

First isolated from Streptomyces verticillus collected from the soil in a coal mine in Japan by Umezawa and colleagues in 1966, bleomycin (BLM) forms a family of glycopeptide antibiotics with antitumour activity [START_REF] Umezawa | New antibiotics, bleomycin A and B[END_REF]. BLM is, therefore, employed in the treatment of, amongst others, Hodgkin's disease, non-Hodgkin's lymphoma and testicular cancer [START_REF] Tounekti | Bleomycin, an Apoptosis-mimetic Drug That Induces Two Types of Cell Death Depending on the Number of Molecules Internalized[END_REF]. BLM is the most commonly used drug combined with chemotherapy against lymphomas, squamous-cell carcinomas, and germ-cell tumours in clinics. BLM is administered intravenously, subcutaneously or intramuscularly, as it is not absorbed from the gut. The primary target for the cytotoxic action of BLM is DNA.

The BLM family comprises at least thirteen small water-soluble glycopeptide antibiotics, all members have an identical core structure but differ in their configuration of sugars and positively charged tails. Some clinically administered forms of BLM are bleomycin sulfate injection under brands named e.g., Blenoxane (Bristol-Myers Squibb) or Bleomycine Bellon (Sanofi). Due to its hydrophilic nature, bleomycin is normally not bound to plasma proteins, and its entry into the cells is limited and primarily mediated by endocytosis [START_REF] Pron | Internalisation of the bleomycin molecules responsible for bleomycin toxicity: A receptor-mediated endocytosis mechanism[END_REF]. Once inside the cell, BLM becomes a very potent drug. Nevertheless, this high intrinsic cytotoxicity is limited because as a large and hydrophilic molecule, BLM is unable to cross cell plasma membranes by free diffusion [START_REF] Mir | Bleomycin: Revival of an old drug[END_REF]Orlowski, 1988;Poddevin, 1991).

Studies indicate that the positively charged tail of BLM might be key to cellular uptake. The BLM require cofactors which are a reduced transition metal (Fe(II) or Cu(I)), oxygen and a oneelectron reductant to generate "activated" bleomycin. Once activated, BLM can catalyse oxidative damage to the ribose or the deoxyribose of nucleotides in the DNA or doublestranded RNA, which further leads to single-and double-stranded DNA breaks and cell apoptosis [START_REF] Chen | Bleomycins: Towards better therapeutics[END_REF]. Those damages are reminiscent of those generated by ionising radiation.

Other main points:

• The eukaryotic cellular responses to BLM administration are complex. They are cellline-and genotype-dependent, with extended cell-cycle arrest, apoptosis and mitotic cell death as the most common outcomes [START_REF] Tounekti | The ratio of single-to double-strand DNA breaks and their absolute values determine cell death pathway[END_REF]. STATE OF THE ART Biophysical-based treatments in cancer therapy -76 -

• In concert with chemical synthesis, biosynthetic genes for BLM have been identified. Some of the proteins in the pathway have been characterised, opening the gate to making libraries of BLM analogues.

• BLM is more toxic in fast-dividing cells as compared with quiescent cells because unrepaired strand breaks mainly affect mitotic cells [START_REF] Tounekti | Bleomycin, an Apoptosis-mimetic Drug That Induces Two Types of Cell Death Depending on the Number of Molecules Internalized[END_REF] BLM is inactivated in normal and tumour tissues by bleomycin hydrolase, a cysteine protease that cleaves the ammonia group from BLM [START_REF] Sebti | Metabolic inactivation of bleomycin analogs by bleomycin hydrolase[END_REF][START_REF] Umezawa | A bleomycin-inactivating enzyme in mouse liver[END_REF]. This enzyme is active in all tissues but is found at deficient activity in the skin and lung, which may explain these being the most common sites of toxicity by BLM. Indeed, the side effects of the BLM are tissue-specific and dose-dependent and encompass lung inflammation which often advances to the lung fibrosis [START_REF] Brömme | Human bleomycin hydrolase: Molecular cloning, sequencing, functional expression, and enzymatic characterization[END_REF][START_REF] Fyfe | Toxicity associated with bleomycin[END_REF].

In clinical practice, side effects of BLM are dose-dependent and include mainly lung fibrosis and skin toxicity (erythema, peeling, hyperkeratosis). These results are probably from the poor expression of bleomycin hydrolase in these tissues, an enzyme responsible for BLM degradation. However, contrary to many anticancer drugs, neither digestive side effects nor immunosuppression nor myelosuppression were reported following BLM administration. In pre-clinical ECT regimens, BLM was first used with electroporation in 1988 to increase its in vitro uptake and thus increase its cytotoxic activity (Orlowski, 1988). Three years later, the internalisation of BLM mediated by electroporation was demonstrated to be dependent on the external concentration of the drug and that as few as 500 BLM molecules were sufficient to trigger cell death (Poddevin, 1991), highlighting the possibility of using doses that spare normal tissues thanks to electroporation, thus avoiding the side effects reported here above. The combination of BLM with EPs was tested on various cell lines and decreased the half-maximal inhibitory concentration (IC50) by up to several thousand folds [START_REF] Jaroszeski | Electrochemotherapy: An emerging drug delivery method for the treatment of cancer[END_REF][START_REF] Larkin | Electrochemotherapy: Aspects of preclinical development and early clinical experience[END_REF]Poddevin, 1991). Similarly, in vivo investigations performed in several species (i.e., mice, rats, rabbits, cats, dogs, horses) revealed that ECT regimens using BLM resulted in tumour regression in various types of both transplanted and spontaneous tumour models [START_REF] Belehradek | Electrochemotherapy of spontaneous mammary tumours in mice[END_REF][START_REF] Čemažar | Electrochemotherapy in Veterinary Oncology[END_REF][START_REF] Gothelf | Electrochemotherapy: Results of cancer treatment using enhanced delivery of bleomycin by electroporation[END_REF][START_REF] Larkin | Electrochemotherapy: Aspects of preclinical development and early clinical experience[END_REF]Mir, 1991a). It is worth noting that the very low dose of BLM used in ECT protocols (ca. 0.5 mg/kg), along with the local delivery of EPs, enables the selective toxicity of the drug toward cancer cells. Indeed, in the treated region, both healthy and cancer cells are permeabilised and consequently will get their DNA cleaved. STATE OF THE ART Biophysical-based treatments in cancer therapy -77 -However, the hundreds of BLM molecules internalised generate enough DNA breaks to drive cell death upon cell division (as is defined as mitotic cell death) but are not sufficient to cause metabolic cell death. Therefore, normal quiescent cells are spared.

Cis-platin

Cis-platin (cis-Pt or cis-diamminedichloridoplatinum (II) (CDDP)), another common antineoplastic agent, possesses a permeabilisation coefficient through the cell plasma membrane by passive diffusion of less than 50%. In contrast, the remainder is transported by carrier molecules [START_REF] Eljack | Mechanisms of cell uptake and toxicity of the anticancer drug cisplatin[END_REF]. Cis-Pt has cytotoxic properties involving DNA transcription and replication interference. Nevertheless, many side effects are observed at the therapeutic dose, including neuronal, cardiac and renal toxicity and bone marrow suppression.

Besides, cis-Pt resistance has been reported as associated with changes in cellular drug uptake, drug efflux, increased detoxification, inhibition of apoptosis and increased DNA repair.

Consequently, electroporation represents a major advantage to avoid side effects and resistance, as much more molecules get access to the cell interior while decreasing the dose administered.

Indeed, in in vitro, the half-maximal inhibitory concentration (IC50) of cis-Pt was decreased by up to 13-fold when using EPs [START_REF] Gehl | Enhancement of cytotoxicity by electropermeabilization: An improved method for screening drugs[END_REF][START_REF] Jaroszeski | Toxicity of anticancer agents mediated by electroporation in vitro[END_REF]. Interestingly, increased cytotoxicity to the drug was also observed in cis-platin-resistant cell lines (Čemažar et al., 1998b). In in vivo, studies also showed an improvement of anticancer activity of ECT with cis-Pt as compared with cis-Pt alone [START_REF] Serša | Antitumor Effectiveness of Electrochemotherapy with cis-Diamminedichloroplatinum(II) in Mice[END_REF], in particular with an intratumoural administration of the drug (Čemažar et al., 1998a). Other platinum (II) analogues have been tested in the context of ECT but cis-Pt appears to be the most efficient when combined with EPs (Čemažar et al., 2006). Cis-Pt is only administrated intratumourally, otherwise, it will generate systemic toxicity (nephropathies, and others).

Other approaches

Other drugs which have been investigated for use in ECT protocols (but not used in the clinical practice of ECT) include:

• Daunorubicin, doxorubicin, etoposide, paclitaxel, fluorouracil, Taxotere, vincristine and methotrexate (Miklavčič, 2014;Orlowski, 1988). However, since they are lipophilic (thus already easily penetrating the cells) highly toxic drugs, no significant enhancement of cytotoxicity was observed when using electroporation.

STATE OF THE ART Biophysical-based treatments in cancer therapy -78 -Contrariwise, the application of EPs concomitant with mitomycin C administration showed a 30 % increase in the efficiency of intravesical chemotherapy in bladder cancer, in comparison to mitomycin C alone [START_REF] Vásquez | Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line: A potential improvement of intravesical chemotherapy in bladder cancer[END_REF].

• Calcium ions uptake: high concentrations of calcium are introduced into the cell cytosol by electroporation (calcium electroporation), inducing cell death in vitro and tumour necrosis in vivo with a difference in sensitivity between different tumour types [START_REF] Frandsen | Direct therapeutic applications of calcium electroporation to effectively induce tumor necrosis[END_REF][START_REF] Frandsen | A review on differences in effects on normal and malignant cells and tissues to electroporation-based therapies: A focus on calcium electroporation[END_REF]. This novel anticancer strategy has recently entered a clinical trial to treat cutaneous metastases [START_REF] Falk | Calcium electroporation for treatment of cutaneous metastases; a randomized double-blinded phase II study, comparing the effect of calcium electroporation with electrochemotherapy[END_REF]. Results of this first clinical trial showed an apparent effect of calcium electroporation comparable to the effect of bleomycin-based ECT, results which were confirmed recently during another double-blinded randomised controlled phase II trial where no serious adverse events were registered and where the complete response was confirmed histologically in both arms (Ágoston et al., 2020).

Calcium, like cis-Pt, is administered intratumourally only and in small tumours.

Adjuvant effects of ECT

Together with the direct cytotoxicity of ECT toward cancer cells, other bystander effects have been reported and contribute to the ECT beneficial outcome. In particular, ECT induces anti-vascular effects at the level of neo-formed blood vessels and stimulates the immune system.

Anti-vascular effects

Serša and colleagues have shown that EPs application on a tumour induces immediately vasoconstriction lasting up to several hours rather than minutes, resulting in an 80 % drop in the tumour blood flow -a phenomenon called "vascular lock" [START_REF] Jarm | Antivascular effects of electrochemotherapy: Implications in treatment of bleeding metastases[END_REF][START_REF] Markelc | Differential Mechanisms Associated with Vascular Disrupting Action of Electrochemotherapy: Intravital Microscopy on the Level of Single Normal and Tumor Blood Vessels[END_REF][START_REF] Serša | Electrochemotherapy with CDDP on LPB sarcoma: Comparison of the anti-tumor effectiveness in immunocompetent and immunodeficient mice[END_REF]. This "vascular lock", by avoiding the drug clearance even after cell membrane resealing, enables a prolonged drug uptake by tumour cells (mainly proposed for low-permeant drugs, such as cis-Pt). This transient vascular lock in the tissues exposed to the EPs is followed by anti-vascular effects resulting from the death of the endothelial vascular cells (provide tumour cells are also killed and thus no longer secrete angiogenic factors…). The "vascular lock" associated with the destruction of the endothelium leads to tumour starvation (lack of oxygen and growth factors) and thus to cancer cell death. Moreover, these anti-vascular effects are of STATE OF THE ART Biophysical-based treatments in cancer therapy -79 -significant interest for treating bleeding tumours as they enable to cease the haemorrhage immediately (Figure 22) [START_REF] Snoj | Limb sparing treatment of bleeding melanoma recurrence by electrochemotherapy[END_REF].

Figure 22| ECT induced vascular lock on a haemorrhagic melanoma metastasis immediately after delivery of the electric pulses Follow-up of bleomycin-mediated ECT on bleeding melanoma metastasis on a leg of a patient treated at the Institute of Oncology Ljubljana. Immediately after delivery of the EPs, the bleeding ceased and did not recur. The lesion developed a crust during a week post-treatment, fell off within three weeks. The tumour reduced in size considerably and remained in partial response during 21 weeks of observation time. [START_REF] Snoj | Limb sparing treatment of bleeding melanoma recurrence by electrochemotherapy[END_REF].

Immune stimulation

There is evidence that the immune system contributes to ECT efficiency. Indeed, in comparison to immunocompetent mice, ECT-mediated tumour regression was dramatically decreased in animals exempt from functional T lymphocytes, either due to the pre-treatment injection of OKT3 mAb [START_REF] Mir | Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses[END_REF] or when treatments were performed in nude mice (Mir, 1991a;Serša et al., 1997a). In other words, blocking the immune system would block the systemic effect of ECT. Moreover, in vivo studies from our group have shown synergistic effects of the combination of ECT and interleukin-2 (IL-2) not only in the enhancement of the local antitumour effect in the treated area but also in the induction of a memory effect (treated animals did not develop tumours of a given type when re-challenged at a later time) [START_REF] Mir | Systemic Antitumor Effects of Electrochemotherapy Combined with Histoincompatible Cells Secreting Interleukin-2[END_REF].

Local oedema was also observed in tumours treated by ECT after intramuscular administration of the drug, suggesting that the EPs delivered to the tumour were the sole responsible ones for the swelling observed (Mir, 1991a). The oedema was more severe in immunocompetent than in immunodeficient mice, consistent with EP-mediated effects that depend on the presence of an intact immune system. The increase of the vascular permeability by oedema probably paved the way for the local infiltrates of dendritic cells (DCs) (Roux et al., 2008) and lymphocytes [START_REF] Mekid | In vivo evolution of tumour cells after the generation of double-strand DNA breaks[END_REF], which were found in treated tumours. ECT treatment of human melanoma led to the maturation of pre-existing tumour-resident Langerhans cells -an epidermal subset of DCs, and their subsequent migration to the tumour-draining lymph node as early as 24 hours after the treatment [START_REF] Gerlini | Dendritic cells recruitment in melanoma metastasis treated by electrochemotherapy[END_REF]. Similarly, an intratumoural recruitment of DCs STATE OF THE ART Biophysical-based treatments in cancer therapy -80 -expressing CD80/CD86 maturation markers 48 hours after the ECT treatment was also detected in immunogenic murine tumours in immunocompetent mice (Roux, 2008).

In in vivo studies, our group have also shown that ECT had high efficiency as it stimulates the immune response, which can destroy the cells that escaped the direct anti-tumoural effect of ECT, either because they were not sufficiently permeabilised by the electric pulses or not surrounded by enough BLM molecules for generating cytotoxicity. ECT thus causes immunogenic cell death (ICD), a phenomenon that occurs partially due to the application of EPs that trigger on their own both an early and physiologically relevant ATP leakage (immuneattractant for DCs) and a calreticulin translocation to the cell surface (a protein activating the DCs). As a consequence, after the bona fide ICD of electroporated cells, cancer antigens can be captured and recognised by DCs and eventually increase antitumour response (Calvet, 2014b).

ECT could be considered as the adjuvant immunogen electrotransfer to peritumoural tissue [START_REF] Serša | Electrochemotherapy of tumors as in situ vaccination boosted by immunogene electrotransfer[END_REF]. The process leads to the local effects and triggers the systematic response against distant metastases -the so-called abscopal effect [START_REF] Postow | Immunologic Correlates of the Abscopal Effect in a Patient with Melanoma[END_REF].

Medical applications and current developments of ECT

Electrochemotherapy of superficial tumours

Solid tumours present several barriers to the targeted delivery of chemotherapies [START_REF] Minchinton | Drug penetration in solid tumours[END_REF]. Treatment of superficial tumours with ECT has shown a steep rise for almost two decades, whose indications range from skin cancers to locally advanced or metastatic neoplasms. Since the release of the ESOPE and especially after its updated version in 2018, consolidated indications include superficial metastatic melanoma, breast cancer, head and neck skin tumours, nonmelanoma skin cancers, and Kaposi sarcoma (Campana et al., 2019b). Moreover, ECT ensures appreciable symptom control in well-selected patients with oropharyngeal cancers. Key aspects favouring the broad acceptance of ECT for the treatment of superficial tumours are the simplicity and versatility of its procedure. Indeed, based on flexible technology, standard ECT (i.e., ECT applied through fixed-geometry electrodes, as shown in Figure 8) represents an easy-to-master procedure that allows treating various cancers. Emerging applications of ECT include treatments of skin metastases from visceral malignancies, cutaneous metastases from STATE OF THE ART Biophysical-based treatments in cancer therapy -81 -hematologic cancers, vulvar carcinoma, and noncancerous skin lesions (Keloid scars). The application of ECT to treat visceral and deep-seated tumours will be discussed thereafter.

Palliative anti-cancer treatment for human patients

With a 5-year survival rate ranging from 98.4% for localised melanoma at diagnosis, over 62.4% for patients with regional lymph nodes being affected at diagnosis or treatment, to only 17.9% for widespread metastatic melanoma [START_REF] Hachey | Therapeutic implications of melanoma heterogeneity[END_REF], effective palliation is ultimately necessary for malignant melanoma patients. For almost three decades since its application in clinics, electrochemotherapy (ECT) was introduced in palliative care to alleviate the burden imposed by tumours, such as cutaneous and subcutaneous malignant melanoma, breast cancer, head and neck cancer with relatively successful local response rates (Campana et al., 2019c;[START_REF] Linnert | Multiple brain metastases -Current management and perspectives for treatment with electrochemotherapy[END_REF][START_REF] Longo | Electrochemotherapy as palliative treatment in patients with advanced head and neck tumours: Outcome analysis in 93 patients treated in a single institution[END_REF]Matthiessen et al., 2012;[START_REF] Pichi | Electrochemotherapy: A well-accepted palliative treatment by patients with head and neck tumours[END_REF].

A recent meta-analysis of ECT in a palliative setting found a complete response (CR) rate of 46.6% and objective response rates (ORR) of 82.2% according to the RECIST, regardless of the tumour type [START_REF] Morley | Electrochemotherapy for the palliative management of cutaneous metastases: A systematic review and meta-analysis[END_REF]. Furthermore, this meta-analysis specified that small tumours were over twice as likely (2.25) to have a complete response than large, and highlighted the need for tailored ECT depending on individual cases.

It is worth reporting the latest use of ECT as palliative care of cancer metastasis in the spinal cord for two human patients (interventional radiology service at the Tenon Hospital, Paris, in collaboration with our group). Two patients received percutaneous image-guided ECT of blastic spine metastases. The first (whose results depicted in Figure 23) had metastatic breast carcinoma, and the second patient had metastatic lung carcinoma. Both patients had previously undergone chemotherapy and received radiation therapy on bone metastases due to severe back pain. Only 24 hours after ECT, enhancement of metastases decreased on contrastenhanced MRI (Figure 23F). The tolerance was excellent, and the post-treatment period was uneventful without reported side effects. The pain was quantified on the visual analogue scale (VAS) which consists of a straight 10 cm line with the endpoints defining extreme limits such as "no pain at all" (0 mm) and "pain as bad as it could be" (100 mm). If before ECT, the VAS was at 65 mm for the first patient and 90 mm for the second, at discharge, the VAS was 0 mm for the first patient and 10 mm for the second.

Treatment of visceral or deep-seated tumours

ECT easily accesses superficial (cutaneous and subcutaneous) tumours. However, with the skin electrodes (fixed geometry, as shown in Figure 8), it is not simple to apply ECT protocols successfully on deep-seated tumours located in internal organs, such as the colon, liver, bone and brain, whose surfaces are often not regular [START_REF] Miklavčič | Electrochemotherapy: Technological advancements for efficient electroporation-based treatment of internal tumors[END_REF]Miklavčič, 2014). Efforts are being put into the development of new electrodes to reach internal tumours with minimally invasive procedures while preserving the efficiency of the ECT [START_REF] Edhemovic | Electrochemotherapy: A new technological approach in treatment of metastases in the liver[END_REF][START_REF] Linnert | Multiple brain metastases -Current management and perspectives for treatment with electrochemotherapy[END_REF][START_REF] Mahmood | Optimizing clinical performance and geometrical robustness of a new electrode device for intracranial tumor electroporation[END_REF]:

• Variable geometry long single needle electrodes, mainly designed to treat liver and bone metastases and soft tissue sarcoma

• Endoluminal electrodes to treat colorectal cancers and oesophageal tumours

• Retractable electrodes for the treatment of brain and liver tumours

The insertion of electrodes is prepared by numerical modelling, proved to be feasible, and is done according to the treatment plan. Since the first reported clinical case of deep-seated tumour ECT [START_REF] Miklavčič | Towards treatment planning and treatment of deep--155 -seated solid tumors by electrochemotherapy[END_REF], these new types of electrodes (Figure 24) have been widely STATE OF THE ART Biophysical-based treatments in cancer therapy -83 -employed in both pre-clinical and clinical studies, primarily in the liver ECT [START_REF] Edhemovic | Electrochemotherapy: A new technological approach in treatment of metastases in the liver[END_REF][START_REF] Fiorentzis | The Potential Use of Electrochemotherapy in the Treatment of Uveal Melanoma: In Vitro Results in 3D Tumor Cultures and In Vivo Results in a Chick Embryo Model[END_REF][START_REF] Simioni | Ablation of soft tissue tumours by long needle variable electrode-geometry electrochemotherapy: final report from a single-arm, singlecentre phase-2 study[END_REF][START_REF] Zmuc | Large Liver Blood Vessels and Bile Ducts Are Not Damaged by Electrochemotherapy with Bleomycin in Pigs[END_REF]. These new configurations of electrodes and ECT procedures have paved the way for the approval of ECT treatment of visceral and deep-seated tumours.

Veterinary oncology

Besides clinical applications in human patients, ECT also finds success in the veterinary oncology [START_REF] Čemažar | Electrochemotherapy in Veterinary Oncology[END_REF]Mir, 1997;[START_REF] Tamzali | Successful treatment of equine sarcoids with cisplatin electrochemotherapy: A retrospective study of 48 cases[END_REF]. An example of a successful ECT in an animal bearing carcinoma is shown in Figure 25. Although ECT efficacy both in human and veterinary oncology was well demonstrated rapidly after the first clinical trial (Mir, 1991b), differences amongst the choice of electrodes, the lack of defined operating procedures, and the use of different pulse generators prevented the widespread adoption of ECT in the clinical setting until the mid-2000s [START_REF] Serša | The state-of-the-art of electrochemotherapy before the ESOPE study; advantages and clinical uses[END_REF], when the development of the first version of standard operating procedure (SOP) has been validated in the European Standard Operating Procedures for ECT (ESOPE) in 2006 (Gehl, 2006;[START_REF] Marty | Electrochemotherapy -An easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: Results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study[END_REF]Mir et al., 2006). Provided with the SOP decision tree to help clinical staff make treatment decisions, this SOP reference describes the recommendations for indication of ECT, pretreatment information, treatment choices, and follow-ups to safely and conveniently treat patients with cutaneous and subcutaneous nodules. As a result, the evaluation and confirmation of treatment response based on ESOPE showed an 85% objective response (OR) rate and a 74% complete response (CR) rate of ECT, regardless of tumour histology, the drug used and route of administration (Campana et al., 2016). In 2018, a new version of SOP for ECT of cutaneous tumours and skin metastasis is updated by a Pan-European expert panel from dermatology, general surgery, head and neck surgery, plastic surgery, and oncology [START_REF] Gehl | Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases[END_REF]. This group concluded that the best tumour control was achieved with i.v. BLM, followed by i.t. cis-Pt and i.t. BLM. ECT procedure can be performed within 30 minutes both in local, locoregional or general anaesthesia -a procedure decided by the treating surgeon. Standardisation of ECT procedure has allowed its implementation in over 150 European cancer centres and has contributed to its diffusion in the clinical practice (Campana, 2019a). In most clinical investigations that focused on the patient's response (in both human and veterinary medicine), STATE OF THE ART Biophysical-based treatments in cancer therapy -85 -the Response Evaluation Criteria in Solid Tumors (RECIST) was applied [START_REF] Coletti | Safety and feasibility of electrochemotherapy in patients with unresectable colorectal liver metastases: A pilot study[END_REF][START_REF] Eisenhauer | New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)[END_REF][START_REF] Milevoj | Healthrelated quality of life in dogs treated with electrochemotherapy and/or interleukin-12 gene electrotransfer[END_REF].

The timeline of clinical ECT is illustrated in

Plasma oncology

The rise of a new physicochemical source for cancer therapy

In the past decade, the most important field of investigation of NTP for medicine is the application in cancer treatment. The anti-cancer potential of NTP was first reported by Fridman and colleagues in 2007 via their in vitro study demonstrating the selective induction of apoptotic in melanoma skin cell lines treated with FE-DBD, suggesting a local and tumour-specific upregulation of the systemic immune response by NTP [START_REF] Fridman | Floating electrode dielectric barrier discharge plasma in air promoting apoptotic behavior in Melanoma skin cancer cell lines[END_REF]. Ten years later, the first clinical trial as proof of concept for NTP treatment of actinic keratoses (in situ squamous cell carcinomas of the skin), achieved encouraging results in five enrolled patients [START_REF] Friedman | Successful treatment of actinic keratoses using nonthermal atmospheric pressure plasma: A case series[END_REF]. NTP has open the door to palliative care of patients whose tumour ulcerations was infected. In some particular cases, also transient tumour remission occurred [START_REF] Metelmann | Head and neck cancer treatment and physical plasma[END_REF]., Metelmann et al., 2018a;[START_REF] Schulz-Von Der Gathen | Visible tumor surface response to physical plasma and apoptotic cell kill in head and neck cancer[END_REF]. NTP is also used in human clinics in conjunction with surgery to treat the margins of the surgical site to destroy residual cells that STATE OF THE ART Biophysical-based treatments in cancer therapy -86 -may not have been removed during surgical intervention [START_REF] Metelmann | Comprehensive clinical plasma medicine: Cold physical plasma for medical application[END_REF]. Plasma oncology could be considered as the newest application of NTP in biomedical research.

Along with fundamental research and pre-clinical investigations in plasma oncology, this recent field of applications of NTP has already found its place in clinics, mostly in Germany and garnered raising interest from investigators for oncology. An example of the very first clinical application of direct NTP treatment in a patient with a locally advanced tumour contaminated with bacteria is shown in Figure 27 (Metelmann, 2018a). This direct NTP treatment reduced microbial load and the resulting typical fetid odour from infected lesions. Plasma treatment with the kINPen® in a 56-year-old male patient having locally advanced squamous cell carcinoma of the oropharynx with tumour ulceration contaminated with bacteria. The kINPen® MED was used every 2 to 3 days with an exposure protocol of 1 minute/cm² treatment, with the jet moved across the tumour surface with a 1 cm separation between the jet and the surface. This NTP treatment, without reported plasmainduced side effects, induced tumour reduction and significantly improved the tumour decontamination, while no lasting remission nor cure could be expected by surgery, radiation or chemotherapy (Metelmann, 2018a). • SteriPlas®: a microwave (2.45 GHz) powered Ar-plasma torch designed and manufactured at ADTEC Europe Ltd., Hounslow, UK in compliance with internationally recognised ISO13485 quality management system Ongoing clinical trials using those CE-marked plasma devices for dermatology are reported in the review of [START_REF] Privat-Maldonado | ROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy[END_REF] To date, NTP technology used in human oncology consists of the direct application of the plasma for palliative care with a few anti-cancer effects reported. This direct NTP treatment remains the strategy of NTP in plasma oncology. Thus, the following sections will exclusively discuss the mechanisms and applications of NTP technology in pre-clinical investigations (plasma oncology) for the potential clinical practice and current challenges.

Mechanisms of biological action of NTP on cancer cells

As mentioned above, from killing bacteria to destroying cancer cells, NTP has emerged in the last twenty-five years as a novel and promising therapy for wound and skin decontamination, promoting wound healing, and controlling wound-resident multi-drug resistant bacteria, dental and cosmetic applications and cancer remission. Many studies are underway worldwide with rapid progress in basic research and in developing useful plasma devices for clinical purposes. Mechanisms coupling the physics and chemistry of NTP to medically-relevant biochemistry and biology are beginning to be understood but much remains to be explored.

It is known that the NTP generated reactive species first interact with the cell membrane, chemically modifying (e.g., oxidising) its lipids within the phospholipid bilayer. The implication of RONS generated by NTP treatment has long been demonstrated as potential anti-cancer agents. One of the first in vitro studies of NTP to generate abundant ROS that induced DNA damages in two human cancer cell lines was conducted at the GREMI (Orléans, France) [START_REF] Vandamme | ROS implication in a new antitumor strategy based on nonthermal plasma[END_REF]. The cytotoxic effect of RONS generated by NTP treatment, as mentioned in section 1.3.2, is in principle attributed by other recent studies to the intracellular redox homeostasis (Graves, 2012b;[START_REF] Privat-Maldonado | ROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy[END_REF]Von Woedtke, 2019;[START_REF] Wolff | Combination of Cold Plasma and Pulsed Electric Fields -A Rationale for Cancer Patients in Palliative Care[END_REF]. Indeed, redox adaptation in cancer development and drug resistance has been studied The vicious cycle of ROS stress in cancer development and progression is described in Figure 28a. Generated from extracellular or intracellular sources, these reactive species, especially ROS, function as a double-edged sword. A moderate increase of ROS may promote cell proliferation and survival, but when reaching the toxic threshold (dashed line in the figure), ROS may overwhelm the cell antioxidant capacity and thus trigger cell death. Under physiological conditions, non-cancerous cells maintain redox homeostasis with a low level of basal ROS by controlling the balance between ROS generation (pro-oxidants) and elimination (antioxidant capacity). This balance ensures tolerance of a certain level of exogenous ROS stress by non-cancerous cells, owing to their "reserve" antioxidant capacity, which can be mobilised to prevent the ROS level from reaching the cytotoxic (cell-death) threshold. On the contrary, the increase in ROS generated from metabolic abnormalities and oncogenic signalling in cancer cells may trigger a redox adaptation response, leading to an upregulation of cell antioxidant capacity and a shift of redox dynamics with high ROS generation and elimination. As a result, cancer cells would be more dependent on the antioxidant system and more vulnerable to further oxidative stress induced by exogenous ROS. They could lose their viability if the ROS level surpasses the cytotoxic threshold (Figure 28b, red bar) [START_REF] Trachootham | Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?[END_REF]. Those exogenous ROS can be ROS-generating agents or compounds that inhibit the antioxidant system or ROS generated by an NTP treatment. 83 . pability is likely to serve as -induced apoptosis, as evidrogen peroxide-induced in the Ras-transformed ere also found to be more athione (GSH), leading to eath 84 , suggesting a crucial urvival. Likewise, studies lanoma cells showed that sion of the GSH syntheed by downregulation of llular depletion of reduced at cells with active c-Myc y upregulating GSH synle that during malignant nic signals both induce cell proliferation through nal factors and promote isms to minimize oxida-G. 4, redox adaptation by enzymes to counteract ems to be a major mechaintain redox homeostasis. aptation mechanism could referentially affect cancer e significant therapeutic x adaptation may involve dox-sensitive transcription r-κB (NF-κB), Nrf2, c-Jun creased expression of anti-OD, catalase, thioredoxin and the GSH antioxidant system. Some of the redoxsensitive transcription factors may also promote the expression of cell-survival molecules, such as the BCL2 family proteins and the Akt survival pathway, conferring drug-resistant capacity (FIG. 4). Furthermore, alteration of the function of cell death factors, such as caspases, may also be involved in redox adaptation 18,22 . These adaptive mechanisms keep the ROS levels within the range that allows cancer cells to escape severe oxidative damage and survive under stress and ROS-mediated mutations. Although in certain cases apoptosis resistance can also be the result of post-translational events 86 , genomic instability seems to be a major mechanism that provides cancer cells with the capacity to alter the expression of gene products that are required for adaptation 87 . Dynamic alterations of numerous genes through genetic or epigenetic mechanisms can accumulate during carcinogenesis without markedly changing the cell's phenotype. This genomic instability allows 'silent' adaptation until it is sufficient to provide a survival advantage in the tumour microenvironment 20,88 . In addition to cell survival, those redox-sensitive transcription factors also regulate the expression of proteins that are involved in Reactive oxygen species (ROS) that are generated from extracellular or intracellular sources can cause DNA damage, which may activate wild-type p53 in normal cells and trigger stress responses and DNA repair to eliminate the ROS-mediated damage to genetic materials. However, in cancer cells with defective p53, ROS-mediated DNA damage would accumulate owing to compromised DNA repair function. In cancer cells with wild-type p53, ROS-mediated DNA mutations or deletion may cause a loss of p53 function, resulting in a defect in DNA repair leading to an accumulation of gene mutations and deletions. This would promote genomic instability leading to activation of oncogenes, aberrant metabolic stress, mitochondrial dysfunction and a decrease in antioxidants. All these events can further increase ROS levels, leading to more DNA damage and genetic instability. Such a vicious cycle can effectively amplify oxidative stress and promote genomic instability and cancer development. Interestingly, some compounds exhibit a potent ability to promote ROS generation in cancer cells and show promising anticancer activity in vitro and in vivo, although the precise mechanisms through which these compounds increase ROS production are poorly understood. Elesclomol (STA-4783), a novel compound that showed therapeutic activity in malignant melanoma and prolonged the progression-free survival time in a Phase II clinical trial 113,114 , seems to exert its cytotoxicity against cancer cells mainly by inducing ROS generation and oxidative damage. Neutralization of ROS by antioxidants suppressed the drug's action 113,114 . Unfortunately, the Phase III clinical trials of elesclomol were recently suspended due to safety concerns. The reason for the increased mortality observed in stage IV metastatic melanoma patients treated with a combination of elesclomol and paclitaxel is yet to be determined. Understanding the underlying mechanisms of the drug's action and toxicity is essential for future development and clinical evaluation.

Some cellular pathways

(BITC), phen raphane) and jugate with GS subsequent ox of GSH can a Buthionine glutamylcyste enzyme for G shown to cau activity in va inhibitors of t sulphasalazine the uptake of a rate-limitin Another t system, whic and is correla resistance 120,1 rational desig that target thi ase 1 (TR1) the metabolism of alkylating agents 91,92 . Thus, aberrant drug metabolism together with the activation of protective cellular mechanisms, can contribute to the drug-resistant phenotype of cancer cells.

Targeting redox alterations in cancer

Therapeutic selectivity is essential in cancer treatment. As cancer cells have elevated ROS generation and are under increased intrinsic oxidative stress, it is conceivable that these malignant cells would be more dependent on antioxidants for cell survival and, therefore, more vulnerable to further oxidative insults induced by ROS-generating agents or by compounds that abrogate the key antioxidant systems in cells. The idea of inducing preferential cancer cell death by a ROS-mediated mechanism based on the different redox states in normal and malignant cells (BOX 2) was proposed a decade ago 93,94 , but its feasibility has only recently gained momentum 14,95,96 . Although ROS-generating agents have been found to be effective in many cases 97,98 , low clinical response and resistance to those agents were also reported 97,99,100 . Elevation of certain transcription factors, antioxidants and survival signals as a result of redox adaptation probably explains the drugresistant phenotype 99,101 . To develop effective therapeutic agents, which are selective and able to overcome drug resistance, it will be extremely important to understand the pros and cons of the different redox-modulating strategies and to reconcile the concepts of intrinsic oxidative stress in cancer cells and their redox adaptation.

Cellular redox homeostasis is maintained by the balance between ROS generation and elimination. Exogenous agents that increase ROS generation or decrease antioxidant capacity will shift the redox balance and result in an overall increase in the level of ROS, which when above a cellular tolerability threshold may induce cell death. Approaches to further increase oxidative stress and kill cancer cells are summarized in FIG. 5.

Promoting ROS production. Electrons leaked from the respiratory complexes in mitochondria are a major 102 Figure 4 | Redox adaptation in cancer development and drug resistance. Severe accumulation of cellular reactive oxygen species (ROS) under various endogenous and exogenous stress stimuli may induce lethal damage in cells that have inadequate stress responses or adaptation. In certain cancer cells, persistent ROS stress may induce adaptive stress responses including activation of redox-sensitive transcription factors, such as nuclear factor κB (NF-κB) and Nrf2, leading to an increase in the expression of ROS-scavenging enzymes, such as superoxide dismutase and glutathione, elevation of survival factors such as BCL2 and MCL1, and inhibition of cell death factors, such as caspases. ROS-mediated DNA mutations or deletions promote genomic instability and thus provide an additional mechanism for stress adaptation. All these events enable cells to survive with the high level of ROS and maintain cellular viability. As these transcription factors also have roles in regulating the expression of genes that are responsible for proliferation, senescence evasion, angiogenesis and metastasis, the redox adaptation processes may promote cancer development. Furthermore, the increase in glutathione during adaptation can enhance the export of certain anticancer drugs and their inactivation. This altered drug metabolism together with enhanced cell survival may [START_REF] Yan | Toward understanding the selective anticancer capacity of cold atmospheric plasma-A model based on aquaporins (Review)[END_REF]. They found that after the NTP treatment, NTPgenerated H2O2 species diffuse into cancer cells significantly faster than in normal homologous cells, resulting in a significant rise of ROS in cancer cells compared with normal cells. Indeed, with the diameter of the pore varied amongst different AQPs, these intrinsic membrane proteins are known to facilitate the transport of free RONS and other small molecules, including carbon dioxide, nitrogen monoxide, ammonia, urea, and glycerol [START_REF] Kruse | The aquaporins[END_REF]. Besides, AQPs 1, 3, and 8 are demonstrated to be involved in the transport of H2O2 in mammalian cells [START_REF] Bienert | Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes[END_REF][START_REF] Wang | Characterization of aquaporin-driven hydrogen peroxide transport[END_REF]. Yet several AQPs have been reported as more abundant at the cell membrane in cancer cells than in non-cancerous cells [START_REF] Verkman | Aquaporins -New players in cancer biology[END_REF], and the rise of intracellular RONS contributed to an increased sensitivity of these cells to NTP treatment.

Besides the expression of AQPs, the diffusion of free radicals is also dependent on the amount of cholesterol in the plasma membrane. Within the same research group, the MD simulation performed by Van der Paal et al. predicted that the lipid order in a phospholipid bilayer decreases upon the addition of lipid peroxidation products, i.e., pore formation occurs when all phospholipids are oxidised ( [START_REF] Van Der Paal | Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress[END_REF] -what would explain the cell membrane permeabilisation observed during (and after) NTP treatment. They also found that cholesterol can protect oxidised membranes against pore formation. Cancer cells have a significantly lower concentration of cholesterol in their plasma membrane (ca. 2-fold lower) than that in the phospholipid bilayer of their non-tumorigenic counterparts [START_REF] Shinitzky | Membrane fluidity in malignancy Adversative and recuperative[END_REF][START_REF] Van Blitterswijk | Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells[END_REF]; they will be more vulnerable to the consequence of lipid peroxidation. These researchers also demonstrated the synergistic effect of electric field and lipid oxidation on the permeability of cell membranes (Yusupov, 2017) -effects that have also been observed by our group, employing MD simulations, for electroporating fields that target oxidatively damaged areas in the plasma membrane (Vernier, 2009), as mentioned above (see section 1.2.2). Therefore, this molecular-level insight is crucial for selectively treating tumours with NTP technology and electroporation-based technology.

The PLASMANT research group also recently investigated the permeabilisation of RONS across native and oxidised phospholipid bilayers. Their MD simulations revealed that the STATE OF THE ART Biophysical-based treatments in cancer therapy -91 -assessed RNS (NO, NO2, N2O4) and O3 could permeate more easily through both native and oxidised membranes in comparison to hydrophilic ROS (OH, HO2, H2O2) [START_REF] Razzokov | Atomic scale understanding of the permeation of plasma species across native and oxidized membranes[END_REF].

More in vitro and in vivo studies and investigations of the plasma chemistry are needed to have relevant concluding remarks with those previous MD simulations.

Amongst the RNS, it has been identified for at least a decade that nitrite anion (NO2 -) can act therapeutically, most probably as a precursor source of nitric oxide [START_REF] Vitturi | Current perspectives and challenges in understanding the role of nitrite as an integral player in nitric oxide biology and therapy[END_REF]. A recent study assumes that whether RONS have a signal-transducing or damaging effect is primarily defined by their quality, being primary or secondary RONS, and only secondly by their quantity [START_REF] Weidinger | Biological Activities of Reactive Oxygen and Nitrogen Species: Oxidative Stress versus Signal Transduction[END_REF]. Girard and colleagues identified in vitro the three long-lived RONS, the H 2 O 2 , NO 2 -and NO 3 -as the main reactive species in plasma-treated PBS responsible for the in vitro anti-cancer effects (Girard et al., 2016). Further details on the complexity of the cellular responses to NTP treatment can be found in recent review articles by [START_REF] Bauer | Mechanisms of Selective Antitumor Action of Cold Atmospheric Plasma-Derived Reactive Oxygen and Nitrogen Species[END_REF]Graves, 2014;[START_REF] Privat-Maldonado | ROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy[END_REF][START_REF] Trachootham | Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?[END_REF]. Other exciting reviews such as [START_REF] Lu | Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects[END_REF], explain the generation, transport, and biological effects of RONS produced in NTP treatment. The review of [START_REF] Semmler | Molecular Mechanisms of the Efficacy of Cold Atmospheric Pressure Plasma (CAP) in Cancer Treatment[END_REF] reports the molecular mechanisms of NTP in oncology, as partially above discussed.

Strategies in pre-clinical NTP treatment

There are two basic strategies in plasma oncology research, schematically illustrated in • Indirect treatment on liquids (plasma-treated liquids) will be used in vitro or administered in tumours for animal studies (e.g., in the form of a subcutaneous injection or intraperitoneal injection, depending on the study's design and model). Schematic illustration adapted from (Yan, 2017) Being directly in the scope of this thesis, the indirect plasma treatment strategy will be emphasised in the following sections. The in vitro and in vivo effects resulting from different major studies of the last decade using indirect strategy will be specially summarised in Table 3 of section 3.3.4 below.

Direct treatment

Yonson and colleagues were, in 2006, the first to investigate the promotion of cell adhesion and proliferation by modifying the surface with a miniature glow discharge plasma torch [START_REF] Yonson | Cell treatment and surface functionalization using a miniature atmospheric pressure glow discharge plasma torch[END_REF]. They studied those effects on a human hepatocellular carcinoma (HepG2), then other adherent and non-adherent cells lines such as melanoma, glioblastoma, and leukaemia cells were used by other investigators (Arndt et al., 2013a;Fridman, 2007;Keidar et al., 2011;[START_REF] Vandamme | Antitumor effect of plasma treatment on u87 glioma xenografts: Preliminary results[END_REF]. As mentioned previously, Vandamme and colleagues from the GREMI not only investigated cancer cell lines but also in an in vivo tumour model [START_REF] Vandamme | ROS implication in a new antitumor strategy based on nonthermal plasma[END_REF]. Their human glioblastoma U87MG bearing nude mice showed a reduction of bioluminescence and tumour volume after treatment with an FE-DBD, as compared to STATE OF THE ART Biophysical-based treatments in cancer therapy -94 -untreated mice. Induction of apoptosis was observed together with an accumulation of cells in their S phase, suggesting an arrest of tumour proliferation. Especially, apoptosis was also observed in the tumour site not being directly exposed to the plasma.

Following those pioneering investigations, other in vitro studies demonstrated the cytotoxic effects of direct NTP treatment on cancer cells. It has been shown that DBD plasma exposed to melanoma cells at doses that did not induce necrosis, induced apoptosis via the production of intracellular ROS [START_REF] Sensenig | Non-thermal plasma induces apoptosis in melanoma cells via production of intracellular reactive oxygen species[END_REF]. The GREMI group displayed in vivo effects of an NTP treatment, alone or in combination with the anti-cancer drug Gemcitabine, in an MIA PaCa2-luc human pancreatic carcinoma transplanted in nude mice (Figure 32) [START_REF] Brullé | Effects of a Non Thermal Plasma Treatment Alone or in Combination with Gemcitabine in a MIA PaCa2-luc Orthotopic Pancreatic Carcinoma Model[END_REF], being the first research group successfully obtaining in vivo anti-tumour effects of NTP.

Figure 32| Example of an in vivo direct NTP treatment of a tumour

Schematics of a fibered plasma (Plasma Gun) setup (upper panel) used to treat human pancreatic carcinoma transplanted in nude mice (lower panel), in combination with gemcitabine, from the first published in vivo anti-cancer study using direct NTP combined with a chemotherapeutic agent [START_REF] Brullé | Effects of a Non Thermal Plasma Treatment Alone or in Combination with Gemcitabine in a MIA PaCa2-luc Orthotopic Pancreatic Carcinoma Model[END_REF].

Another effect of NTP treatment, the induction of immunogenic cell death (ICD) has also been reported in vitro using direct plasma treatment strategy [START_REF] Lin | Nanosecond-pulsed dbd plasma-generated reactive oxygen species trigger immunogenic cell death in a549 lung carcinoma cells through intracellular oxidative stress[END_REF][START_REF] Lin | Non-thermal plasma induces immunogenic cell death in vivo in murine CT26 colorectal tumors[END_REF][START_REF] Miller | Why Target Immune Cells for Plasma Treatment of Cancer[END_REF] but also by the indirect strategy (see Table 3). Direct plasma treatment was also used to permeabilise the HeLa cell membrane e.g., using a plasma torch (Leduc et al., 2009) or the plasma jet [START_REF] Vijayarangan | New insights on molecular internalization and drug delivery following plasma jet exposures[END_REF].

To date, in vivo studies on small animals with direct plasma treatments often employed plasma jets exposed directly to tumours. As this is not in the scope of this thesis, those studies will not be reported but overall, the induction of apoptosis in the tumour tissue and/or the STATE OF THE ART Biophysical-based treatments in cancer therapy -95 -increase in the ratio of the apoptotic to anti-apoptotic proteins expression has usually been observed.

Indirect treatment

The most commonly used liquids for indirect NTP treatment are cell culture media saline solutions, buffered saline solutions (e.g., phosphate buffered solution or PBS), Ringer's lactate solution or water. The resulting plasma-treated liquids (PTL) were adopted by NTP scientific community under different terms: plasma-irradiated medium, plasma-activated liquid (PAL), plasma-activated (PAM) or plasma-treated medium (PTM), plasma-activated PBS (P-A PBS) or plasma-treated PBS (P-T PBS or pPBS). Until now, no consensus has been reached yet. Our group and collaborators employed the term "plasma-treated liquids" or PTL as referred to all types of liquids being used to be treated with NTP.

As illustrated in Figure 31b, the properties or at least especially the long-lived RONS of the NTP can be transferred to a liquid which can be stored, transported and used in in vitro as well as in vivo studies [START_REF] Jablonowski | Research on plasma medicine-relevant plasmaliquid interaction: What happened in the past five years?[END_REF][START_REF] Yan | Controlling plasma stimulated media in cancer treatment application[END_REF]. Those plasma-treated liquids have been demonstrated as inducers of physiological outputs in cells and tissues. The anti-cancer potential of the PTLs has first been reported using cell culture media, such as DMEM or RPMI.

However, the use of these complex cell culture media has raised some issues. Indeed, their composition, rich in amino acids, vitamins, sugars and inorganic salts, has a strong influence on the reactive species generated in the liquids and their stability. One of the standard components of cell culture media is the pyruvate, which is the output of the glycolysis, has been known as a potent antioxidant and has been demonstrated to be a ROS scavenger [START_REF] Desagher | Pyruvate Protects Neurons against Hydrogen Peroxide-Induced Toxicity[END_REF][START_REF] Jagtap | Sodium pyruvate protects against H2O2 mediated apoptosis in human neuroblastoma cell line-SK-N-MC[END_REF][START_REF] Tauffenberger | Lactate and pyruvate promote oxidative stress resistance through hormetic ROS signaling[END_REF]. Several studies highlighted that pyruvate mitigated the effectiveness of PTLs on cells by inducing oxidative stress resistance, thus reducing the cytotoxicity of PTLs treatment [START_REF] Bergemann | Pyruvate as a cell-protective agent during cold atmospheric plasma treatment in vitro: Impact on basic research for selective killing of tumor cells[END_REF][START_REF] Tornin | Pyruvate Plays a Main Role in the Antitumoral Selectivity of Cold Atmospheric Plasma in -163 -Osteosarcoma[END_REF]. Some studies also demonstrated that culture media components such as cysteine, tryptophan, phenylalanine or tyrosine were the main targets of effective RONS present in the PTL, yet the degradation of those amino acids due to NTP would impact the stability of the RONS [START_REF] Chauvin | Analysis of reactive oxygen and nitrogen species generated in three liquid media by low temperature helium plasma jet[END_REF][START_REF] Yan | Toward understanding the selective anticancer capacity of cold atmospheric plasma-A model based on aquaporins (Review)[END_REF].

Therefore, other liquids with a more straightforward composition (without RONS scavengers), such as saline solutions (isotonic NaCl or Ringer's lactate solution (RLS)), buffered saline solution (PBS) or water, were proposed in various studies as more relevant liquids to Those various aforementioned in vitro studies of plasma-treated liquids, together with a very few numbers of in vivo studies, presumed a common cytotoxicity mechanism mediated by RONS, and more specifically by hydrogen peroxide (H2O2), as H2O2 is the main component of long-lived RONS present in plasma-treated liquids with anti-cancer potential. As discussed in the previous section, very high concentrations of H2O2 in the cell induce exhaustion of its antioxidant mechanisms and, ultimately, its energy metabolism. However, the level of toxicity generated by the presence of H2O2 remains cell line-dependent. Most cancerous cell lines are affected by oxidative stress, yet some lines and study models show increased cell proliferation.

The latter effect could be defended by the redox adaptation of cancer cells under persistent ROS stress, processes by which cancer cells maintain their redox homeostasis, as described above (see section 3.3.2). Henceforth, this stimulation of cancer development could eventually explain the lack of success in vivo investigations on the anti-cancer effectiveness of NTP treatment as a single treatment (especially those employing an indirect strategy).

Consequently, efforts have been allocated to combining NTP with other therapies, such as chemotherapy, to improve the treatment effectiveness, as presented by some studies in Table 3. Indeed, some studies claimed the efficacy of NTP treatment when an anti-cancer drug was combined. Also, since there is a synergistic effect between electric pulses and the lipid oxidation by ROS (see section 3.3.2), results of in vitro combining NTP and electric pulses have been reported (Table 3).

Nevertheless, there have been very few in vivo studies with positive antitumour effects for almost a decade of investigating anticancer therapy using NTP. Only nine in vivo studies (compared with about forty in vitro studies) reported anti-tumour results of plasma-treated liquids. Moreover, for all of those investigations, none to very few had similar treatment parameters, from the used plasma devices to the studied in vivo models and treatment protocols. As discussed in previous sections, besides the three CE marked plasma devices for clinical practice, all other plasma devices are in-lab-built. Despite their practical applications and reported anti-cancer effects, very inadequate to no physical, technical and biological description is fully detailed and reported.

STATE OF THE ART Biophysical-based treatments in cancer therapy -101 -Despite all the advantages above and limits of ECT and NTP in cancer treatment, there are, up to date, very few studies investigating the combination of those two therapies to outperform each monotherapy. At the beginning of this thesis, there was no in vitro study that examined the combination of ECT and indirect NTP, and only three years later, some studies investigating the combination of direct or indirect NTP with chemotherapy or PEFs or the combined chemotherapy + PEFs (thus ECT) have been published.

To the best of my knowledge, the only in vivo study undertaken to compare the anti-tumour effectiveness of ECT with direct NTP strategy was conducted by Daeschlein and colleagues in the B16 melanoma mouse model [START_REF] Daeschlein | Comparison between cold plasma, electrochemotherapy and combined therapy in a melanoma mouse model[END_REF]. By applying the plasma jet (APPJ) and DBD plasma sources directly to the tumour surface (when the tumour size reaches 8 mm Ø), they compare the effects of the NTP direct treatment with ECT alone or in combination with ECT. In both cases, NTP treatment was repeated daily for five days. ECT was performed with i.v.

BLM at a field strength of 1000 V/cm using the Cliniporator® (IGEA, Carpi, Italy). When combined with ECT, a one-shot treatment, plasma treatment was subsequently applied until day 5. Their results showed that: both plasma sources as a single treatment displayed a significant delay of the tumour growth, which proved less effective than ECT alone; plasma jet combined with ECT significantly improved per cent mouse survival, with significant superiority compared with ECT.

Current challenges for research & development in plasma medicine

Plasma-treated liquids have broadened how NTP can be applied by providing a tool for cancer treatment, demonstrating their efficiency in various cancer cell lines eradication in different in vitro studies and in a few in vivo investigations. Unlike electrochemotherapy, an established anti-cancer treatment modality (established operating procedure, as described in Chapter 1.2), there are still current challenges for research and development in plasma medicine before achieving clinical practice.

The issues mentioned above regarding the reproducibility and relevance of in vitro and especially in vivo studies could be resolved, at least in a significant part, by establishing a better specification and systematisation of NTP devices for biomedical applications as regards clinical practice. Indeed, it might be convenient if plasma devices could be manufactured and used by considering the purpose of the application. Undeniably, the NTP devices used for treatment close to the medical target should be limited to a temperature below 40°C. In contrast, a STATE OF THE ART Biophysical-based treatments in cancer therapy -102 -treatment that would require thermal damage should be performed with devices more comparable to the electrosurgical plasma devices, etc.

A further approach to improving plasma characterisation in vitro is identifying a technical target that is representative of the human body -the clinical target-and identifying plasmatarget interactions and their effects on plasma characteristics. This issue was evoked last year by [START_REF] Judée | Plasma gun for medical applications: Engineering an equivalent electrical target of the human body and deciphering relevant electrical parameters[END_REF], the same year of the publication of my first article, which in part deals with this issue, providing an excellent solution to experimentally deliver direct plasma treatment to models possessing the electrical characteristics of the human body. This article represents the first chapter of the results section.

RESEARCH HYPOTHESES AND OBJECTIVES

Although electrochemotherapy (ECT) is an established and standardised targeted anticancer treatment in human and veterinary medicine, given its nature, it still has some inherent drawbacks, including erythema, superficial epidermal erosion, muscle contraction and relative discomfort due to the application of intense pulsed electric fields (PEFs), thus the necessity of administrating myorelaxant and anaesthesia (local, loco-regional or general) [START_REF] Yarmush | Electroporation-Based Technologies for Medicine: Principles, Applications, and Challenges[END_REF].

On the other hand, non-thermal plasma (NTP) and plasma-treated liquids give particular interest of their rich source of reactive oxygen and nitrogen species (RONS). The newest field of research and application of NTP in medicine, and one of the main foci of my thesis, is plasma oncology. Indeed, as introduced in state of the art, NTP being used either as direct therapy or as indirect therapy displays selective anti-cancer effects when allowing cell permeabilisation [START_REF] Cramariuc | Corona discharge in electroporation of cell membranes[END_REF][START_REF] Jinno | Investigation of plasma induced electrical and chemical factors and their contribution processes to plasma gene transfection[END_REF]Leduc, 2009;Sasaki et al., 2016;Tanaka et al., 2017a). One of the molecular mechanisms involved in the efficacy of NTP in oncology is lipid peroxidation by free radicals, which leads to pore formation in the cell membrane, hence facilitating the diffusion of RONS into the cell (Figure 30). Recent studies suggested possible synergy between PEFs and NTP on the permeability of cell membranes via lipid oxidation, an effect that has also been demonstrated using MD simulations (Yusupov, 2017). Since PEFs allow cell electropermeabilisation (Mir, 1988;Rols et al., 2002;Vernier, 2009) and electric pulses (electroporating fields) target oxidatively damaged areas, if the plasma membranes are oxidised, they are more prone to become permeabilised when the cells are exposed to short and intense electric pulses.

To date, there are, to the best of our knowledge, only nine in vivo studies exploring the anti-tumour effects of plasma-treated liquids (see Table 3). Only recently, some pioneer studies are investigating the combination of NTP and µsPEFs in different in vitro cell models [START_REF] Griseti | Pulsed Electric Field Treatment Enhances the Cytotoxicity of Plasma-Activated Liquids in a Three-Dimensional Human Colorectal Cancer Cell Model[END_REF][START_REF] Von Woedtke | Cell stimulation versus cell death induced by sequential treatments with pulsed electric fields and cold atmospheric pressure plasma[END_REF]Wolff et al., 2020) or for bacterial inactivation [START_REF] Zhang | Synergistic antibacterial effects of treatments with low temperature plasma jet and pulsed electric fields[END_REF] or the combination of direct NTP with ECT in vivo [START_REF] Daeschlein | Comparison between cold plasma, electrochemotherapy and combined therapy in a melanoma mouse model[END_REF]. Most of this knowledge was not yet published when this thesis was initiated. Because of the background of the team in the in vitro and in vivo cell electroporation, and because of the potential of collaboration with two groups of experts in the NTP production and application, we sought to address hypothetical cumulative effects, maybe additive or synergistic responses of the combination of NTP and ECT via respective impact on the phospholipid bilayer of the cell membranes.

My thesis's objectives for three years of research were:

1) Study possible synergistic effects of µsPEFs (thus ECT) and NTP on the cell membrane permeabilisation (thus cytotoxicity)

2) Improve the outcome of ECT by:

• Enhancing the electropermeabilisation level of the cell membrane • Reducing the dose of anti-cancer drugs (bleomycin)

• or reducing the µsPEF amplitude without altering the electropermeabilisation effects 3) Develop quantitative and qualitative studies of the indirect plasma treatment via the studies of plasma-treated liquids (their pH and their stability over different storage conditions, the chemical analysis of long-lived RONS present in those plasma-treated liquids) 4) Enhance our understanding of the electroporation mechanisms Thus, this thesis sought to improve electrochemotherapy (ECT) by employing non-thermal plasma (NTP) medicine. Antitumour ECT consists of the extraordinary sensitisation of the effect of certain anticancer drugs (in particular bleomycin, a typical example of molecules unable to enter the cell cytosol) by very short and local electrical pulses (8 pulses at a pulse length of 100 microseconds), delivered to the treated tumour nodules. These impulses transiently permeabilise the cells without killing them. ECT is particularly effective on treated tumour nodules and has very few side effects. Nevertheless, ECT is known to cause contractions and requires anaesthesia. It is essential to explore several methods (already demonstrated in vitro) to reduce these undesirable effects and make ECT more comfortable for the patient while always being effective. Therefore, we investigated in vivo the safety and efficiency of our NTP multijet device, then the in vitro enhancement of the cell membrane permeabilisation level when combining pPBS and µsPEFs at very low amplitude and finally, the in vivo anti-tumour effect of the combined treatment with pPBS and ECT on small animal, applying a field strength lower than that used in conventional ECT.

RESULTS

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RESULTS

RESULTS

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Characterisation of a novel NTP device for stable and reproducible applications in both pre-clinic and clinic

Some of the most critical problems in plasma medicine are plasma devices coupled electrically to targets, and different targets have different impedances (as termed "bioimpedances" of biological targets). Measurements of the biomedical effects of plasma on one type of system will partly depend on the different electrical properties of the targets, obscuring the differences in biological responses. Furthermore, results from various in vitro or in vivo tests will not be the same as applications on human targets.

To obtain a stable NTP system for reproducible applications in both pre-clinic and clinic, we first characterised our plasma devices to control the bio-impedances of different targets, ranging from in vitro to in vivo models and humans. We designed working conditions and conception of the NTP device for applications in both pre-clinical and clinical studies, using a Plasma Gun designed by the GREMI, which has a more stable reactor (than other plasma devices from the GREMI) with a grounded electrode.

This study is published in IEEE Transactions on Radiation and Plasma Medical Sciences This study was also investigated in vivo using a newly developed NTP multijet device based on the Plasma Gun to explore the toxicity of the NTP multijet treatments on mice skin. Those results were presented in the second section of this chapter (see section 1.2 of the Results).

They were used to establish clinical assay and treatments on human patients since there is no electrical difference between the animal models and the patient, thanks to our proposed method. The characteristics of this NTP multijet device are described in the second paper (article #2, see section 2 of the Results).

RESULTS

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Mimicking of human body electrical characteristics for easier translation of plasma biomedical studies to clinical applications (article #1)

In this paper, we proposed to add a simple compensating circuit between targets and ground to make the electrical properties of all analysed systems (in vitro, in vivo and human patients) as similar as possible, at least from the electrical point of view. We then showed results for various in vitro and in vivo target exposures using a typical He plasma jet device.

Measurements include voltage-current characteristics, optical emission spectra for key reactive species and the composition of liquid solutions exposed to the plasma.

The addition of the compensation circuit is shown to significantly reduce the differences observed between different targets (the differences between in vitro targets, mouse model targets and human targets). Abstract-Nonthermal plasma (NTP) medical applications are now well established but the translation from in vitro plasma effects to in vivo effects remains far from being intuitive. Among various possible reasons, the translation may be disturbed by a loose control over the electrical characteristics of the different targets (e.g., cell culture dish, animal models) met during the development process, a parameter generally neglected and often unmentioned in papers. The aim of this article is to raise consciousness on how target electrical parameters (e.g., conductivity, electric potential. . . ) play a major role in determining plasma treatment conditions. This effect is of particular relevance in plasma medicine where we move from treating small in vitro samples to humans, passing by animal models. The plasma conditions on targets with different electrical characteristics are compared by means of electrical measurements, optical emission spectroscopy, and basic liquid analysis. Commonly tested in vitro targets induce the generation of a plasma significantly different from that produced in contact with a human body. We demonstrate how by means of a basic and easy to implement electrical circuit it is possible to "compensate" the electrical differences between in vitro models, mice and human body and in such a way to reach more reproducible treatment conditions between in vitro and in vivo targets. The proposed method for the control of the target electrical parameters could greatly favor the transition from in vitro and in vivo models to patients for many plasma devices developed for biomedical applications.

My contribution

Index Terms-Atmospheric pressure plasma, compensation circuit, plasma medicine, potential, targets.

I. INTRODUCTION

T HE ANTITUMOUR effect of nonthermal plasmas (NTP) is now well established. The list of studies demonstrating plasma action on tumour cells in vitro is too long CNRS/Université d'Orléans, 45067 Orléans, France (e-mail: augusto.stancampiano@univ-orleans.fr; sebastien.dozias@univ-orleans.fr; jean-michel.pouvesle@univ-orleans.fr; eric.robert@univ-orleans.fr).

T.-H. Chung and L. M. Mir are with VTA, CNRS, Univ. Paris-Sud, Gustave Roussy, Université Paris-Saclay, 94800 Villejuif, France (e-mail: thai-hoa.chung@gustaveroussy.fr; luis.mir@cnrs.fr).

Color versions of one or more of the figures in this article are available online at http://ieeexplore.ieee.org.

Digital Object Identifier 10.1109/TRPMS.2019.2936667 to be reported here, but we can estimate that more than 50 cell lines of most types of cancers have already been tested [1]- [3]. Similarly, the number of studies demonstrating other in vitro plasma medical applications, such as wound healing and decontamination, is nowadays significant and constantly increasing [4]. Nevertheless, studies involving patients are still limited and the exact plasma action mode is far from being fully understood [5], [6]. In general, the translation from in vitro plasma effects to in vivo effects remains far from being intuitive. This task is even more challenging due to the physical and chemical complexity of NTP interactions with liquids/cells/tissues, which depend on a number of variables, such as, for example, the applied voltage, pulse duration, pulse repetition frequency, gas mixture, distance to the target, time of exposure, protocols of application, and physical and chemical characteristics evolution of the target during the NTP delivery. Amongst these parameters, the electrical characteristics of the target are often neglected and usually go unmentioned, especially in particles which are more focused on the biological effects of the plasma. Nevertheless, as already reported in the literature, the target nature can strongly influence the plasma characteristics [7]- [11]. Physically, during a direct NTP treatment, the target (e.g., the medium bathing the cells in culture or the living treated tissue) is part of a "transient electrical circuit" that is formed between the high voltage electrode and the ground (physical electrical connection to the earth). This means that target electrical parameters (e.g., conductivity, potential, total impedance. . . ) play a major role in determining treatment results. Even with all the other parameters fixed, different targets can lead to very different plasma characteristics, and therefore, very different plasma effects. In this article, we demonstrate that even the same target (water solution in a multiwell plate) but under different electrical conditions [grounded potential (GR) or at floating potential (FP)] or set on different supports (dielectric plate versus metallic plate) can lead to very different treatment results.

The variation of the NTP treatment conditions due to target characteristics is of extreme relevance, especially in the plasma medicine field where we move from treating small in vitro samples to humans passing by various animal models.

The standard approach for medical validation that foresees the sequence in vitro model → in vivo model → patients may not be the best option if applied in a straightforward manner to plasma medical studies. The plasma generated on a small in vitro sample, characterized by a high conductivity 2469-7311 c 2019 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission.

See https://www.ieee.org/publications/rights/index.html for more information.

Authorized licensed use limited to: Universita degli Studi di Bologna. Downloaded on November 08,2020 at 12:52:25 UTC from IEEE Xplore. Restrictions apply. and a low capacitance, can definitively not be compared with the one produced on a human body, with typically a lower conductivity and a much higher capacitance. This in turn ends up potentially affecting the reliability of the results achieved on in vitro and even in vivo models and their possibility to be translated to patients beside plenty of biological reasons that already make the passage far from being trivial. Very recently, the issue has been partially addressed by some studies proposing feedback control systems based on either the real time tuning of the thermal effect and the intensity of plasma jets [11] or on the dynamic response of cancer cells to NTP based on mathematical model developed starting from experimental results [12]. Another proposed approach is based on the development of a basic material target that mimics the same dielectric properties of the human body and that could be used for the study of the plasma properties in more realistic operating conditions [13].

An improved control over plasma treatment conditions will also help to better investigate the impact of physical and electrical stimuli possibly induced by plasma. Within the recent past, the importance of electrostimulation at weak currents and the impact of the plasma-associated electric field were being rediscovered and investigated [14], [15].

In this article, we propose and successfully test a simple and versatile solution to reduce the degree of variation encountered in NTP studies translating from in vitro and animal models to human body. The main idea is to compensate the electrical differences between the models and the human body by means of a small and affordable electrical circuit that can be applied on both animal and in vitro models (see Fig. 1). The approach is significantly different from those found in the literature as it is based on a noninvasive modification of the targets, with special attention to common biomedical models. Acting on the target electrical characteristics rather than on the plasma source control, the compensation method tackles the issue from a completely different angle and is potentially complementary to feedback-based control systems as those already proposed by Gidon et al. [11] and Lyu et al. [12]. Moreover, being based on a human body equivalent circuit derived from international standards for safety evaluation of electrical equipment, this approach could encourage the development of plasma sources complying with the requirements for basic safety and essential performance [16]. The same circuit may also be valuable for modeling studies trying to simulate targets close to those in actual plasma medical treatments.

II. MATERIALS AND METHODS

A. Plasma Source

The atmospheric plasma jet used in this article is a Plasma gun (PG) already described in detail in [8]. Briefly, the PG is a coaxial DBD reactor with a quartz capillary flushed with helium and powered by a micropulsed high voltage generator. The 12 cm long capillary was tapered at the outlet (Ø in = 1.5 mm, Ø ext = 3 mm). The plasma source was operated in two sets of operating conditions. In the first part, concerning only in vitro experiments, the source was flushed with a helium flow of 1 slm, powered by 4 µs duration voltage pulses of +12 kV peak with a 1 kHz repetition rate and maintained at a distance of 10 mm from the target. In the second half of the work that included in vitro, animal targets and humans, the PG was operated at 0.5 slm of helium, +10 kV, 1 kHz, and at 15 mm from the target. The different sets of parameters were selected in order to on the one side emphasise reactive species production in in vitro liquid samples (so to ease their detection) and on the other side to limit the current crossing in vivo targets.

B. In Vitro Targets

To represent in vitro targets we used a common 24-multiwell plate (Nunclon Delta Surface, Thermo Fisher Scientific, DK) and a bigger custom-made well with a rectangular base (W 2 cm × L 5 cm × H 0.9 cm, material: PVC). The liquid adopted for the test was high purity water (distilled, conductivity < 1µS, by Chem-lab) with dissolved NaCl (Fisher Scientific, U.K.) to adjust its conductivity to the desired values of 10 and 20 mS/cm (reasonable values for common culture medium as those adopted in electroporation studies).

The conductivity of the solution was measured by means of a liquid conductivity probe (InLab Conductivity Probes 51344030, Mettler Toledo). A volume of 1-3 or 9 mL was introduced, respectively, in the multiwell plate and the custom well before each experiment. The multiwell plate was either positioned on a conductive plate (aluminium, 3 mm thick) electrically connected to ground or on a dielectric plate [low density polyethylene (LDPE), 15 cm thick] that was itself positioned on the conductive plate (Fig. 2). Both plates were chosen large enough to completely cover all the multiwell plate bottom.

Floating Potential (FP): The liquid at FP was not in contact with any conductive material.

Ground Potential (GR): The liquid in the multiwell plate was connected to ground by means of a small stainless-steel plate [3.6 × 7 × 0.5 mm; Fig. 3(a)] while in the custom-made well grounding was processed through a stainless-steel plate of bigger dimensions (19 × 8.1 × 1 mm). Both connection plates were positioned inside the liquid close to the wall of the well, so to be as far as possible from the plasma impinging point at the centre of the well. While possible, electrolysis was regarded as negligible in this configuration as no visible bubbling or erosion of the electrode was detected in any of the investigated conditions. Evaporation accounted for a liquid volume variation lower that 3% and was therefore considered negligible in this case.

C. In Vivo Targets

To represent in vivo targets we adopted mice (BALB/cJ) with or without tumour (CT26 murine colon carcinoma subcutaneously at 500.000 cells/100 µL on each flank, eight days before treatment). Treated flank of the mice was shaved and depilated the day before the treatment. Mice were anesthetised and positioned on a metal heating plate connected to the ground to maintain a constant body temperature of the mice during the treatment [Fig. 3(b)]. To represent the human body, the authors volunteered as samples. The chosen treated area was the fingertips of the index finger of the one hand [Fig. 3(c)]. Ground connection was realized by means of a hand-hold metal cylinder. No other grounded surfaces were in contact with the person during tests.

D. Electrical Measurements

Voltage measurements were performed by means of a high voltage passive probe (Tektronix P6015A) on the high voltage cable connecting the PG and the generator. Current measurements were made by a current passive probe (Pearson 6585). For tests comparing in vitro and in vivo targets with human body, the current probe was positioned on the cable connecting the target to the ground. For tests comparing in vitro targets in different electrical conditions, since no ground connection was available for the cases at FP, the current probe was positioned around the plasma source capillary (5 cm away from the outlet).

E. Reactive Species in Liquid Evaluation

Semi-quantitative chemical analyses of peroxide, nitrate, and nitrite concentrations produced in the plasma treated liquid were performed to support the understanding of the influence of the target electrical conditions. For these semi-quantitative measurements Quantofix test strips (Macherey-Nagel GmbH & Co. KG, DE) were used. Tests were repeated in triplicate.

F. Optical Emission Spectroscopy

Plasma spectra were acquired by means of a spectrometer (MayaPro2000, integration time 200 ms, 8 averaged spectra for each acquisition) connected to a 1 mm in diameter VIS and UV optical fibre ( 14• full angle aperture). The optical fibre inlet was positioned horizontally 2 mm away from the plasma jet axis. The fibre vertical position was varied by means of a millimetric manual displacement system in order to scan the emission along the jet axis. All acquisitions were done within 10 s from the start of the treatment on a new sample. The spectra were analyzed to extract the line intensity (a.u.) with no sensitivity correction for the chosen reference lines associated with species significant from a biological point of view: NO* (2363 nm), OH* (3088 nm), N 2 * (B-A) (775 nm), and O* (777.2 nm).

G. Compensation Circuit

The human body is a very complex system and presents electrical characteristics that can greatly vary from one person to another due to various factors as, for example, age, size, sex, and health conditions. Moreover, the total body impedance is frequency dependent on and vary with the characteristics and position of the electrodes/contact points [17]- [19]. Even the same person may present very different total impedances if is connected to the ground through his bare feet, one hand or not at all. For safety reasons, in general in medical practice patient are unlikely to be completely insulated (at FP) and are more often connected to GP or to the return electrode of the electrical medical instrument in use (e.g., in electro-surgery) [20], [21]. In this article, we decided to adopt as a reference circuit for the human body the electrical model (see Fig. 1) reported in the International Standard IEC 60601-1 for medical electrical equipment [22]. This standard has been chosen as internationally recognized and widely adopted [16], [23]. In practice, the circuit is realized starting from the one proposed in the IEC 60601-1 (Fig. 1) and reducing both resistors (becoming 100 and 9.1 k ) so to have some margin to take into account the target impedance [Fig. 3(d)]. In first approximation, due to the short duration of the current pulse (0.7 µs) with respect to the characteristic time of the circuit (RC time constant = 1.5 µs) we can assume the 150 pF capacitance to be as a closed circuit. In this way, we can calculate the equivalent impedance as the results of resistors in parallel. The equivalent impedance of the human body circuit (Fig. 1) after the simplification results in 909 while that of the compensation circuit, without taking into account the variable resistor, is 99 . The difference between the two circuits after the simplification is therefore of 810

. If we position the compensation circuit in series between the target and the ground, we will ideally end up with three resistors connected in series: the target, the variable resistor, and the equivalent impedance of 99 calculated above. In order to make the target-compensation circuit look similar to the human body circuit, it is sufficient to adjust the variable resistor so that summed with the target natural impedance (that can be measured or estimated), will give a 810 impedance. The proposed method is certainly affected by strong approximations but on the other hand presents a very simple structure and is easy to implement. Moreover, being composed of widely available electrical components, the total cost of the circuit results very low and its dimension can be further reduced starting from this first prototype [Fig. 3(e)].

III. RESULTS AND DISCUSSION

A. To Ground or Not to Ground

As already mentioned, the electrical characteristics of in vitro targets are often neglected or considered as marginal. In this first part of the study, we demonstrate how even by positioning the same liquid (1 mL salt water at 10 mS/cm), in the same 24-multiwell plate, but on a conductive grounded plate (e.g., biosafety cabinet) or on a dielectric plate (e.g., standard lab table) we can greatly influence the treatment conditions. Considering the cases with the liquid at FP, we see that moving the plate from a dielectric support to a conductive one significantly modify the current going through the plasma in the capillary (Fig. 4). For these FP cases, the current peak (28 mA on dielectric plate and 35 mA on conductive plate) that takes place after the impact of the primary plasma front and the formation of a conductive channel between the high voltage electrode inside the capillary and the target [8] is limited by the capacitance associated to the in vitro target. Therefore, increasing this capacitance by moving the plate from a dielectric plate to a conductive grounded plate may increase the associated current.

A variation of the treatment conditions moving from one support to another is also testified by the analysis of the treated liquid (Table I) where we measure that a significantly greater amount of peroxides and nitrates are produced when the support is conductive. Therefore, we are led to believe that two biological in vitro assays, performed on the one inside a biosafety cabinet and the other on a dielectric table would supposedly produce different results.

The same test was repeated with the liquid set at GR. In this case, the recorded currents resulted virtually identical between the two supporting plates (Fig. 4). Grounding the liquid is like connecting a resistor in parallel to the capacitance as we already discussed above. Since the conductivity of the tested liquid (as well as that of the most common culture media) is relatively high, the equivalent resistance results small. This in turn favors the passage of a greater conduction current compared to the cases at FP (Fig. 4) and greatly reduces the impact of the capacitance associated with the plate underneath. These observations were confirmed by the liquid analysis where the shift from FP to GR induced the production of higher concentrations of reactive species (Table I) and hindered the influence of the supporting plate. In fact no significant difference could be detected between the two grounded cases.

The differences between the FP and the GR reactive species production in liquid can potentially be attributed also to different mechanism of charges accumulation on the liquid surface (reduced in GR cases) and the influence of induced current on the liquid chemistry.

Also optical emission spectroscopy (OES) acquisitions (Fig. 5) show a significant higher intensity emission for the GR cases (since they were identical, only one is reported for the simplicity) compared to the cases at FP. Emission of the NO* system and nitrogen first positive system N 2 (B-A) almost only occur for the GR cases. This is in agreement with already published studies on the PG comparing free jet condition with those impinging on a grounded metallic target [8].

The differences between the FP and the GR cases are particularly emphasized in proximity of the plasma source outlet. While the FP cases exhibit a constant decrease of the emission intensity moving away from the outlet, the GR cases present a peak for the NO*, OH*, and N 2 (B-A) at 11 mm from the liquid surface (4 mm from the capillary, see Fig. 5). This is probably due to fluid dynamic reasons related to the mixing of the helium flow with the surrounding air. Interestingly, for some species [OH* and N2 (B-A)] there is an evident difference between the FP and the GR cases also in close proximity of the target surface. While the FP present nearly no emission in close proximity to the target surface, the GR cases show some significant emission. This is probably the Fig. 6. Voltage (V) and current (I) waveforms during direct treatment on grounded human body and on grounded salt water (10 mS/cm) in 24-multiwell plate with and without compensation circuit. Gap distance 15 mm. consequence of a secondary ionization front propagating from the target to the high voltage electrode as already reported in the previous studies [8] and that may be more energetic in the GR cases in virtue of a higher degree of ionization in the noticed plasma channel left by the primary ionization front. It must be recalled that while reactive species with relatively long lifetime (e.g., NO*, O 3 , and H 2 O 2 ) generated in the plasma channel can be transported to the liquid surface by the gas flow, other with submillisecond lifetime such as the OH* can reach the target only if generated in close proximity to the interface [24].

In this perspective, it is important to underline how the emission spectra of the in vitro GR case results significantly more similar to that of a human body than both in vitro FP cases. Several reactive species playing important roles in plasma medical applications such as in our case NO* for the promotion and regulation of wound healing [25], may not be produced on an in vitro FP target or are produced in significant lower amounts when compared to a human body target (Fig. 5).

These results suggest that grounding in vitro samples may be a good option to achieve more realistic operating conditions in plasma medical studies. Summing up this first part of results, we may conclude that grounding in vitro models may beneficially affect the plasma treatment making it more stable and independent of the supporting plate nature while increasing the amount of reactive species, especially the nitrogen ones, produced both in gas and in liquid phase.

B. Electrical Compensation of Models

In the second part of the study, the proposed compensation circuit described in the materials and methods was assessed. As mentioned, the goal of the circuit is to adjust the total impedance of the investigated models to match that of a human body. As a first step, the current on the ground connection of a human body treated by the PG (Fig. 6) was recorded. This measurement was adopted as a reference for later tests. Then, the current going through 3 mL of salt water (10 mS/cm) in a 24-multiwell plate and connected to the ground was measured. The same was repeated for the same setup but after the connection of the compensation circuit between the liquid and the ground. As shown in Fig. 6, the current peak going through a liquid sample (not compensated) is significantly higher than that through a human body. This is due to the considerably lower resistance and capacitance characterizing the in vitro sample. The introduction of the compensation circuit greatly reduces these differences so that the current measured on compensated in vitro targets is much closer to that measured on a human body.

Since the recorded current depends on the characteristics of the plasma, we can assume that with the compensation circuit, the current generated on the in vitro sample being more similar to the one generated on a patient, the plasma effects would thus be more similar. This is partially confirmed by the OES results (Fig. 5) where it is observed that for the emission associated to NO* the compensation circuit helps the plasma generated on the in vitro sample to resemble that on the human body. The emissions associated to the other species are less affected but on the other hand no negative effect is observed due to the introduction of the compensation circuit. Especially, it is worth noting that if compared to the more common condition featuring an in vitro sample at FP, the proposed condition with a grounded and compensated sample significantly helps the generation of a plasma similar to the one found when treating a human body.

Only for the human body and GR cases, we observe a significant production of nitrogen reactive species, absent in the FP cases. The same holds for OH* that looks similar, as well as for the magnitude of the O* emission which is the same.

The same tests were repeated with similar results (data not shown) changing the geometry (use of the custom-made well) or the liquid conductivity (20 mS/cm instead of 10 mS/cm). Interestingly, the circuit, originally designed to make the in vitro samples to behave more similar to the human body, may also be used to homogenize the behavior of different in vitro samples (e.g., different well or liquid) from an electrical point of view. This is confirmed in Table II where the compensation leads to the production of similar amounts of reactive species for two different well geometries and liquid volumes. In this case, not only the compensation circuit was adapted but also the treatment time so to maintain a constant ratio of 5 min treatment every 3 mL of liquid. Such setting of the ratio between treatment duration and liquid volume to reach the same reactive species concentration is very often used but may not be accurate enough as expected without the compensation circuit. This was measured with our set up for the compensated cases for which moving from 5 to 15 min of treatment when increasing the liquid volume, resulted in a drastic change of the reactive species concentration (first two rows of Table II).

As a further validation of the compensation method, the same approach was repeated on animal targets. Due to a lack of supporting literature concerning equivalent electrical circuit for mice and the complexity of effectuating accurate measurements of the mice properties, in this case we approached the compensation through successive iterations. The best results in this case were achieved assuming a mouse equivalent impedance of 200 . This is certainly an approximation that may be affected by several factors, such as skin condition and mouse to ground connection (in this case processed through the metal plate supporting the mouse). Preliminary results show that the electrical characteristics of a mouse are, as expected, already closer to those of a human body than those of an in vitro sample. Even if on a smaller scale, also in this case, the addition of the compensation circuit contributed to increase the similarity both in the rising and falling front of the current waveform (Fig. 7). The same experiment was repeated on mice with grafted tumours (Fig. 8). For this target, the compensation resulted less satisfactory probably in virtue of the different resistivity of the tissues exposed to the plasma (healthy skin and tissues versus a thinner skin and a more conductive tumour mass below the skin). Indeed, tumours present a conductivity that is usually higher than the healthy tissue [26], meaning that the equivalent circuit also will be different. Perhaps it should have to be compared to the reference circuit for a human body affected by the same pathology.

IV. CONCLUSION

In the first part of this article, the importance of taking into account and control the electrical characteristics of the in vitro samples commonly employed in plasma biomedical studies was demonstrated. When the target is at FP, even the nature of the plate supporting the sample can influence the treatment. For example, different concentrations of reactive species in the treated medium can be the result of treating the sample on a dielectric table rather than inside a biosafety metallic cabinet. For this reason, it may be advisable to impose the potential of the target to ground level in order to reduce the influence of the surroundings and increase the reproducibility of the experiments. Nevertheless, a well-controlled FP condition can still be more appropriate for specific cases where, for example, the risk of contamination does not allow the use of submerged electrodes into the liquid.

In the second part of the study, we proposed a new method aimed at the reduction of the degree of variation encountered in NTP studies moving from in vitro/animal models to human body. Keeping in mind that the final target of plasma medicine studies are human patients, we propose to adopt a small and affordable electrical circuit to compensate the electrical differences between models and human body and, as result, have the same NTP effects on very different targets including humans.

The use of the compensation method was proven to lead to not only closer current waveforms through different targets but also to a closer reactive species generation in liquid solutions. Also, emission spectra analysis confirmed that the plasma characteristics on a compensated in vitro sample are closer to that found on a human body if compared with those generated on a more common FP configuration.

Therefore, results confirm that the circuit helps maintaining the same treatment effects from one target to another.

Moreover, the mimicking circuit could also help to keep the same treatment conditions when there is a change from one in vitro support to another (e.g., from a well plate type to another).

In spite of the promising results, the compensation circuit alone is clearly not sufficient to transform in vitro samples into perfect replica of a human body. Other factors not related to the electrical properties such as surface evaporation and morphology inherently induce differences between models and human body. For these aspects, other studies have proposed other methods to mimic various human body properties [27]. Is not to be excluded that in the future these methods could be implemented with the electrical compensation method here described to provide even better results.

In the meantime, while being not perfect, the proposed compensation method could significantly improve the representativity of in vitro plasma experiments in a practical and simple manner that at present show no drawback that could prevent its use.

Therefore, we hope that this solution would help to optimize the transition from preclinical to clinical studies in the plasma medicine field.

RESULTS

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In vivo toxicity of direct NTP multi-jet treatment on mice skin

All the in vivo experimentations respected the following animal models and protocol:

• Male Swiss nude mice and inbred female immunocompetent C57Bl/6J mice (7-week-old at an average weight of 19 g) were purchased at the animal facilities of our Institute Gustave Roussy (Villejuif, France) and Janvier laboratory (Saint-Berthevin, France), respectively. Animals were housed at specific pathogen-free animal housing facilities of the Institute Gustave Roussy for seven days before • Before experimentation, mice were permanently identified by single-colour toe tattooing using green paste (Ketchum Animal Tatto Ink Paste, Ketchum Manufacturing Inc., Canada).

• For the immunocompetent C57Bl/6J mice bearing subcutaneous (s.c.) tumours: To facilitate inoculation and avoid obstruction with treatments, the fur on the right flank was removed from mice by dry shaving.

Aim of the study

To the purpose mentioned in the Research Hypotheses and Objectives, an in vivo experimentation was proposed to investigate the safety of the NTP multijet designed by the GREMI for its potential in skincare and treatments. This project collaborated with the GREMI and the Service of Infectious Diseases of the Orléans Regional Hospital, which provides care for skin issues (primarily infection) patients.

RESULTS

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Materials and methods

Treatment parameters

Two different modes of exposure to NTP multijet on the skin were studied: the "multijet" mode, where the distance between the NTP output and the target (mouse skin surface) was fixed at 5 mm (dplasma-skin = 5 mm) (upper panel of Figure 33 and Error! Reference source not found.), and the "diffuse" mode where dplasma-skin = 2 mm (lower panel of Figure 33 and Error! Reference source not found.). Those distances were controlled thanks to customised 3D-printed spacers (not shown on those figures). In the "diffuse" mode, due to the proximity of the plasma jets to the skin surface, the NTP treatment formed a homogeneous "cloud" (similar to what was observed with low-pressure glow discharge plasma) in comparison to the "multi-jet" mode of exposure where five distinguished single jets were seen on the skin.

The NTP device was set at 11 kV with a helium flow rate of one standard litre per minute (1 slm). Various plasma parameters and exposure times were studied: 500 Hz versus 1 kHz versus 2kHz, 1-minute plasma exposure versus three times of 1-minute exposure with at least a pause of 1 minute between each treatment versus 3 minutes of continuous plasma exposure.

Animal model and experimental protocol

• Male Swiss nude mice and female C57Bl/6J mice (see description above), three mice per group.

• A day before the experiment, mice were depilated with depilatory cream (Veet® hair removal cream) and identified as described above.

• On the experiment day, mice were anaesthetised with Isoflurane (Isoflurin® 1000 mg.g-1, Axience, FR) and were treated with NTP multijet under different treatment parameters described above.

• The potential skin effects due to thermal heating during plasma-surface interaction were monitored by thermal imaging using an IR camera (FLIR i7) during the treatment and up to a minute post-treatment, and by photographs of the treatment surface on mice skin immediately after the plasma treatment and by a follow-up of the treated zone of each mouse (as well as the weight and behaviour of each mouse, according to end-point criteria) up to two weeks post-treatment.

RESULTS

-111 -

Results of in vivo toxicity of direct treatment with NTP multijet

The experimental protocol and selected results of this in vivo study on two mice strains are shown in Figure 33 and Error! Reference source not found.. The results presented here were from the plasma treatment where the most damage effects on the skin were observed, i.e., with the plasma treatment using HV pulses at 2 kHz for a treatment time of 3 minutes continuously or three times of 1-minute treatment. Other plasma parameters (500 Hz and 1 kHz) and treatment times showed no to very mild damage to the skin after the treatment. When applying the most aggressive treatment protocol on mice skin, a continuous exposure of NTP set at 2 kHz for 3 minutes (Figure 33), the skin surface temperature during (and after) the plasma treatment (monitored with thermal imaging) showed a mean increase of 12,5°C for the multi-jet exposure mode and 15,5°C for the diffuse mode, with the highest surface temperature recorded during the treatment at 44°C for the multi-jet mode and at 47°C for the diffuse mode. However, as soon as the plasma treatment ceased, the temperature on the skin surface gradually dropped. After 30 seconds post-treatment, it was recorded at ca. 32-34°C for both plasma exposure modes of this treatment condition. The site exposed to the plasma was slightly red but dry with no blisters, except for one mouse in the group "multi-jet"

RESULTS

-112 -treatment where a pale colour spot was observed in the red zone, resembling a blister but without fluid (Figure 33 upper panel, second range of figures from the top). This pale colour spot was widened at D+1 post-treatment and formed a reddish scab a few days later.

Similar thermal skin damage effects were observed on mice of other groups of treatment (Figure 33 lower panel and Error! Reference source not found.), in particular in four to five visible reddish spots on the skin of mice receiving the multijet plasma treatment (in a geometry reminiscent of the individual jets applied on the skin), as compared with the skin of mice receiving the "diffuse" mode where no trace of single jets was observed but a zone representing the plasma-treated area, with milder thermal effects. We also observed that nude mice skin was more affected under the same plasma treatment than C75Bl/6 mice skin.

Moreover, the treatment with plasma being set at 500 Hz gave more muscle contraction in mice (muscle contraction of the leg was observed when plasma treatment was on the thigh) compared to other HV pulses (1 kHz or 2 kHz).

Given that the mouse thermal-neutral point (TNP) is ca. 29°C in the light phase and ca.

33°C in the dark phase (Škop et al., 2020), the results of different treatment protocols indicated -besides the safety measurements of the temperature of the skin during and after the treatment -that direct NTP treatment was tolerable for those two mice strains with the maintenance of skin integrity -crucial to ensure mice physiology. Indeed, the red zone on the skin of all treatments described above was never bled, and it quickly formed dry red scabs the day following the treatment, which scabbed over the following week. All mice remained healthy all along with the experimentation (via monitoring mice's fur, behaviour, and weight).

As the outcome of those in vivo experimentations on the skin toxicity under NTP multijet treatments, we proposed optimal protocols to the Infectious Diseases Service of the Orléans Regional Hospital as: 1) Use diffuse mode while knowing that the appearance of individual jets is acceptable (they are almost obligatorily formed in case of skins or wounds with an irregular surface)

2) Dose escalation: 500 Hz then 1 kHz; if the tolerance is excellent and if the efficacy is insufficient, the 2 kHz should also be considered because the recommendations My contribution to this paper, as the first author, consists in the conceptualisation, the methodology, the data acquisition and validation, the data analysis and visualisation, the data interpretation, and the writing (original draft preparation, review and editing).

RESULTS

-115 - After the post-treatment, the cell layer was harvested in TripLE Express solution, and the cell membrane permeabilisation was analysed by flow cytometry. Graphical abstract adapted from (Chung et al., 2020).

Introduction

Electrochemotherapy (ECT) is a non-thermal, safe, and e cient tumour treatment [1][2][3][4] that is currently used in more than 150 clinics in the European Union and abroad, together with its application in veterinary oncology for treatment of metastases as well as primary tumours [5,6]. ECT is based on the combination of otherwise non-or low-permeant drugs possessing a high intrinsic cytotoxicity (e.g., hydrophilic molecules such as bleomycin or cisplatin) with the local application of a train of eight short and intense monopolar electric pulses (EPs), yet nontoxic [7,8]. The applied EPs create a transient transmembrane potential di↵erence that causes changes in the cell membrane structure and transiently permeabilise its phospholipid bilayer [7]. This biophysical process, which is named reversible electroporation or reversible electropermeabilisation, allows for the penetration of the chemotherapeutic agent inside the cell to generate irreversible DNA damages. ECT selectively kills the tumour cells at the low doses of the chemotherapeutic agents used, since bleomycin (BLM, of 1415 Da) at low doses is only toxic for the cells dividing in the volume treated by the EPs. Overall, no serious negative e↵ects that are related to the application of ECT on patients have ever been reported. Nevertheless, one of the main drawbacks of ECT application are muscles contraction with discomfort sensations associated with repeated electrical stimulation, mainly linked to the characteristics of the high-amplitude electric pulses used. Indeed, these EPs depolarise the neurons in the treated area and can, therefore, generate action potentials, either in the musculo-excitatory nerves or in the sensory nerves, imposing the use of at least a local anaesthesia during the treatment [9]. Recent clinical studies have reported that the painful sensation that is associated to ECT can last longer for locally advanced and metastatic soft tissue sarcomas [10] and large cutaneous recurrences of breast cancer [11].

The main objective of the present study was, therefore, to determine new conditions for ECT, devoid of these side e↵ects. We were interested in reducing the electric field strength of the classical 100 microseconds pulses used in ECT, without reducing the permeabilisation of the cell membrane. We suggest a combined treatment of µsPEFs with non-thermal plasma (NTP) to outperform ECT since cell electropermeabilisation is characterised by lipids oxidation at the time of the electric pulses delivery [12]. Indeed, NTP can be a source of reactive species favouring the lipid oxidation reactions. Over the last decade, several studies in the plasma medicine field have pointed out the use of plasmas at atmospheric pressure in oncology, as plasmas o↵er the possibility to achieve cell membrane permeabilisation [13,14] as well as to selectively kill cancer cells without a↵ecting normal cells [15][16][17]. Nowadays, NTP are broadly used not only in preclinical development of anticancer therapies, including malignant melanoma, ovarian, colorectal, liver, lung, hepatoma, breast, and brain cancers [18], but also in clinical studies [19]. The virtue of NTP relies on the abundant production of reactive species that were primarily generated upon plasma-air interactions: reactive oxygen species (ROS), such as superoxide (O 2 ¯), hydroxyl radicals (OH ), atomic oxygen (O 2 ), singlet delta oxygen ( 1 O 2 ), ozone (O 3 ), and hydrogen peroxide (H 2 O 2 ), as well as reactive nitrogen species (RNS), such as nitric oxide (NO), nitrogen dioxide (NO 2 ), nitrogen trioxide (NO 3 ), nitrous oxide (N 2 O), dinitrogen tetroxide (N 2 O 4 ), and also positive ions, such as dinitrogen (N 2 + ) [20]. Two general NTP strategies are defined for cancer treatment: direct treatment and indirect treatment. The first approach consists of a direct treatment of cancer cells or tumours with the NTP source, where the gaseous plasma species and the plasma-induced electric field have direct actions on the surface of the targets. The second approach implicates an indirect treatment, where the plasma source is used to treat liquids (cell culture media, water, or physiological solutions) and the biological targets are subsequently exposed to the plasma-treated liquids [16,[21][22][23][24]. Some of the long-lived ROS and RNS (also known as RONS) that are generated in the plasma-treated liquids (such as hydrogen peroxide, nitrite and nitrate) are known to play a major role in the oxidation of phospholipid bilayers of the cell membrane [25,26]. We speculate that the NTP caused oxidative stress might be an important factor in augmenting the e cacy of the electroporation-based therapies. Thus, the improvement of cell membrane permeabilisation by the combination of indirect plasma treatment and µsPEFs was explored. Furthermore, we were interested in eventual di↵erent responses of two di↵erent cell lines to the proposed combined treatment. The study was especially focused on malignant melanoma cells, a very aggressive skin cancer, which is one of the main targets of ECT [8,[27][28][29] and plasma medicine [17,30]. For this purpose, we used a novel NTP setup that was able to create multiple plasma jets and, therefore, o↵er a larger and more homogeneous surface treatment. We also implemented a compensation circuit to change the electrical impedance of in vitro targets to that of a reference model for the human body based on our recent studies on the sample influence on plasma characteristics [31]. This setup was used for the plasma treatment of phosphate-bu↵ered saline (PBS) with Ca 2+ /Mg 2+ (PBS + / + ). The responses to the combined treatment of the plasma-treated PBS + / + (or pPBS) with pulsed electric fields of di↵erent strengths were investigated while using adherent DC-3F Chinese hamster lung fibroblasts and adherent malignant B16-F10 murine melanoma cells. Cell membrane permeabilisation was monitored by flow cytometry. The obtained results demonstrate the great potential of the combination of indirect NTP application and µsPEF delivery for cancer treatment.

Results

We tested the cytotoxic e↵ect of pPBS alone to define favourable initial conditions under which cells would not be excessively harmed by the pPBS prior to the assessment of the e↵ect of the combined treatment (pPBS and EPs). The first assay combining pPBS produced at +7 kV and µsPEF with an amplitude of 1100 V/cm was performed in DC-3F fibroblasts. The results, as shown in Appendix A as a proof of concept, give an initial evaluation of the potential e↵ect of indirect plasma treatment and external electric field pulses. Following these preliminary results, the combined e↵ect of pPBS with PEF was investigated in more detail after the pPBS was further characterised and the experimental conditions were optimised and shown to be reproducible.

Evaluation of the Reactive Species in the Plasma-Treated PBS + / +

We explored the conditions where the NTP multi-jet source used for the preparation of pPBS would be stable, easy to apply, and its characteristics reproducible over all the treatments of the PBS + / + , as well as the chemical composition of the resulting pPBS reproducible.

Reactor Electrical Characteristics

3 mL of PBS + / + were treated by the NTP that was produced with the multi-jet source. Di↵erent treatment times between 1 and 20 min. were studied. The reactor was driven by positive high voltage pulses of either +7 kV or +11 kV peak amplitudes. For both voltage amplitudes, the electrical current of the discharge was monitored continuously during the treatment. The electrical measurements (Figure 1) revealed that the treatment corresponding to +11 kV peak voltage was more stable than that of +7 kV peak voltage over the whole treatment duration. In the frame of this analysis, high-definition videos of the NTP multi-jet operated at +7 kV and at +11 kV (see Videos S1 and S2 in Supplementary Materials) were also recorded, furthermore supporting the higher stability of the NTP multi-jet at +11 kV. Thus, this value was chosen as the operating voltage to produce plasma-treated PBS + / + .

Characterisation of the Plasma-Treated PBS + / +

The concentration of hydrogen peroxide, which is a key player for the peroxidation of lipids [21,32], was first assessed in pPBS prepared while using the NTP setup 1 described in the Materials and Methods. The concentration of H 2 O 2 increased almost linearly with the treatment time for both voltage amplitudes studied here, i.e., +7 and +11 kV. On top of that, at +11 kV, the concentration of H 2 O 2 in the pPBS is up to three-fold higher than that in the pPBS that results from a treatment at +7kV (Figure 2a).

After the collection of the proof of concept (Appendix A) and the optimisation of the NTP setup 1, as described in the Materials and Methods (resulting in NTP setup 2), we performed a precise dosimetry of the predominantly stable secondary RONS generated in the pPBS (H 2 O 2 , NO 2 , and NO 3 ) as a function of the plasma treatment time (Figure 2b). The reactive radicals accumulation in the pPBS was time-dependant, being the highest when the plasma treatment was the longest (20 min, see Figure 2). Finally, the pH and the conductivity ( ) of the sham (PBS + / + exposed to only the helium flow (no plasma) and compensated for the evaporation with distilled water) and the pPBS after 20 min. of treatment at either +7 kV or +11 kV were also evaluated (Table 1). When compared to the sham, the pPBS displayed a slightly reduced pH and increased conductivity at both +7 kV and +11 kV. The pPBS produced with the plasma source at +11 kV displayed a lower pH and a higher conductivity than that generated at +7 kV. The plasma treatment time of 20 min. was selected as the condition to use for the preparation of pPBS for all of the following experiments and pPBS was therefore further analysed. The storage temperature and stability overtime of pPBS were investigated (Figure 3). No significant degradation of the previously mentioned RONS was observed over 14 days for a storage temperature of +4 C.

Cancers 2020, 1, x 5 of 21

Finally, the pH and the conductivity (σ) of the sham (PBS + /+ exposed to only the helium flow (no plasma) and compensated for the evaporation with distilled water) and the pPBS after 20 min. of treatment at either +7 kV or +11 kV were also evaluated (Table 1). When compared to the sham, the pPBS displayed a slightly reduced pH and increased conductivity at both +7 kV and +11 kV. The pPBS produced with the plasma source at +11 kV displayed a lower pH and a higher conductivity than that generated at +7 kV. The plasma treatment time of 20 min. was selected as the condition to use for the preparation of pPBS for all of the following experiments and pPBS was therefore further analysed. The storage temperature and stability overtime of pPBS were investigated (Figure 3). No significant degradation of the previously mentioned RONS was observed over 14 days for a storage temperature of +4 °C. Altogether, the above results assist in the definition of the optimal NTP multi-jet parameters for the production of the pPBS used in our experiments with cells. Thus, the voltage amplitude of +11kV and the plasma treatment time of 20 min. were used for the chemical activation of the PBS + /+, which was subsequently stored at +4 °C for up to 14 days.

Investigation of the Effects of the Combined Treatment on DC-3F Chinese Hamster Lung Fibroblasts

Adherent DC-3F Chinese hamster lung fibroblasts were exposed to µsPEF of various amplitudes, 0 V/cm (i.e., no PEF), 600 V/cm, and 1100 V/cm. The µsPEFs were combined with different pre-and/or post-treatments either with sham or with pPBS, as described in Figure 4. Altogether, the above results assist in the definition of the optimal NTP multi-jet parameters for the production of the pPBS used in our experiments with cells. Thus, the voltage amplitude of +11kV and the plasma treatment time of 20 min. were used for the chemical activation of the PBS + / + , which was subsequently stored at +4 C for up to 14 days.

Investigation of the E↵ects of the Combined Treatment on DC-3F Chinese Hamster Lung Fibroblasts

Adherent DC-3F Chinese hamster lung fibroblasts were exposed to µsPEF of various amplitudes, 0 V/cm (i.e., no PEF), 600 V/cm, and 1100 V/cm. The µsPEFs were combined with di↵erent pre-and/or post-treatments either with sham or with pPBS, as described in Figure 4.

The permeabilisation of the cell membrane was analysed by flow cytometry while using YO-PRO ® -1 iodide. This approach allows for the determination of the percentage of permeabilised cells (Figure 5a) and the intracellular fluorescence per cell (Figure 5b). In the conditions where no PEF was applied to the cell monolayer (0 V/cm), a slight increase of the YO-PRO ® -1 uptake was observed in the cells treated while using protocols 2, 4, or 6, as compared to those that were treated with sham. The increase in the intracellular fluorescence intensity of the dye was significant in the case of protocols 2 and 6. When a µsPEF of 1100 V/cm was applied, the percentage of permeabilised cells increased in all of the groups and no significant increase in the percentage of electropermeabilised Cancers 2020, 12, 219 6 of 21 DC-3F cells was caused by the pPBS treatment, regardless of the treatment protocol (Figure 5a). However, we remarked a significant increase of up to 2.4-fold of the intracellular fluorescence in cells that were treated while using protocol 5, when compared to all of the other protocols and, in particular, to the control (Figure 5b). At 600 V/cm, the results were just the opposite: with respect to the PEF alone, no significant enhancement of the intracellular fluorescence of YO-PRO ® -1 iodide uptake was caused by the pPBS, regardless of the treatment protocol, but the percentage of electropermeabilised cells displayed a significant increase in the population of cells that were treated under protocols 3, 5, and 6 as compared to the control. Indeed, while only ca. 30 to 35% of cells treated while using sham were permeabilised at 600 V/cm, the population of permeabilised cells treated using protocol 6 was increased by almost 1.7-fold (+69%) and that of the cells treated using protocol 5 was the double (2-fold enhancement). There is no statistically significant di↵erence between protocols 5 and 6. Remarkably, for protocol 5 at 600 V/cm, c.a. 60 to 70% of the treated cells were permeabilised, reaching the same percentage of permeabilised cells as that of the cells in the control that was only exposed to µsPEFs at 1100 V/cm. The permeabilisation of the cell membrane was analysed by flow cytometry while using YO-PRO ® -1 iodide. This approach allows for the determination of the percentage of permeabilised cells (Figure 5a) and the intracellular fluorescence per cell (Figure 5b). In the conditions where no PEF was applied to the cell monolayer (0 V/cm), a slight increase of the YO-PRO ® -1 uptake was observed in the cells treated while using protocols 2, 4, or 6, as compared to those that were treated with sham. The increase in the intracellular fluorescence intensity of the dye was significant in the case of protocols 2 and 6. When a µsPEF of 1100 V/cm was applied, the percentage of permeabilised cells increased in all of the groups and no significant increase in the percentage of electropermeabilised DC-3F cells was caused by the pPBS treatment, regardless of the treatment protocol (Figure 5a). However, we remarked a significant increase of up to 2.4-fold of the intracellular fluorescence in cells that were treated while using protocol 5, when compared to all of the other protocols and, in particular, to the control (Figure 5b). At 600 V/cm, the results were just the opposite: with respect to the PEF alone, no significant enhancement of the intracellular fluorescence of YO-PRO ® -1 iodide uptake was caused by the pPBS, regardless of the treatment protocol, but the percentage of electropermeabilised cells displayed a significant increase in the population of cells that were treated under protocols 3, 5, and 6 as compared to the control. Indeed, while only ca. 30 to 35% of cells treated while using sham were permeabilised at 600 V/cm, the population of permeabilised cells treated using protocol 6 was increased by almost 1.7fold (+69%) and that of the cells treated using protocol 5 was the double (2-fold enhancement). There is no statistically significant difference between protocols 5 and 6. Remarkably, for protocol 5 at 600 V/cm, c.a. 60 to 70% of the treated cells were permeabilised, reaching the same percentage of permeabilised cells as that of the cells in the control that was only exposed to µsPEFs at 1100 V/cm. We investigated the effect of the combined treatment on B16-F10 melanoma cells while using the same seven protocols of the previous section (Figure 6). Even without any PEF applied, a significant We investigated the e↵ect of the combined treatment on B16-F10 melanoma cells while using the same seven protocols of the previous section (Figure 6). Even without any PEF applied, a significant increase of the intracellular fluorescence intensity of the dye was detected for protocols 2, 4, and especially protocol 6. For this protocol 6, even the percentage of permeabilised cells displayed a significant two-fold enhancement as compared to the control. Using PEFs at 1100 V/cm, the percentage of electropermeabilised cells was not statistically di↵erent from the control without pPBS, except for protocol 4, which was significantly lower. However, with protocols 5 and 6, a significant increase of up to 2.66-fold of the intracellular fluorescence of YO-PRO ® -1 iodide was observed as compared to the control. When applying a 600 V/cm PEF, the pre-and post-treatment of cells with pPBS (protocols 5 and 6) induced a significant enhancement of the cell membrane electropermeabilisation, both in the percentage of electropermeabilised cells (up to a 1.8-fold enhancement) and in the fluorescence intensity per cell (up to a two-fold enhancement). There is no statistically significant di↵erence between protocols 5 and 6, both inducing strong cell permeabilisation increase, reaching the same percentage of permeabilised cells as that of the cells that were exposed to 1100 V/cm in the absence of pPBS. We also observed a significant enhancement of the YO-PRO ® -1 iodide intracellular fluorescence in the cells that were treated at 600 V/cm while using protocol 4, i.e., with only a pre-treatment with pPBS for 20 min. The two previous sections show different behaviours of the two cell lines, particularly in the case of the median intracellular fluorescence while using pPBS and µsPEFs of 600 V/cm amplitude. With the B16-F10 cells being apparently more sensitive to the µsPEF than the DC-3F cells, we decided to investigate the consequences of the application of the seven protocols using µsPEF of only 500 V/cm amplitude. It was also of interest to explore the consequences of using µsPEFs of high field amplitude, as for instance 1400 V/cm, anticipating a larger cell permeability. In this last case, the YO-PRO ® -1 iodide concentration was reduced to 1µM (instead of 2 µM) to avoid a saturation of the flow cytometer signals.

Once more, when no PEF was applied, simple treatment of the B16-F10 cells with pPBS induced a statistically significant increase, not only in the percentage of permeabilised cells but also in the intracellular fluorescence of YO-PRO ® -1 iodide. Regarding the percentage of permeabilised cells that were treated with pPBS alone, we observed significant increases when compared to the control: twofold using protocol 2, 2.2-fold using protocol 4, and 2.8-fold using protocol 6 (Figure 7a). Concerning the intracellular fluorescence of YO-PRO ® -1 iodide (Figure 7b), we observed a significant 1.7-fold enhancement while using protocol 6, which also showed statistically significant differences with The two previous sections show di↵erent behaviours of the two cell lines, particularly in the case of the median intracellular fluorescence while using pPBS and µsPEFs of 600 V/cm amplitude. With the B16-F10 cells being apparently more sensitive to the µsPEF than the DC-3F cells, we decided to investigate the consequences of the application of the seven protocols using µsPEF of only 500 V/cm amplitude. It was also of interest to explore the consequences of using µsPEFs of high field amplitude, as for instance 1400 V/cm, anticipating a larger cell permeability. In this last case, the YO-PRO ® -1 iodide concentration was reduced to 1µM (instead of 2 µM) to avoid a saturation of the flow cytometer signals.

Once more, when no PEF was applied, simple treatment of the B16-F10 cells with pPBS induced a statistically significant increase, not only in the percentage of permeabilised cells but also in the intracellular fluorescence of YO-PRO ® -1 iodide. Regarding the percentage of permeabilised cells that were treated with pPBS alone, we observed significant increases when compared to the control: two-fold using protocol 2, 2.2-fold using protocol 4, and 2.8-fold using protocol 6 (Figure 7a). Concerning the intracellular fluorescence of YO-PRO ® -1 iodide (Figure 7b), we observed a significant 1.7-fold enhancement while using protocol 6, which also showed statistically significant di↵erences with protocols 2 and 4 (there is no statistical di↵erence between protocols 2 and 4 as compared to the control).

Cancers 2020, 1, x 9 of 21 between protocols 5 and 6 (i.e., between a total contact time of 20 and 30 min. between the cells and the pPBS) was found in the intracellular fluorescence intensity. Figure 8 illustrates the observed differences in the membrane permeabilisation level of B16-F10 melanoma cells due to the combined treatment. The fluorescence threshold for permeabilised cells was determined from the cells that were treated with sham (the control cells exposed to untreated PBS + /+) and not exposed to PEF (Figure 8a). We noticed that, even though no PEF was applied (0 V/cm), a slight shift towards higher values of the fluorescence per cell (indicating an increase in membrane permeability) was already present in the population of cells that were treated while using protocol 6 (pre-and post-treatment with pPBS) (Figure 8e when compared to Figure 8a). This corresponds to the significant enhancement of membrane permeabilisation that was observed in Figures 6a and7a induced by pPBS alone. This shift was larger when low amplitude µsPEFs were applied to the cells using protocol 6 at 500 V/cm (Figure 8f) and 600 V/cm (Figure 8g). At 1100 V/cm (Figure 8h), this shift was much larger and actually the population of positive cells constituted a separate peak. The very high level of fluorescence brought by the PEFs at 1100V/cm indicates a high permeabilisation level, being in agreement with the statistically significant enhancement of the membrane electropermeabilisation levels that were observed in Figures 6 and7. At 1400 V/cm, when compared to the control, a significant enhancement of the cell membrane permeabilisation level was observed in cells that were treated while using protocols 1, 3, 5, and 6 in what regards the number of permeabilised cells (up to a 1.5-fold enhancement) (Figure 7a) and while using protocols 3, 4, 5, and 6 in what concerns the intracellular fluorescence of YO-PRO ® -1 iodide entering the cells (up to a 2.2-fold enhancement) (Figure 7b). No statistically significant di↵erence between protocols 5 and 6 was found at 1400 V/cm. Concerning the PEFs at 500 V/cm, all of the combinations (except protocol 1) resulted in a significant enhancement of the percentage of electropermeabilised cells with respect to the control. The intracellular fluorescence intensity also significantly increased while using protocols 3, 5, and 6 as compared to the control. At this very low µsPEF amplitude and with these cells sensitive to the pPBS alone, a statistically significant di↵erence between protocols 5 and 6 (i.e., between a total contact time of 20 and 30 min. between the cells and the pPBS) was found in the intracellular fluorescence intensity.

Figure 8 illustrates the observed di↵erences in the membrane permeabilisation level of B16-F10 melanoma cells due to the combined treatment. The fluorescence threshold for permeabilised cells was determined from the cells that were treated with sham (the control cells exposed to untreated PBS + / + ) and not exposed to PEF (Figure 8a). We noticed that, even though no PEF was applied (0 V/cm), a slight shift towards higher values of the fluorescence per cell (indicating an increase in membrane permeability) was already present in the population of cells that were treated while using protocol 6 (pre-and post-treatment with pPBS) (Figure 8e when compared to Figure 8a). This corresponds to the significant enhancement of membrane permeabilisation that was observed in Figures 6a and7a Cancers 2020, 12, 219 9 of 21 induced by pPBS alone. This shift was larger when low amplitude µsPEFs were applied to the cells using protocol 6 at 500 V/cm (Figure 8f) and 600 V/cm (Figure 8g). At 1100 V/cm (Figure 8h), this shift was much larger and actually the population of positive cells constituted a separate peak. The very high level of fluorescence brought by the PEFs at 1100V/cm indicates a high permeabilisation level, being in agreement with the statistically significant enhancement of the membrane electropermeabilisation levels that were observed in Figures 6 and7.

Discussion

In this study, the potential effects of a treatment combining plasma-treated PBS + /+ (pPBS) and monopolar microsecond pulsed electric fields (µsPEFs) on two different cell lines were investigated. The indirect use of the NTP, by means of the application of pPBS, allowed for us to treat the cells in a very homogenous way. The use of parallel plate electrodes encompassing the whole cell monolayer also brought very homogeneous conditions for the cells' treatment by the µsPEFs. Finally, great care was put on the cell culture conditions before the application of the pPBS and/or the µsPEFs in order to have homogeneous subconfluently (at ca. 80% density) monolayers. Therefore, all of the conditions were gathered to make possible comparisons between the various protocols that were used in our study.

The combination of the µsPEF and the pPBS aimed at the reduction of the applied electric field strength (without decreasing the cells electropermeabilisation) and/or the enhancement of the electropermeabilisation level of the cell membrane. The range of intensities of the µsPEF that was used in our study has already been demonstrated as non-cytotoxic [33,34]. The work reported in the results section was performed with a setup optimised, as described in the results section and in [31] (NTP setup 2), after establishing the proof of concept (Appendix A) with a new NTP multi-jet setup that was recently developed by our group to homogeneously treat large surfaces (NTP setup 1). The NTP setup 2 produced pPBS with reproducible characteristics due to well-controlled exposure of the PBS + /+ and allowed for us to examine possible differences in the pPBS effects on the two different cell lines used in this study. Our results indicate that the DC-3F cells are less sensitive than the B16-F10 cells to the pPBS alone. Nevertheless, a statistically significant increase in the intracellular concentration of the YO-PRO ® -1 could be detected in DC-3F cells, even though the pPBS treatment did not suffice for significantly increasing the percentage of permeabilised cells. A low µsPEF intensity (600 V/cm) combined with a pre-and post-treatment of cells with pPBS favours the permeabilisation of cell membrane and doubles the population of permeabilised cells (as compared to the exposure to only µsPEFs of the same intensity). Furthermore, this combined treatment allows for reaching the same percentage of permeabilised cells as a treatment with µsPEFs alone at 1100 V/cm. However, the level of the

Discussion

In this study, the potential e↵ects of a treatment combining plasma-treated PBS + / + (pPBS) and monopolar microsecond pulsed electric fields (µsPEFs) on two di↵erent cell lines were investigated. The indirect use of the NTP, by means of the application of pPBS, allowed for us to treat the cells in a very homogenous way. The use of parallel plate electrodes encompassing the whole cell monolayer also brought very homogeneous conditions for the cells' treatment by the µsPEFs. Finally, great care was put on the cell culture conditions before the application of the pPBS and/or the µsPEFs in order to have homogeneous subconfluently (at ca. 80% density) monolayers. Therefore, all of the conditions were gathered to make possible comparisons between the various protocols that were used in our study.

The combination of the µsPEF and the pPBS aimed at the reduction of the applied electric field strength (without decreasing the cells electropermeabilisation) and/or the enhancement of the electropermeabilisation level of the cell membrane. The range of intensities of the µsPEF that was used in our study has already been demonstrated as non-cytotoxic [33,34]. The work reported in the results section was performed with a setup optimised, as described in the results section and in [31] (NTP setup 2), after establishing the proof of concept (Appendix A) with a new NTP multi-jet setup that was recently developed by our group to homogeneously treat large surfaces (NTP setup 1). The NTP setup 2 produced pPBS with reproducible characteristics due to well-controlled exposure of the PBS + / + and allowed for us to examine possible di↵erences in the pPBS e↵ects on the two di↵erent cell lines used in this study. Our results indicate that the DC-3F cells are less sensitive than the B16-F10 cells to the pPBS alone. Nevertheless, a statistically significant increase in the intracellular concentration of the YO-PRO ® -1 could be detected in DC-3F cells, even though the pPBS treatment did not su ce for significantly increasing the percentage of permeabilised cells. A low µsPEF intensity (600 V/cm) combined with a pre-and post-treatment of cells with pPBS favours the permeabilisation of cell membrane and doubles the population of permeabilised cells (as compared to the exposure to only µsPEFs of the same intensity). Furthermore, this combined treatment allows for reaching the same percentage of permeabilised cells as a treatment with µsPEFs alone at 1100 V/cm. However, the level of the intracellular fluorescence is not comparable: this level is much lower at 600 V/cm (even in combination with the pPBS) than at 1100 V/cm. At 1100 V/cm, a further increase, significant (over two-fold enhancement), in the intensity of the fluorescence per cell was observed when the µsPEFs were combined with a pre-and post-treatment of the cells with pPBS. All of these increments reveal an increase of the level of electropermeabilisation by the pPBS.

For the comparison with the DC-3F cells, experiments were also performed with the "cancerous" B16-F10 melanoma cells whose size is larger than that of DC-3F cells. This cell size di↵erence is known to play an important role in the cell membrane electropermeabilisation level [35,36]. Therefore, B16-F10 cells were expected to be more sensitive than the DC-3F cells when exposed to µsPEF of the same amplitude. This was indeed observed at 1100 V/cm, since the median intracellular fluorescence of the B16-F10 cells (about 1.8 ⇥ 10 6 a.u.) was higher than that of the DC-3F cells (0.8 ⇥ 10 6 a.u.), in the absence of pPBS application (Figures 5b and6b, control conditions). When applying PEFs at low field amplitudes (600 V/cm), this consequence was also noticed (median intracellular fluorescence of 0.55 ⇥ 10 6 a.u. for the B16-F10 cells versus 0.22 ⇥ 10 6 a.u. for the DC-3F cells). These data also point out the increase of the median intracellular fluorescence of both cell lines with higher PEFs intensities. This trend is also observed when the individual flow cytometry diagrams are analysed (Figure 8c versus Figures 8d and8g versus Figure 8h). The results on the percentage of permeabilised cells also show that B16-F10 cells seem to be more sensitive to both treatments. With a single treatment with pPBS (protocol 6, i.e., 30 min. of total contact time between cells and pPBS) without any PEF application, the percentage of permeabilised B16-F10 cells (Figures 6a and7a) is significantly higher than the untreated cells (two-fold enhancement), while no statistical di↵erence was found in the case of the DC-3F cells (Figure 5a). At 1100 V/cm for both cells, when the percentage of the µsPEF-permeabilised cells was already very high, this percentage was not increased by the pPBS, regardless of the treatment protocol. Interestingly, when the percentage of µsPEF-permeabilised cells was low (at 600 V/cm for the DC-3F cells and 500 V/cm for the B16-F10 cells-see below), the application of the pPBS resulted in an increase of the percentage of permeabilised cells. Moreover, in a first approximation, the longer the total treatment time with the pPBS, the higher that increase. In the case when the B16-F10 cells were exposed to 1400 V/cm, protocols 5 and 6 again revealed an increase in this percentage, as well as an increase in the intracellular fluorescence intensity, caused by the pPBS application. We can speculate that the increase by pPBS in the cells permeabilisation at 1400 V/cm could be related to the occurrence of longer or irreversible electroporation, which would cause a larger uptake of the YO-PRO ® -1 iodide by the electroporated cells. To conclude, a pre-and post-treatment of cells with pPBS enhances the membrane electropermeabilisation level for most of the combined treatment protocols with PEFs of 600 to 1400 V/cm amplitude. The oxidative stress that was generated within the phospholipid bilayer of the cells by the radicals brought by the pPBS could mediate this e↵ect. Moreover, a period of 10 min. of pre-treatment of cells with pPBS (protocol 5) is su cient for starting to generate these e↵ects.

Experiments were repeated at 500 V/cm with the malignant B16-F10 melanoma cells to be under the same extremely low permeabilisation levels by PEF alone as those of the DC-3F cells that were treated at 600 V/cm. Interestingly, even at this low µsPEF amplitude, 10 min. of pre-treatment with pPBS (protocol 3) could already achieve an increase in both the percentage of the µsPEF-permeabilised cells and the intracellular uptake of the dye. A combination of both pre-and post-treatment with pPBS (protocols 5 and 6) strongly induced these e↵ects. Especially, 30 min. of total contact time between the cells and pPBS (protocol 6) resulted in the same percentage of µsPEF-permeabilised cells as the controls µsPEF only treated at 1100 V/cm or 1400 V/cm.

Cancers 2020, 12,219 It is worth mentioning that pPBS should a↵ect all of the cells, although slightly, because radicals are present throughout the whole volume of the pPBS in contact with the cells, while µsPEF, particularly at low field amplitudes, should only strongly a↵ect part of the cells (those whose size and geometry allows for locally generating a su ciently high transmembrane voltage di↵erence). In this respect, we can underline that, regardless of the µsPEF strength applied, it was never observed an entire electropermeabilisation of the cell population for both cell lines studied (i.e., the percentage of electropermeabilised cells did not reach 100% in any of the cell lines that were tested at any treatment condition). This result agrees with most of the in vitro electroporation studies, where the viability of most of the cells is sought [34,35].

Multiple research groups discussed the critical influence of both cell type and cancer type on the cell sensitivity to indirect plasma treatment [16,17,37,38]. On the other hand, it has been documented that di↵erent cell types have di↵erent responses to oxidative stress [39] or PEFs/electrochemotherapy/electroporation-based therapies [35,36,40,41]. From the "electropermeabilisation" point of view, the most important factor is the size of the cells. Microscope images easily show that the B16-F10 cells are larger than the DC-3F cells. Therefore, it was expected that, in this study, for the B16-F10 cells, lower field amplitudes (500 V/cm) would be necessary to electropermeabilise them to the same extent as the DC-3F cells at 600 V/cm. What we could not anticipate was the increased sensitivity of the B16-F10 cells to the pPBS alone. While the various protocols with pPBS in the absence of PEFs only resulted in a higher intracellular uptake of the YO-PRO ® -1 for the DC-3F cells, significant di↵erences with respect to the controls were achieved in the case of the B16-F10 cells, concerning both the percentage of permeabilised cells and the intracellular YO-PRO ® -1 concentration. There is no obvious reason for such a di↵erence.

Importantly, the cell-dependant phenomena that were observed in this study have to be also discussed along with the "plasma-treated PBS" e↵ects. We demonstrate here that the radicals of the pPBS contribute to the enhancement of the cell membrane permeabilisation level in the combined treatment. This observation is in agreement with the study of Vernier and colleagues, who, while using molecular dynamics (MD) simulations and experiments with living cells, demonstrated that electroporating fields target oxidatively damaged areas in the cell membrane [42]. Yusupov and colleagues also used MD simulations to demonstrate that oxidation of the lipids in a phospholipid bilayer lowers the permeation free energy barriers of the ROS, which can further enhance the action of the ROS and also result in a drop of the electric field threshold needed for pore formation (electroporation), with respect to the potential facilitation of the pPBS e↵ects by the PEFs. Their study also highlights that the lipid oxidation by plasma generated ROS synergistically enhances this e↵ect [43].

Regarding the NTP multi-jet that was used in the present study to produce pPBS, there is evidence that the e cacy of plasma-treated liquids depends on the generated RONS concentration, which depends on the operating conditions used for liquid treatment [18,24]. Consolidated data were achieved while using an optimised NTP setup that delivers a stable peak current under very precise geometrical conditions. It is interesting to note that the e↵ects were similar to those that were achieved in the proof of concept (Appendix A) while using a non-optimised setup. In fact, with both setups, the accumulation of H 2 O 2 in pPBS reached similar levels (ca. 1.4 mM, see Figure 2a,b), which might explain why the increase in the cell permeabilisation level was similar. This fact reinforces the implication of the pPBS radicals in the improvement of the cell membrane permeabilisation by the µsPEF. Moreover, the resulted pPBS can remain stable at +4 C for later use over a long period (at least 14 days), which facilitates its application and stock production, o↵ering extensive advantages for biomedical purposes as compared to recent studies [44][45][46] It is worth mentioning that there are contradicting observations as to whether the conductivity of an external medium impacts the e ciency of the reversible cell membrane permeabilisation that is caused by PEFs [33,47,48]. However, a study from our group investigating the same DC-3F cell line and µsPEFs, demonstrated that media of lower conductivity induced more e cient reversible permeabilisation [33]. Our results indicate that the observed e↵ects are not linked to the conductivity of the liquids, but rather to the radicals presence, since the pPBS has a higher conductivity than that of the sham (see Table 1) and yet induces more permeabilisation e↵ects on the cell membrane.

As previously mentioned, when cells were treated with pPBS before and after low amplitude µsPEFs, the percentage of permeabilised cells reached values similar to those of cells that were exposed to a classical electroporation field amplitude (1100 V/cm). The combined treatment, thus, opens the possibility to reduce the amplitude of the EP used in ECT, one of the goals of the present study. However, formal proofs have to be brought. Indeed, as demonstrated by the low uptake of the YO-PRO ® -1 iodide by the cells exposed to the combined treatment at 600 V/cm (DC-3F cells) or 500 V/cm (B16-F10 cells) when compared to the uptake by the cells electropermeabilised by the µsPEFs alone at 1100 V/cm, the intensity of the fluorescent dye in cells that were treated by the combined treatment is low. Nevertheless, because of the large e cacy of BLM once inside the cells (500 molecules are su cient to kill the dividing cells [49]), the percentage of permeabilised cells is more important than the level of permeabilisation (as quantified by the median intracellular fluorescence). As a matter of fact, in 1988, we already published that the same DC-3F cells, which were exposed to pulses leading to the reversible permeabilisation of 98% of the cells according to Lucifer Yellow uptake (a permeabilisation marker of ca. 450 Da, slightly smaller than the YO-PRO ® -1 Iodide) also lead to 98% of cell killing in the presence of BLM [7]. In any case, the combined treatment that is explored in this paper is interesting as the µsPEF amplitudes could be greatly reduced during ECT or other electroporation-based therapies (to mitigate their side e↵ects), or the used anticancer drug concentration could be reduced if it is chosen to maintain a high µsPEF amplitude.

Materials and Methods

Unless specified otherwise, all of the reagents were purchased from Life Technologies, Courtaboeuf, France.

Cell Culture

DC-3F Chinese hamster lung fibroblasts [50] and B16-F10 murine melanoma cells [51], all mycoplasma-free, were cultured in Minimum Essential Medium (MEM, 31095-029) and Dulbecco's Modified Eagle Medium (DMEM, High Glucose, GlutaMAX Supplement, pyruvate, 31966-021), respectively. All of the media were supplemented with 10% foetal bovine serum (FBS, F7524), 100 U•mL 1 penicillin and 100 mg•mL 1 streptomycin (15140-122). The adherent cells were propagated at 37 C in a 95% humidity atmosphere containing 5% CO 2 (HERAcell 240i incubator CO 2 , Thermo Fisher Scientific, Courtaboeuf, FR) and then passaged upon confluency (every two days at a 1:10 dilution or every three days at a 1:30 dilution) while using TrypLE™ Express (12604-013). The cells were routinely checked for mycoplasma contamination via polymerase chain reaction (PCR). Cell viability was assessed while using trypan blue exclusion dye method (Trypan Blue Solution, T8154) with the Countess ™ II FL Automated Cell Counter (Invitrogen, Thermo Fisher Scientific, Courtaboeuf, FR) and only viable cells were considered. The NTP multi-jet used in this study is based on a Plasma Gun (PG) device, which was described previously [31]. Briefly, the PG is a coaxial dielectric barrier discharge (DBD) reactor that consists of a quartz capillary tube flushed with helium and a microsecond-pulses high voltage (µs-pulses HV) generator powers it. As compared to the classical configuration, this new version presents two reactor zones (Figure 9a). The first zone is located inside the µs-pulsed HV generator, where a high voltage electrode (hollow metallic tube) is placed into a glass tube. At the outlet of the glass tube, a flexible dielectric tube is mounted containing a floating-potential electrode (conductive wire of ca. 1 mm 2 section) inside it. The dielectric tube goes out from the first reactor zone by connecting it with the second reactor zone. This zone of the plasma device (the applicator) is essentially a second coaxial DBD reactor that is made of a PTFE (Polytetrafluoroethylene) body (shown in white in Figure 9) with the floating electrode being placed in its centre and a grounded ring electrode on the outside. The outlet of the second reactor zone has five micro-orifices (Ø int = 800 µm) producing, thus, five distinct plasma jets and covering a larger liquid area during the treatment (Figure 9b). Four of the orifices are disposed at the corner of a 4.2 mm square with the fifth orifice located at the crossing of the square diagonals. For all of the experiments, helium (99.9999% pure, Air Liquide, FR) at a fixed flow rate of 1 slm (standard litre per minute) was used as the operating gas. The NTP source was powered by high voltage pulses with duration of 4 µs (measured at half height) and a peak value of either +7 or +11 kV at a repetition rate of 2 kHz. The applied voltage was measured while using a high-voltage passive probe (Tektronix P6015A), while the current was determined by measuring the voltage drop on the resistor in the compensation circuit with another high-voltage passive probe (Tektronix TPP1000, Beaverton, OR, USA).

A non-optimised setup 1 was used for cytotoxicity assessment and the proof of concept that is reported in Appendix A (NTP setup 1). It used a stainless steel plate as ground electrode with no compensation circuit and a three-dimensional (3D) printed spacer to fix the gap distance between the plasma output orifices and the surface of the PBS. Based on our concomitant recent work [31], the NTP multi-jet source was optimised (NTP setup 2). It consists of (i) a new and more stable reactor, (ii) a ring shaped wire electrode (stainless-steel wire Ø = 1mm) placed in the bottom of the well (instead of the plane electrode used in the setup 1), which allows for avoiding liquid leakage from the well and reducing the number of discharges that formed between the plasma jets and the grounded electrode, and (iii) a compensation circuit designed to impose the total target impedance to a reference model mimicking the human body impedance [31], which would ease the translation of the results to animal models or human body (Figure 9, note that to allow for better visualisation of the plasma multi-jets, the spacer is not represented in the simplified scheme of Figure 9a and not in use in Figure 9b).

Cancers 2020, 1, x 14 of 21 per minute) was used as the operating gas. The NTP source was powered by high voltage pulses with duration of 4 µs (measured at half height) and a peak value of either +7 or +11 kV at a repetition rate of 2 kHz. The applied voltage was measured while using a high-voltage passive probe (Tektronix P6015A), while the current was determined by measuring the voltage drop on the resistor in the compensation circuit with another high-voltage passive probe (Tektronix TPP1000, Beaverton, OR, USA).

A non-optimised setup 1 was used for cytotoxicity assessment and the proof of concept that is reported in Appendix A (NTP setup 1). It used a stainless steel plate as ground electrode with no compensation circuit and a three-dimensional (3D) printed spacer to fix the gap distance between the plasma output orifices and the surface of the PBS. Based on our concomitant recent work [31], the NTP multi-jet source was optimised (NTP setup 2). It consists of (i) a new and more stable reactor, (ii) a ring shaped wire electrode (stainless-steel wire Ø = 1mm) placed in the bottom of the well (instead of the plane electrode used in the setup 1), which allows for avoiding liquid leakage from the well and reducing the number of discharges that formed between the plasma jets and the grounded electrode, and (iii) a compensation circuit designed to impose the total target impedance to a reference model mimicking the human body impedance [31], which would ease the translation of the results to animal models or human body (Figure 9, note that to allow for better visualisation of the plasma multi-jets, the spacer is not represented in the simplified scheme of Figure 9a and not in use in Figure 9b).

Preparation of the Plasma-Treated PBS + /+

For the preparation of the liquids used in the indirect plasma treatment with the NTP multi-jet device, Dulbecco's phosphate-buffered saline with Calcium and Magnesium (DPBS, Ca ++ , Mg ++ , 14040-133), termed PBS + /+, was chosen, as it is an appropriate buffer for adherent cells. Each well in 24multiwell plates (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 142475) was filled with 3 mL of PBS + /+. The distance between the liquid surface and the plasma multi-jet output orifices was maintained at 4 mm thanks to a customised 3D-printed spacer. PBS + /+ was exposed to the NTP multi-jet for different times, varying between 1 and 20 min. A digital hygrometer/thermometer (Velleman ® Home, BE) was used to monitor the humidity and the room temperature in the working area. Water loss due to evaporation was compensated by distilled water addition in the pPBS at the end of the plasma treatment to maintain the osmolality of the plasma-treated PBS + /+ (pPBS). Depending on the room temperature, 600 to 700 µL of sterile water were added per well when PBS + /+ was plasma-treated for 20 min.

Preparation of the Plasma-Treated PBS + / +

For the preparation of the liquids used in the indirect plasma treatment with the NTP multi-jet device, Dulbecco's phosphate-bu↵ered saline with Calcium and Magnesium (DPBS, Ca ++ , Mg ++ , 14040-133), termed PBS + / + , was chosen, as it is an appropriate bu↵er for adherent cells. Each well in 24-multiwell plates (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 142475) was filled with 3 mL of PBS + / + . The distance between the liquid surface and the plasma multi-jet output orifices was maintained at 4 mm thanks to a customised 3D-printed spacer. PBS + / + was exposed to the NTP multi-jet for di↵erent times, varying between 1 and 20 min. A digital hygrometer/thermometer (Velleman ® Home, BE) was used to monitor the humidity and the room temperature in the working area. Water loss due to evaporation was compensated by distilled water addition in the pPBS at the end of the plasma treatment to maintain the osmolality of the plasma-treated PBS + / + (pPBS). Depending on the room temperature, 600 to 700 µL of sterile water were added per well when PBS + / + was plasma-treated for 20 min.

Plasma-treated PBS + / + Characterisation

The concentration of peroxide (H 2 O 2 ) in pPBS was determined while using titanium (IV) oxysulfate (TiOSO 4 ). H 2 O 2 reacts with TiOSO 4 to produce pertitanic acid, which is yellow [22] and detectable by a spectrophotometer. The absorbance was measured at 407 nm while using a spectrophotometer multi-plate reader (Infinite ® M200 PRO Tecan). The quantification of nitrite (NO 2 ) and nitrate (NO 3 ) in pPBS was performed within an hour after its treatment with the NTP multi-jet using nitrate/nitrite colorimetric assay kits (Cayman Chemical, Interchim, FR), according to the supplier's instructions and using the same spectrophotometric system was used. The pH and the conductivity of the treated-liquids were also measured while using specific probes for liquid analysis (InLab Micro Pro and Seven Compact Duo, by Mettler-Toledo). Semi-quantitative chemical analyses of peroxide, nitrate, and nitrite concentrations produced in pPBS were also assessed after each pPBS preparation while using Quantofix ® test strips (Macherey-Nagel GmbH & Co. KG, Düren, DE) to ensure the quality of each preparation, which also revealed the reproducibility of the pPBS preparation.

For the evaluation of the RONS stability under storage at +4 C, the initial pPBS volume (3 mL) was divided into four samples of equal volume. The concentration of the reactive species (H 2 O 2 , NO 2 -, and NO 3 -) in one of the samples was measured in the day of plasma treatment (day 0) and used as a reference (100%). The concentration in the other three samples was measured after 1, 7, and 14 days of storage at +4 C. The relative concentration of the reactive species after storage at +4 C is, thus, given in relation to the concentration measured at day 0.

Adherent Cells Electropulsation Setup

We used the system described in [34] for the electropulsation of the adherent cell monolayer. More precisely, as displayed in Figure 10, an in-house built mould of PDMS (polydimethylsiloxane, SYNGARD™ 184 Silicone Elastomer, DE) with an empty 2 cm 2 rectangle was inserted in a Ø 35 mm Petri dish (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 353001) to obtain a 2 cm 2 surface for cell growth. One day before the experiment, 600 µL of cell suspension were added to the defined area of each Petri dish at a density of 2.20 ⇥ 10 5 cells•mL 1 (i.e., 1.32 ⇥ 10 5 cells/600 µL) for DC-3F fibroblasts or 1.80 ⇥ 10 5 cells•mL 1 (i.e., 1.08 ⇥ 10 5 cells/600 µL) for B16-F10 melanoma cells, in respect to their growing speed and morphology (malignant B16-F10 murine melanoma cells grow much faster than DC-3F Chinese hamster lung fibroblasts). These seeding densities are based on our previous observation and experience, as they appear to be suitable for obtaining a homogenous cell layer at ca. 80% confluency (not entirely dense) after 24 h of cell culture. Di↵erent pre-treatment protocols were tested on the day of the experiment. Afterwards, the electric pulses were applied on the cell layer by means of an in-house built electrode configuration consisting of two parallel stainless-steel plates (2 mm thick), fixed in the PDMS mould, and distant of 6 mm (Figure 10). The electrodes and the custom moulding PDMS were designed to ensure the entire exposition of the cell monolayer to EPs. To generate microsecond pulsed electric fields (µsPEFs), the Cliniporator™ (IGEA, Carpi, IT) was used to deliver eight consecutive square-wave electric pulses of 100 µs duration, at a repetition frequency of 1 Hz, and di↵erent field strengths (500, 600, 1100, and 1400 V/cm).

an in-house built electrode configuration consisting of two parallel stainless-steel plates (2 mm thick), fixed in the PDMS mould, and distant of 6 mm (Figure 10). The electrodes and the custom moulding PDMS were designed to ensure the entire exposition of the cell monolayer to EPs. To generate microsecond pulsed electric fields (µsPEFs), the Cliniporator™ (IGEA, Carpi, IT) was used to deliver eight consecutive square-wave electric pulses of 100 µs duration, at a repetition frequency of 1 Hz, and different field strengths (500, 600, 1100, and 1400 V/cm).

Combined Treatment

The combined treatment associated µsPEFs and pPBS to treat cells. Sham (PBS + / + not treated with the plasma multi-jet) was used as a control for all of the experiments. Before exposing the cells to the sham or the pPBS, YO-PRO ® -1 Iodide (629.04 Da) (Life Technologies, Y3603) was added to these media at a final working concentration of 2 µM (unless otherwise specified). In the day of experiment, the medium above the cells was removed and the cell layer was washed twice with PBS + / + . 500 µL of the YO-PRO ® -1-containing pre-treatment liquid (sham or pPBS) was added on the cells for di↵erent incubation times (10 or 20 min.), and then µsPEFs were delivered (strengths ranging between 500 and 1400 V/cm). Afterwards, the electrodes were removed from the PDMS support and the pre-treatment liquid was then replaced by the YO-PRO ® -1-containing post-treatment liquid (sham or pPBS) for 10 min.

In the absence of PEF (0 V/cm), the pre-treatment time was fixed at 20 min. In the presence of µsPEFs, the control condition (cells pre-and post-treated using Sham) was carried out with 20 min. of pre-treatment.

Evaluation of Cell Membrane Electropermeabilisation Induced by the Combined Treatment

The eventual permeabilisation of the cells that were treated by the combined treatment was investigated by fluorescent nucleic acids stain YO-PRO ® -1 Iodide uptake. YO-PRO ® -1 is a non-permeant dye that is frequently used as an indicator for permeabilisation. Indeed, when the cell membrane is permeabilised, YO-PRO ® -1 can enter the cell and intercalates with nucleic acids, which results in a strong green fluorescence signal that can be detected by flow cytometry.

After the post treatment, the treated liquid above the cell monolayer was removed and the cells were harvested while using TrypLE™ Express Enzyme dissociation (400 µL per Petri dish). The cell suspension was then analysed by flow cytometry (C6 flow cytometer, BD Accuri, San Jose, California, US). 10 000 events were recorded, the YO-PRO ® -1 uptake (cell permeabilisation) was evaluated while using green fluorescence channel (excitation 488 nm, emission 530/30 nm). More precisely, the percentage of fluorescent/permeabilised cells as well as the mean and median fluorescence intensity per cell (level of permeabilisation of each cell) were assessed. The cell membrane permeabilisation threshold was fixed for all of the samples based on that of cells treated with Sham and not exposed to PEF.

Statistical Analysis

The experiments were performed at least three times independently, i.e., at least over three di↵erent days. In addition, each parameter set was performed in triplicate, which resulted in a total of at least nine replicates for each parameter set. The outliers were identified and removed while using the Grubb's method (Alpha = 0.05). To study the significance of di↵erences, one-way ANOVA, Cancers 2020, 12, 219 16 of 21 followed by Bonferroni's multiple comparison tests, were performed while using Prism (GraphPad Software, La Jolla, CA, US). Statistical significance levels were associated with p-values of <0.05 (*), <0.01 (**), <0.001 (***) and <0.0001 (****).

Conclusions

The present study demonstrates that the application of plasma-treated PBS + / + that is produced by the newly-developed NTP multi-jet source could e↵ectively enhance the µsPEFs induced cell membrane permeabilisation, both in terms of the percentage of permeabilised cells and the intracellular content of a permeabilisation marker. These e↵ects occur, even at very low µsPEF amplitudes, and the results are cell-dependant. The malignant B16-F10 murine melanoma cells are more sensitive to the e↵ect of this combined treatment than the DC-3F Chinese hamster lung fibroblasts.

These very promising results underline the great potential of a combined ECT and indirect NTP treatment for anticancer therapies. Investigation and in-depth physical, chemical, and biological understanding of those e↵ects will be fundamental for in vivo studies and clinical trials, which might open up new ways for the implementation of guided cancer therapies while using indirect NTP treatment and ECT in the future.

TrypLE™ Express Enzyme solution and suspended in their culture medium. Cells were counted without Trypan Blue Stain and seeded out, in appropriate dilutions, 250 cells per well of a 6-multiwell plate (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 140675). After 5 days of cell culture, the medium above the cells was discarded, cells were washed carefully twice with PBS. To each well, 2 mL of colony fixation-staining solution (crystal violet 0.2% (wt/vol), formaldehyde 3.7% (vol/vol) and ethanol 20% (vol/vol) in H 2 O) was added and left for 2 to 5 min. before being removed carefully. Stained cells were rinsed by immersing the plates in a sink filled with tap water. The plates were dried at room temperature, then the resulting clones were counted for each treatment condition. The viability, reported as a percentage of survived and proliferated adherent cells, was referred to the number of clones in the sham condition.

Appendix A.1.

Results and Discussion

In order to ensure that the pPBS alone does not significantly a↵ect cell growth and death within a "sublethal" contact time with the cells, cells were incubated with pPBS for a large incubation time and the cell viability after the treatment was assessed via a cytotoxicity assay. The survival curves of cells in percentage of viable colonies with respect to the sham condition (Figure A1) show that the viability of DC-3F cells was preserved when being in contact with pPBS for 1 to 15 min. When the cells were incubated in pPBS for longer, up to 30 min., the long-term cytotoxic e↵ect was limited, with a maximal loss of viability of 40%. For even longer durations, cytotoxicity was larger. These results demonstrate that the long-term cytotoxicity of pPBS depends on time of contact with the cells, as reported in other works [52]. As up to 30 min. of exposure to the pPBS resulted in a tolerable range of cytotoxic, in the remaining work of our study, the maximal duration of treatment of the cells to the pPBS was fixed to 30 min. We thus considered this incubation time window as the most appropriate to combine with µsPEFs for a sublethal and e cient cell reversible electropermeabilisation.

Cancers 2020, 1, x 18 of 21 incubated in pPBS for longer, up to 30 min., the long-term cytotoxic effect was limited, with a maximal loss of viability of 40%. For even longer durations, cytotoxicity was larger. These results demonstrate that the long-term cytotoxicity of pPBS depends on time of contact with the cells, as reported in other works [52]. As up to 30 min. of exposure to the pPBS resulted in a tolerable range of cytotoxic, in the remaining work of our study, the maximal duration of treatment of the cells to the pPBS was fixed to 30 min. We thus considered this incubation time window as the most appropriate to combine with µsPEFs for a sublethal and efficient cell reversible electropermeabilisation. of YO-PRO ® -1 Iodide (present in all the treatment liquids), indicating membrane permeabilisation (Figure A2). This e↵ect is significantly greater in cells pre-treated with pPBS than in control cells pre-treated with sham (1.68-fold enhancement).

The treatment of DC-3F cells with pPBS combined with an EP at 1100 V/cm intensity has thus given a proof of concept that pPBS favoured the membrane electropermeabilisation.

RESULTS

-116 -3 Preserving the anti-cancer efficacy of plasma-treated liquids over time: a prerequisite for their clinical application (manuscript to be submitted)

Over the last decade, there has been an increasing interest in non-thermal plasma (NTP) uses in medical studies and applications, especially plasma-treated liquids in oncology, in combining treatment to enhance the efficacy of established procedures monotherapies. To develop new anti-cancer modalities or treatments, there was a primordial need to understand the stability, components, and pH over different storage conditions of those plasma-treated liquids.

After the characterisation of the abovementioned NTP multijet device for stable and reproducible applications in both pre-clinic and clinic (see section 1 of the Results), and after preliminary assay demonstrating the stability of the plasma-treated PBS (or pPBS), under certain conditions, to use for in vitro experiments (see section 2 of the Results and Annexe of the second paper), we here addressed a thorough study of chemical stability of different pPBS for optimal production and uses. This study, presented in a manuscript hereunder, is under final review by authors before its submission.

My contribution to this study, as the second co-author, consists in the coordination of the experiments between the LPGP and the Gustave Roussy Institute (including the planning and the management of materials) in the in vitro experiments performed at the Gustave Roussy Institute (conceptualisation, methodology, data acquisition and validation, data analysis and visualisation, data interpretation), and in writing (original draft preparation, review and editing of the whole paper).

Introduction

Over the last decades, plasma pharmacy has been an emerging research field within plasma medicine, especially when it comes to cancer treatment 1-3 . Numerous publications show that plasmatreated solutions (PTS) can be an effective anti-cancer agent against different types of tumor cells, including ovarian cancer, cervical cancer, pancreatic cancer, glioblastoma, colon cancer and melanoma, both in vitro and in vivo 4-9 .

Several cold atmospheric pressure plasma (CAPP) sources have been used for the treatment of liquids, including radiofrequency discharges, dielectric barrier discharges (DBD) and atmospheric pressure plasma jets (APPJs) 9-1213 . Furthermore, a variety of solutions have been subjected to plasma treatment aiming their transformation into efficient anti-cancer agents, namely water, phosphatebuffered saline (PBS), ringer lactate and cell culture media 12,14-19 The anti-cancer properties of such PTS are mainly due to the delivery of long-lived reactive oxygen and nitrogen species (RONS) that can be generated in or transferred into the liquid phase 17 . Despite their complex chemical composition, it is now widely accepted that hydrogen peroxide (H2O2) is the main species responsible for their cytotoxic effects. However, synergistic effects with nitrite (NO2 -), nitrate (NO3 -) and the pH of the PTS have also been reported 14,20,21 . Furthermore, some RONS (such as peroxynitrite (ONOO -), nitric oxide (NO), nitrogen dioxide (NO2) and hydroxyl radical ( • OH)) have been identified as responsible for the oxidation of phospholipid bilayers of the cell membrane upon contact with PTS, which could lead to cell membrane permeabilization 22,23 .

To be considered as effective anti-cancer agents, it is essential that these PTS maintain their anticancer properties over time. One key parameter for the conservation of PTS, as well as of any other agent, is the storage temperature. Shen et al. investigated the evolution of plasma-treated water when stored at 4 different temperatures (+25, +4, -20 and -80 °C) over 30 days in terms of H2O2, NO2 -and NO3 -concentrations as well as its bactericidal activity 24 . They found that the bactericidal ability of plasma-treated water increased with decreasing storage temperature. Besides, they also found that the concentrations of H2O2, NO2 -and NO3 -decreased over time for all storage temperatures except for -80 °C. Contrariwise, Judée et al., who used plasma-activated medium (PAM) to treat colon adenocarcinoma multicellular tumor spheroid (MCTS), found that, when the PAM was stored at +4 °C or -80 °C, it retained its genotoxic activity, which was not the case for the PAM stored at +37 °C or -20 °C16 . Thus, they suggested that H2O2 concentration in PAM remained stable during at least 7 days of storage at +4 °C and -80 °C, while storage at +37 °C and -20 °C certainly decomposed H2O2. Yan et al. showed that while the H2O2 was stable in plasma-treated PBS stored at +8 °C and -25 °C for up to 3 days, its concentration was reduced as a function of storage time (and for the same storage conditions) when the PTS was cell culture medium 25 . These discrepancies between reports can be attributed to both the different types of plasmas used for the treatment of the solutions and to the different treated solutions. Indeed, different working conditions such as gas flow rate, gas composition, treatment distance, room conditions (e.g. humidity and temperature) and high voltage amplitude result in the production/transfer of different reactive species in/into the liquid, the composition of which also plays a major role in its final complex chemistry 9,26,27 . Besides the fluctuation of the RONS concentration, the chemical reactivity of PTS is also highly dependent on the final pH of the solution after plasma treatment. The pH of the PTS could be one of the crucial parameters that may explain the choice of the different storage temperatures proposed by the aforementioned authors. Indeed, not only the acidic pH of PTS may contribute alongside RONS to their cytotoxic capacity, but it is also an important parameter for the stability of RONS 21,28 .

The present work is devoted to the determination of pertinent storage conditions to preserve the cytotoxic effects of plasma-treated PBS(Ca 2+ /Mg 2+ ) (pPBS). Thus, the chemical stability of pPBS was assessed here, in terms of the H2O2, NO2 -and NO3 -concentrations as a function of storage time and temperature for up to 75 days after production by plasma treatment. The chemical mechanisms leading to the degradation of these RONS in certain storage conditions of the PTS and the catalytic role of the acidic pH were also investigated. Finally, the impact of the appropriate conservation of PTS on their anti-cancer efficacy on two different cell lines was also assessed in vitro, by quantifying the viability and the membrane permeabilization of the cells following treatment with those pPBS. Indeed, understanding the physicochemical properties and the anti-tumor activity of PTS before and after the storage is crucial for their practical application as anti-cancer agents.

Materials and Methods

Unless otherwise specified, all the reagents for biological experiments were purchased from Life Technologies (Courtaboeuf, France) and all the reagents for the dosimetry investigations of RONS were purchased from Sigma-Aldrich (France).

Experimental setup

The conceptual view of the plasma reactor used in this study is depicted in Figure 1a. It is based on the Plasma Gun (PG) device, which was described previously in detail 29-31 Briefly, it consists of a high voltage electrode inserted inside a quartz dielectric tube, and driven by high voltage pulses with an amplitude of 11 kV (FWHM of ~2.3 μs) at a repetition rate of 2 kHz provided by a custom-made power supply. A second electrode is placed outside the dielectric tube and is connected to the ground. Thus, a coaxial dielectric barrier discharge (DBD) is formed. The working gas used was pure helium (Alphagaz 1 He, >99.9999%, type S11, Air Liquide, France) at a flow rate of 1 standard litre per minute (SLM), regulated by a digital flowmeter (Vögtlin Instruments, ALTO Instruments, France). The main plasma reactor, where the plasma is produced, is located inside the power supply. At the reactor's orifice, a flexible dielectric tube is mounted, containing a floating-potential electrode (conductive wire of ca. 1 mm 2 section). The dielectric tube connects the main reactor with a second plasma reactor. This second part of the plasma device, the applicator, is essentially a second coaxial DBD reactor that is made of a PTFE (polytetrafluoroethylene) body with the floating electrode placed in its centre and a grounded electrode on the outside. The outlet of the second plasma reactor has five micro-orifices (Øint = 800 µm), producing, thus, five distinct plasma jets covering a larger liquid surface area during the plasma treatment. Four of the orifices are disposed at the corner of a 4.2 mm square, with the fifth orifice located at the crossing of the square diagonals. The liquid treated by the plasma, here PBS(Ca 2+ /Mg 2+ ), is placed in wells of an x24 well plate and is connected to the ground thanks to a ring-shaped wire electrode (stainless-steel wire Ø = 1 mm) located at the bottom of the wells. The distance between the applicator orifices and the surface of the liquid was maintained at 4 mm thanks to a customized 3D-printed spacer. Between the electrode and the ground, it was placed a compensation circuit designed to impose a total target impedance equal to that of a human body standard model, as presented in our previous work 31 . This grants additional control of the process parameters (which always include the target impedance) and eases the possible future translation of the results to a direct plasma application on animal models and human patients.

Electrical characteristics

The applied voltage was measured with a high-voltage passive probe (Lecroy, PPE 20 kV, France), while the current passing through the solution and reaching the ring electrode was determined by measuring the voltage drop on the resistor in the compensation circuit with another high-voltage passive probe (Lecroy, PPE 6 kV, France). As shown in Figure 1b), the current pulse has an amplitude of 0.2 A and a width at half a maximum of ~1.5 μs. The current waveform exhibits a sharp current spike generated by the contact of the plasma plume with the liquid, followed by a broader component typically measured in the presence of conductive targets, but that is absent in the free jet operation 32 . Here, the compensation circuit is useful to monitor the real power delivered to the liquid solution.

pPBS production, mimicking solutions preparation and their storage procedure

The procedure followed to produce plasma-treated PBS(Ca 2+ /Mg 2+ ), or pPBS, is depicted schematically in Figure 1c. More precisely, 3 mL of Dulbecco's phosphate-buffered saline (PBS) containing Ca 2+ and Mg 2+ (DPBS, Ca ++ , Mg ++ , 14040-133), named PBS(Ca 2+ /Mg 2+ ), were placed in individual wells of 24-multiwell plates (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 142475, France) and treated by the plasma for 20 minutes (except if stated differently). Afterwards, the amount of water evaporated during the plasma treatment (ca. 700 μL) was compensated by adding 700 μL of sterile water. Subsequently, the pPBS was divided and placed into several Eppendorf tubes (0.5 mL each). On the day of the plasma treatment (D0), and within 2 hours after the PBS treatment, the absolute concentrations of H2O2, NO2 -and NO3 -(see Figure 2), as well as the pH value of the pPBS, were measured. Note that it has been verified that the RONS concentrations remain the same between immediately after () and a few hours after the plasma treatment, if the pPBS is kept at +4 °C or + 20 °C. The remaining Eppendorf tubes were stored at different storage temperatures considered in this study: +20 °C, +4 °C, -20 °C and -80 °C. For the preparation of mimicking solutions, we used untreated PBS(Ca 2+ /Mg 2+ ), or uPBS, into which we added pure concentrations of H2O2, NO2 -and NO3 -as those measured in pPBS ([H2O2] = 3 mM, [NO2 -] = 1.5 mM and [NO3 -] = 0.75 mM). The pH of the mimicking solutions was adjusted to the same value as that measured in pPBS. The concentration of the reactive species (H2O2, NO2 -and NO3 -) and the value of the pH were measured, both in pPBS and in mimicking solutions, after 1 (24 hours after the treatment at D0), 7, 14, 21 and 75 days of storage (Figure 1c). Finally, the degradation of the RONS and the modification of the pH due to the storage time or/and temperature were quantified with the initial values measured on the day of the plasma treatment (for pPBS) or the day of preparation (for the mimicking solutions), within 2 hours after it (D0).

Quantification of hydrogen peroxide (H2O2), nitrite (NO2 -) and nitrate (NO3 -)

The concentration of hydrogen peroxide in pPBS was determined using titanium oxysulfate, as previously described 9 . The absorbance was measured at 407 nm using a well plate reader (Infinite 627® M200 PRO Tecan, France). The quantification of nitrite and nitrate was performed using the nitrate/nitrite colorimetric assay kit (Cayman) according to the supplier's instructions, and the absorbance was measured at 540 nm using the same spectrophotometric system.

pH detection

The pH was measured in the pPBS using a SevenEasy™ pH meter S20 fitted with an InLab ® 639 Microelectrode (Mettler Toledo, France). For monitoring the pH during the freezing process (slow (by using isopropanol-filled containers) towards -80 °C, or fast (by liquid nitrogen) and then stored at -20 °C), two different electrophoretic color markers were used as pH indicators: bromophenol blue and thymol blue. While the first color marker changes from blue at pH 4.6 to yellow at pH 3.0, the second transitions from yellow at pH 2.8 to red at pH 1.2. Thymol blue was provided by Sigma-Aldrich, while Bromophenol blue was purchased from Bertin (France). The pH of the mimicking solutions (Figures 8 and13) was modified using hydrochloric acid.

Dismutation of H2O2 by pyruvate

The contribution of H2O2 to the degradation of NO2

-was quantified by using 5 mM and 50 mM of sodium pyruvate, a known H2O2 scavenger (Thermo-Fischer Scientific, France). The pyruvate was added to pPBS after the plasma treatment.

Cell culture

DC-3F Chinese hamster lung fibroblasts 33 and highly tumorigenic murine fibrosarcoma LPB cells 34 , all mycoplasma-free, were cultured as adherent cells in Minimum Essential Medium (MEM, 31095-029) and Roswell Park Memorial Institute 1640 Medium (RPMI Medium, 21875-034), respectively. All media were supplemented with 10% fetal bovine serum (FBS, F7524), 100 U.mL -1 penicillin and 100 mg.mL -1 streptomycin (15140-122). Adherent cells were spread at 37 °C in a 95% humidity atmosphere containing 5% CO2 (HERAcell 240i incubator CO2, Thermo Scientific, France) and passaged upon confluency (every two days at a 1:10 dilution or every three days at a 1:30 dilution) using TrypLE™ Express Enzyme solution (12604-013). Short-term cell viability for routine sub-culturing was assessed by the trypan blue exclusion dye method (Trypan Blue Solution, T8154) with a Countess™ II FL Automated Cell Counter (Invitrogen, France), considering only viable cells. Cells were regularly checked for mycoplasma contamination via polymerase chain reaction (PCR) (not detailed).

For all assays on those cell lines (cytotoxicity and cell membrane permeabilization), 300 µL of cell suspension at a density of 1.80 × 10 5 cells.mL -1 were used to seed each well of 48-well plates (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 150687, France), 24 hours before the experiment (i.e. at D-1), allowing to reach c.a. 80 to 90% confluence of the cell layer on the day of the experiment (D0). Just before any assay, the medium above the cells was gently removed by aspiration and the cell layer was washed twice with PBS(Ca 2+ /Mg 2+ ). For sham conditions, all procedures remained identical to pPBS conditions, but untreated PBS(Ca 2+ /Mg 2+ ) (non-exposed to the plasma), or uPBS, previously stored under the same storage conditions as the pPBS, was used instead.

Cell viability assessment by clonogenic assay

The cytotoxicity of pPBS, previously stored under different storage conditions, was determined employing the clonogenic assay for DC-3F cells. LPB cells do not form colonies and, therefore, the assessment of their viability could not be performed using this precise test. Adherent DC-3F cells were incubated with 200 µL of pPBS per well for 10 minutes (note that the pPBS put in contact with the cells was always at room temperature, and had just been unfrozen or warmed up a few minutes before, if applicable). Afterwards, pPBS was gently removed from the cell layer and cells in each well were detached by 100 μL TrypLE™ Express and suspended in its culture medium. A serial dilution of the single-cell suspensions was performed for cell cloning as previously reported 31 . Concretely, based on the total number of cells counted in the single-cell suspension in sham (for D0) or sham previouslystored at +4 °C (for D1 and D21), the single-cell suspension of every sample was diluted in complete medium at a final concentration of 125 cells.mL -1 in a final volume of 8 mL. Cells were then distributed in 3 wells of a 6-multiwell plate (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 140675, France) by pouring 250 cells in 2 mL per well. This seeding allows individual (and surviving) cells to form discrete colonies over 5 days of cell culture at 37 °C in a 5% CO2 humidified cell incubator. After 5 days of cell culture, colonies were fixed and stained with a solution of formaldehyde (3.7% vol/vol) containing crystal violet (0.2% wt/vol) in H2O. The number of clones for each condition was counted. Colonies containing more than 50 cells were counted as "normal size colonies", and those containing from c.a. 20 to fewer than 50 cells were counted as "small colonies". The viability was then normalized to the number of clones in the corresponding sham sample.

Cell membrane permeability assessment by flow cytometry analysis

The eventual permeabilization of the cells treated by the pPBS was investigated using the fluorescent nucleic acids stain YO-PRO ® -1 Iodide uptake (629.04 Da). The non-permeant YO-PRO ® -1 dye is commonly used as an indicator for cell membrane permeabilization. Indeed, YO-PRO ® -1 can enter the cell only when the cell membrane is permeabilized, and, once in the cells, it intercalates with nucleic acids, resulting in a strong green fluorescence signal which is detectable by flow cytometry. On the day of the experiment, the medium above the cells was removed and the cell layer was washed twice with PBS(Ca 2+ /Mg 2+ ). The two cell lines were then incubated for 1 hour with the mixture of pPBS (previously-stored under different storage conditions) and YO-PRO ® -1 Iodide at 2 µM (200 µL per well). After the treatment, the liquid above the cell monolayer was gently removed and the cells were harvested using TrypLE™ Express Enzyme dissociation (200 μL per well). The cell suspension was immediately analyzed by flow cytometry (C6 flow cytometer, BD Accuri, San Jose, California, US) with 10 000 events recorded per sample within a selected gate that excludes cellular debris. The YO-PRO ® -1 uptake (cell permeabilization) was evaluated using green fluorescence channels (excitation 488 nm, emission 530/30 nm). More precisely, we assessed the median of the fluorescence intensity per cell (an indication of the average level of permeabilization of the cells).

Statistical analysis

Unless otherwise specified, all the results corresponding to the chemical composition of the pPBS are expressed as mean values ± standard deviation (SD) of three independent experiments. All the results corresponding to the effect of the pPBS on the cancer cells are expressed as mean values ± SD of three independent experiments, with each time point in at least four independent samples (quadruplicate). The significance level, or p-value, was calculated using the Student's t-test. Statistical significance levels found between the values of different groups were associated with p-values of > 0.05 (NS), < 0.05 (*), < 0.01 (**), < 0.001 (***) and < 0.0001 (****).

Results

RONS (H2O2, NO2 -and NO3 -) degradation as a function of storage time and temperature

As previously observed 9 , the anti-cancer properties of PTS are mainly due to the combined action of H2O2, NO2

-and NO3 -, and in some cases also with the acidic pH 21 . Thus, the chemical stability of these RONS in the plasma-treated PBS(Ca 2+ /Mg 2+ ), hereafter termed pPBS, was assessed as a function of the storage time and temperature. The concentration of these reactive species in pPBS within 2h from plasma treatment (D0) is depicted in Figure 2. At first, the degradation of H2O2 in the pPBS was investigated (see Figure 3). When the pPBS was stored at room temperature (around +20 °C) or +4 °C, no significant degradation of H2O2 was observed for up to 75 days of storage concerning the initial concentration obtained just after the plasma treatment (at D0, Figure 2). Also, no significant difference was observed between these two storage temperatures, in terms of H2O2 stability over time. On the contrary, when the pPBS was stored at -20 °C or -80 °C, the concentration of H2O2 decreased over time, even after just 1 day of storage (24 hours after the treatment at D0). The maximum degradation was measured after 7 days of storage, when the concentration of H2O2 was found to be ~20% and ~30% lower than its initial concentration when stored at -80 °C and -20 °C, respectively. No further significant degradation was observed after this period of storage (>D7). However, it must be stressed that the difference (~10% points) between these two storage temperatures (-20 °C and -80 °C) is statistically significant. As previously reported 31 , also in the present experimental conditions, the concentration of RONS (H2O2, NO2 -and NO3 -) in the pPBS increases almost linearly with the plasma treatment time (see Figure 4). To evaluate the effect of the total concentration of these reactive species on the degradation of H2O2, PBS(Ca 2+ /Mg 2+ ) was plasma-treated for 1, 2.5, 5, 10 and 20 minutes and then stored at +4 °C and -20 °C (see Figure 5). These two storage temperatures were chosen because they induce the lowest and the highest degradation of H2O2 over storage time, respectively (see Figure 3). The ratio of the concentrations of H2O2 and NO2 -varies between 0.5 and 2 for the plasma treatment times considered here (see Figure SF1). Our results unveil, on the one hand, that, independently of the plasma treatment time, and, thus, of the initial concentrations of the RONS studied here, H2O2 is always significantly degraded when the pPBS is stored at -20 °C (~20-35% degradation, as compared to the initial concentration of H2O2 measured at D0), except in some cases for only one day of storage (D1) where there is no degradation. This degradation is more pronounced after 7 days (D7) than on the first day (D1) of storage. On the other hand, the concentration of H2O2 is stable (>85% concerning the H2O2 concentration at D0) over 21 days (D21), when the pPBS is stored at +4 °C. The concentration of H2O2 in % is calculated concerning the initial concentration of H2O2 measured on the day of the plasma treatment (D0), within 2 hours from it. For the 20 minutes plasma treatment time, the data are the mean ± SD of 9 (for +4 °C) and 17 to 18 independent experiments (for -20 °C), while for 10 minutes plasma treatment time, of 7 to 10 independent experiments (for both storage temperatures). For all the other plasma treatment times (1, 2.5 and 5 minutes), the data are the mean ± SD of 2 to 5 independent experiments (for both storage temperatures).

Although H2O2 has been described as a key player in pPBS-induced reduction of cancer cells viability 20 , its synergistic effect with reactive nitrogen species such as NO2 -has also been reported 9,21 . To conclude on the overall degradation of the pPBS as a function of the storage time and temperature, the concentration of these reactive nitrogen species was also monitored (see Figure 6). We found that both NO2 -and NO3 -are stable for up to 21 days when the pPBS is stored at +4 °C or +20 °C. For longer storage times (75 days), the concentration of NO2 -decreases by ~35-40% at these storage temperatures. As for H2O2, there is no significant difference between storing at room temperature (around +20 °C) or in the fridge (+4 °C). When the pPBS is stored at -20 °C, we observe a massive degradation of NO2 -, over ~90%, occurring even from the first day of storage. The degradation at -80 °C is also significant

Impact on the degradation of RONS of their initial concentration and the pH of the solution

To understand the mechanisms of the pPBS degradation due to storage time or/and temperature, we first isolated the reactive species considered in this work (H2O2, NO2 -and NO3 -) by preparing mimicking solutions of those species in PBS(Ca 2+ /Mg 2+ ) with concentrations equivalent to those produced by a 20 minutes plasma treatment ([H2O2] = 3 mM, [NO2 -] = 1.5 mM and [NO3 -] = 0.75 mM). Two different mimicking solutions were prepared: the first contained these reactive species concentrations in PBS(Ca 2+ /Mg 2+ ) of pH = 7.1, while the second contained the same reactive species concentrations in PBS(Ca 2+ /Mg 2+ ) of pH = 6.1 (equal to that of pPBS). For the following cases, there was no degradation over 21 days of storage at any of the storage temperatures studied here: 1) H2O2 alone, 2) NO2 -alone, 3) NO3 -alone, 4) NO2 -+ NO3 -, 5) Η2Ο2 + NO3 -in mimicking solutions of both pH = 7.1 and pH = 6.1 (see Figures SF2 (for 1), 2) and 3)), SF3 (for 4)) and SF4 (for 5))). In the case of H2O2 + NO2 - in mimicking solutions of pH = 7.1, presented in Figure SF5, the evolution over storage time at different storage temperatures of the reactive species is similar to the case of H2O2 + NO2 -+ NO3 -also at pH = 7.1 (see Figure 7). For these cases, the degradation over storage time of H2O2 in the mimicking solutions simulates its degradation in pPBS with a slightly delayed evolution, i.e., about -30% after 21 days instead of only 7 days of storage (see Figure 3). This time-shift is even more pronounced for the NO2 - degradation, as the effect of the storage temperature observed after the first day of storage for the pPBS is only observed after 75 days of storage for the mimicking solutions (compare Figures 6a and7). As so, the degradation of both H2O2 and NO2 -is similar in pPBS or mimicking solutions after 75 days of storage. Contrariwise, the degradation of the solutions containing H2O2 + NO2 -or H2O2 + NO2 -+ NO3 -, but at a pH = 6.1, simulates closely the degradation of the plasma-treated solution, as shown in Figures SF5 and8, respectively. Finally, mimicking PBS(Ca 2+ /Mg 2+ ) solutions with different concentrations of H2O2 or NO2 -and pH = 7.1 were also investigated (see blue and red points in Figure 7), to study the effect of these RONS initial concentrations on their degradation. On the one hand, we observe that for a doubled concentration of H2O2, the degradation of H2O2 is slightly smaller and the degradation of NO2 -slightly higher. On the other hand, with twice the concentration of NO2 -, the degradation of H2O2 is significantly higher and that of NO2 -significantly lower, especially when the solution is stored at -80 °C. This means that the stoichiometry of the reaction between H2O2 and NO2 -inducing their degradation should be of 1:1, as, after a 20 min plasma treatment, the concentration of H2O2 in pPBS is about double of that of NO2 -(see Figures 2 andSF1). As a whole, these experiments highlight the great importance of both the reactive species concentration and the pH of the solution on the degradation over time of these RONS concentration in pPBS. 3 and6) and in mimicking solutions of H2O2, NO2 -and NO3 -in PBS(Ca 2+ /Mg 2+ ) of pH = 6.1 (blue symbols), as a function of the storage time, when stored at -20 °C (squares) and -80 °C (down triangles). The concentration in % is calculated with the initial concentration of a) H2O2 or b) NO2 -measured on the day of preparation of the solutions (D0). For the mimicking solutions stored at -20 °C and -80 °C, the data are the mean ± SD of 3 and 2 independent experiments, respectively.

To underline the essential role of H2O2 on the NO2 -oxidation in pPBS, due to storage time and/or storage temperature, pyruvate, a known H2O2 scavenger, was used. PTS containing or not 5 mM or 50 mM of sodium pyruvate (added after the plasma treatment) were stored at -20 °C and the concentration of H2O2, NO2 -and NO3 -was measured after 1, 7, 14 and 21 days of storage (see Figures 9 for NO2 -and NO3 -and SF6 for H2O2). By adding pyruvate, we were able to scavenge most (5 mM, 90% reduction) or virtually all (50 mM, below the detection limit) of the H2O2 in pPBS and, thus, to reduce its concentration by at least 90% (Figure SF6). As depicted in Figure 9, the addition of pyruvate partially prevents the degradation of NO2 -. More specifically, the quenching of 90% of the H2O2 present in pPBS resulting from the addition of 5 mM pyruvate led to a decrease of the degradation of NO2 -from ~85-95% (without pyruvate) to ~35-50% (with 5 mM pyruvate) upon storage at -20 °C. This NO2 -degradation that is still observed when pPBS with 5 mM pyruvate is stored at -20 °C is due to the remaining concentration of H2O2. Indeed, according to Figure SF7, even such small concentrations of H2O2 can oxidize ~20-30% of the NO2 -. From Figure SF7, it can also be concluded that the reaction between H2O2 and NO2 -is not catalytic and, again, that its stoichiometry is 1:1. Collectively, the results with the mimicking solutions at pH = 6.1 (Figure 8) and those with the addition of sodium pyruvate (Figure 9) suggest that H2O2 is by far the main oxidizer of NO2 -in our experimental conditions of very low pH (~2-3), confirming previously published results on the nitrous acid oxidation in aqueous aerosols 35 . Finally, as shown in Figure 9, the concentration of NO3 -increases inversely proportional to the decrease of the concentration of NO2 -, both with and without pyruvate, highlighting that NO3 -is a product of the peroxidation of NO2 -.

Impact of the freezing rate on the degradation of RONS

As stated before, the degradation of the RONS studied here (H2O2, NO2 -and NO3 -) occurs mainly for storage temperatures requiring the freezing of the pPBS solution (-20 °C and -80 °C). To better understand the impact of the freezing process, the degradation of the chemical components of the solution as a function of its freezing rate was studied. As so, pPBS solutions were fast frozen by liquid nitrogen and then stored at -20 °C (see open squares in Figure 10), or slowly frozen in isopropanolfilled containers when stored at -80 °C (see full circles

Acidification of the solution during its freezing

The pH of pPBS was monitored as a function of the storage time and temperature. We found that the plasma treatment results in an acidification of the PBS(Ca 2+ /Mg 2+ ), from an initial pH of 7.1 (± 0.1) to a pH of 6.2 (± 0.5). This pH value stays relatively stable (± 0.3, see Table 1) independently of the storage time or temperature (note that for the cases of storage at -20 °C and -80 °C, the pH was measured after unfreezing the solutions of pPBS). Nevertheless, as previously reported 36 , the pH value of the solutions is affected during the freezing process. Thus, the variation of the pH of the pPBS during the freezing towards -20 °C and -80 °C was monitored. As depicted in Figures 12 andSF8, the pH of the still unfrozen solution changes throughout the first hour of freezing. Using two different pH indicators (bromophenol blue and thymol blue), the final pH value of the frozen pPBS is estimated to be around 2.5 and 0.75 for storage at -20 °C and -80 °C, respectively. The pH reaches values lower than 3 in less than 1 hour. Figure SF9 shows the acidification of untreated PBS(Ca 2+ /Mg 2+ ), uPBS, with different initial pH values (from 1 to 7), as a result of the freezing of the solutions. Both pH color markers indicate that freezing of uPBS, when its initial pH is around 6 or less, results in substantial acidification of the solution. Moreover, the acidification, due to freezing, is rather similar for the uPBS with an initial pH of 6 than the pPBS (compare Figures SF8 andSF9). Finally, the acidification of both uPBS for all the initial pH values considered here and pPBS is similar when stored at -20 °C or -80 °C. It is interesting to note that, after unfreezing the solutions, both pH color markers indicate that the initial pH is retrieved (Figure SF8), confirming the pH values measured with the pH meter (Table 1).

Table 1. pH of the pPBS measured on the day of the plasma treatment (D0), within 2 hours from it, and after storage at different temperatures (+20, +4, -20 and -80 °C) for different periods (1, 7, 14, 21 and 75 days). The data are the mean ± SD of 3 (for D1, D7, D14 and D21) or 2 (for D75) independent experiments. Having established that the freezing of the solution decreases its pH, regardless if it is uPBS or pPBS, we then studied the effect of this acidification on the concentration of the three RONS considered here. Figure 13 shows the concentration of H2O2, NO2 -and NO3 -6 hours after the preparation of mimicking solutions of PBS(Ca 2+ /Mg 2+ ) with different pH values and maintained at room temperature. As it can be seen in Figure 13, we should consider a pH value of 5 as a lower threshold for the stability of these reactive species. For pH values lower than 5, the acidic environment of the solution results in the degradation of H2O2 and favors the oxidation of NO2 -and its partial transformation into NO3 -. These data are coherent with both the calculated pH values of the frozen pPBS solutions determined with the help of the pH indicators (~2-3) and our measurements of the concentration of the reactive species in pPBS stored at -20 °C or -80 °C. Indeed, similarly to after storage at -20 °C or -80 °C, at a pH of ~2-3, we observe a massive degradation of the NO2 -and a modest degradation for the H2O2. The degradation of NO2 -presented in Figure 13 is even greater than after storing at -20 °C or -80 °C. This might be due to the higher temperature of the solutions considered in Figure 13(room temperature), which could favor the reactions of degradation of H2O2 and NO2 -.

Application on cancer cells

Cell viability post pPBS treatment

The cytotoxicity of the pPBS stored in different conditions was studied on adherent DC-3F cells for a treatment time (time of incubation in pPBS) of 10 minutes, using the clonogenic assay. The results are presented in Figure 14 and summarised in Table 2. Firstly, we observed that the pPBS is highly effective in reducing the viability of the cancer cells, even after only 10 minutes of treatment. Considering a total colonies count (normal and small colonies), the treatment of DC-3F cancer cells with pPBS at D0 (freshly produced and maintained at room temperature for a few hours) reduced their viability by 60 % (see Figure 14 and Table 2). Regarding the normal size colonies, there is only 15% of cell viability (or 85% of cytotoxicity) after treatment with freshly prepared pPBS (D0). Subsequently, the ability of pPBS to preserve its anti-cancer capability was evaluated when stored for up to 21 days at +4 °C and -20 °C. Our results show that, even after 21 days of storage at +4 °C, the pPBS fully retained its cytotoxic capacity (only ca. 14.0 to 16.6% and ca. 40 to 50% of normal and total colonies are formed, respectively). Contrariwise, when the pPBS is stored at -20 °C, it is much less effective in inducing cancer cell death (even after only 1 day of storage). This loss of efficacy is more pronounced regarding the normal colonies than the small colonies and for longer storage periods. When using pPBS stored for 21 days at -20 °C, the cells retained their viability and formed 3-fold more colonies in normal size, as compared with those being treated with pPBS stored at +4 °C for 21 days or at -20 °C for 1 day. However, the reduction of the viability of small colonies does not exhibit a significant difference between the different storage conditions. It is interesting to note, though, that there is a 30-75% increase in the number of small colonies when using pPBS stored for 1 day at -20 °C compared to all the other treatment conditions, unveiling a transient cytostatic effect. As previously discussed, while the degradation of NO2 -is already massive after only 1 day of storage (85%), the decrease of the H2O2 concentration at D1 is not as significant (20-25%) and it is lower than after 21 days of storage (30-35%). Considering a total colony count, the cancer cells are highly resistant to the treatment with pPBS stored for 21 days at -20 °C (an increase of ca. 1.4 to 2.2-fold of total colonies, as compared with all the other treatment conditions). Collectively, these results show both a cytostatic and a cytotoxic effect of the pPBS, and are in full agreement with our observations regarding the concentration of the reactive species as a function of storage time and temperature. Indeed, at the storage conditions where H2O2 and NO2 -are stable (+4 °C), the pPBS preserves its cytotoxic capacity over time, while at the storage conditions where these reactive species are degraded (-20 °C), the pPBS becomes less effective against tumor cells, especially over time of storage.

Storage conditions

Normal colonies (%) Small colonies (%) Table 2. Cytotoxicity of pPBS stored at different storage conditions as a function of colonies formed after being treated with pPBS for 10 minutes and immediately cultured for 5 days at 37 °C in a 5% CO2 humidified cell incubator. Visible colonies were stained, counted and normalized to the number of clones in the corresponding sham, expressed as 100%. Colonies containing more than 50 cells were counted as "normal size colonies", and those containing from c.a. 20 to fewer than 50 cells were counted as "small colonies". The data are the mean ± SD of 3 independent experiments (each in quadruplicate).

Cell membrane permeabilization post pPBS treatment

The effect of pPBS stored in different conditions on the cell membrane permeabilization was also studied on two different types of cancer cell lines: DC-3F and LPB cells. The results are presented in Figure 15. First of all, we observed that the cellular membrane of both cancer cell lines is significantly permeabilized after 1 hour of contact with pPBS. This effect is in agreement with what our group has previously reported 31 . Contrary to the effect of the storage time and temperature on the viability of the cancer cells, here the pPBS stored at -20 °C is equally capable of permeabilizing the cells as those stored at +4 °C, for both cell lines and for all the different storage times studied. Interestingly, when the pPBS is stored for 21 days (both at +4 °C and -20 °C), it is still able to permeabilize the DC-3F cells but not the LPB cells. Indeed, even in conditions where the concentration of the RONS studied here is preserved (+4 °C), the pPBS is no longer effective in permeabilizing LPB cells after 21 days of storage. Collectively, these results unveil that the NO2 -, that is massively degraded upon storage at -20 °C but not at +4 °C, does not play an important role in the permeabilization of these cancer cell lines. This could mean that H2O2 is the main driver of the cellular membrane permeabilization due to pPBS. As the H2O2 in the pPBS is further degraded from 1 to 21 days of storage (+10 percentage points), its concentration at D21 might be under the threshold required for permeabilizing LPB cells. However, the presence of another reactive species in pPBS (that is not studied here), responsible for the permeabilization of LPB cells, which is degraded over 21 days of storage, cannot be excluded.

Discussion

The application of cold atmospheric-pressure plasmas (CAPPs) in cancer therapy has been lately one of the central research topics in the domain of plasma medicine. Two methods of plasma cancer treatment have mainly been used: direct and indirect plasma treatments 9,37 , each one with its advantages and drawbacks 21 . One of the main advantages of indirect plasma treatment is the possibility of production and storage of extensive quantities of plasma-treated solutions (PTS), minimizing the time and equipment constraints that direct plasma treatment requires. However, to be considered as efficient anti-cancer agents, these PTS should retain their anti-cancer effects over time. This study aimed to bring new insights on the chemical stability of plasma-treated PBS(Ca 2+ /Mg 2+ ), pPBS, when stored for a considerable time at different temperatures, and to determine whether the pPBS can preserve its anticancer properties over storage.

At first, the reactive oxygen and nitrogen species (RONS) that are mainly responsible for the anticancer effects of pPBS, i.e. H2O2, NO2 -and NO3 -9 , were monitored as a function of storage time and temperature. On one hand, we found that these three reactive species are relatively stable both at around +20 °C (room temperature) and +4 °C (fridge) for a significant period of storage (up to 75 days). Contrariwise, when stored at -20 °C and -80 °C, the absolute concentration of both H2O2 and NO2 - decreases significantly. This degradation is much more pronounced for NO2 -, and at -20 °C for both reactive species. On the other hand, we witnessed an increase in the concentration of NO3 -when the pPBS was stored at -20 °C or -80 °C. Regardless of the storage conditions, the total concentration of the reactive nitrogen species studied here, i.e. [NO2 -+ NO3 -], remains nearly constant. Thus, when the concentration of NO2 -decreases, that of NO3 -increases almost inversely proportionally. Similar behavior of the concentration of each of these three reactive species was also observed when, instead of pPBS, untreated PBS(Ca 2+ /Mg 2+ ), uPBS, containing the same concentrations of H2O2, NO2 -and NO3 - and the same pH as in pPBS (named mimicking solution) was stored at -20 °C and -80 °C (Figure 8). Interestingly, when the initial pH of the mimicking solution was not altered (pH of uPBS = 7.1, while pH of pPBS = 6.1), the degradation of H2O2 and NO2 -and the increase of NO3 -was much less profound (Figure 7). Additionally, excessive concentrations of H2O2 or NO2 -in the mimicking solution further degraded the NO2 -or H2O2, respectively. Finally, the addition of 5 mM or 50 mM of pyruvate, a known H2O2 scavenger, in pPBS after the plasma treatment partially prevented the degradation of NO2 -(Figure 9). Collectively, these results show that the main driver of the NO2 -degradation when the pPBS is stored at -20 °C or -80 °C is the H2O2 (which is also degraded), while a key role in that degradation is also played by the pH of the solution before its storage.

To investigate the role of the pH on the chemical stability of the pPBS, mimicking solutions were prepared containing the same concentrations of reactive species as in pPBS, but with different pH values, from highly acidic (pH = 1) to neutral (pH = 7). We found that the three reactive species studied here are stable for 6 hours for a pH greater than 5, while in more acidic environments, their concentration either decreases (strongly for NO2 -and moderately for H2O2) or increases (strongly for NO3 -). Similarly, Lukes et al. showed that,under acidic conditions (pH = 3.3), the concentration of H2O2 and NO2 -decreases during the post-treatment period (300 minutes), while the concentration of NO3 - increases 38,39 . As reported by Anbar in 1954 28 , the oxidation of nitrite (NO2 -) to nitrate (NO3 -), in the presence of H2O2, takes place rapidly when the pH of the solution is highly acidic (see equation 1). Another chemical reaction that could degrade NO2 -is its oxidation by dissolved oxygen that takes place also in acidic environments 40 . Nevertheless, we can consider that dissolved oxygen is not playing an important role in our experimental conditions as uPBS containing only NO2 -(without H2O2) did not exhibit any degradation upon storage at -20 °C or -80 °C, and uPBS containing both species and an adequate initial pH (6.1) simulate well the chemical evolution of pPBS. Interestingly, the pH of our PTS, pPBS, is much higher than that required for the NO2 -oxidation to take place. On top of that, as reported in other works, also in our case the pH appears to remain stable regardless of the storage period or temperature of the solution (Table 1) 41 . Given that the degradation of H2O2 and NO2 -is only observed, in our case, at the storage temperatures of -20 °C or -80 °C, one can wonder what are the mechanisms that are triggered by the freezing of the solution.

𝐻 2 𝑂 2 + 𝑁𝑂 2 -+ 𝐻 + → 𝑁𝑂 3 -+ 𝐻 + + 𝐻 2 𝑂 (1)
As it was previously reported, freezing of phosphate buffer solutions can result in the gradual decrease of the pH of the part of the solution that remains in the liquid state during the freezing process 42-44 . These pH variations in partially frozen solutions are dependent upon the eutectic temperatures and the concentration and solubility of its various components. Thus, as water is removed from the liquid phase (during freezing), the concentration of the various salts increases in the remaining solution. To verify these observations in our experimental conditions, two pH indicators (bromophenol blue and thymol blue) were added to uPBS of different pH values. Subsequently, these solutions (uPBS + pH indicators) were stored at -20 °C or -80 °C. Photographs of the Eppendorf tubes containing these solutions unveiled the gradual acidification of the uPBS when stored at both -20 °C and -80 °C (Figure SF9). The final pH value of the frozen solutions was estimated to be around 2-3 for solutions of initial pH of 6 or less. When mimicking solutions of initial pH = 7 were frozen, their acidification was less significant (Figures SF8 andSF9). Similar results were obtained when pPBS containing the pH indicators were frozen (Figures 12 andSF8). Additionally, it is interesting to notice that no significant difference was observed regarding the solution acidification between storage at -20 °C and -80 °C, even if the acidification is more pronounced at -80 °C. Collectively, our results confirm that, when frozen, even uPBS becomes highly acidic. This acidification, which is related only to uPBS composition and to the storage temperature, and is likely not affected by the enriched chemistry of pPBS due to plasma treatment, favors, as previously discussed, the subsequent oxidation of NO2 -by H2O2 into NO3 -(see equation 1).

Having established that the storage of the solutions (uPBS or pPBS) at -20 °C or -80 °C leads to the degradation of H2O2 and NO2 -due to their acidification during freezing, we tried to explain the difference in this degradation observed between these two storage temperatures. Indeed, our results showed that, for all the cases studied here (both pPBS and mimicking solutions), the degradation of the solution is less significant when the freezing temperature is lower (-80 °C). It was previously reported that an important factor of the pPBS freezing that affects the concentration of the reactive species studied here (H2O2, NO2 -and NO3 -) is the freezing rate of the solution 40,45 . However, in our case, that degradation is not affected by the freezing rate (Figures 10 and11). Recently, though, Alpana et al. 42 reported that the eutectic temperature of Dulbecco's PBS is -23.7 ± 0.3 °C (similar values were also found by Han et al. 46 ). This means that when stored at -20 °C, the storage temperature at which the most considerable degradation of H2O2 and NO2 -is observed, the temperature of the solutions is above the eutectic point of the saline buffer solutions used in this work. Contrariwise, when the solutions are stored at -80 °C, their temperature is far below this point. Taking this into consideration and that the freezing of the solution results in an acidic pH (either at -20 °C or -80 °C), we propose that the oxidation of NO2 -by H2O2, and, thus, their mutual degradation, is favored at -20 °C compared to -80 °C because these reactive species co-exist for a longer time in an acidic pH in a solution partially frozen when stored at -20 °C than at -80 °C. Besides that, it has been determined that the rate constant of the reaction described in equation 2 decreases with decreasing temperature at low pH (around 2) 47 . Equation 2 also highlights that, as discussed before, the conversion of NO2 -into NO3 -by oxidation by H2O2 increases with the increase of the concentration of the reactants and the decrease of the pH. It is worth noting that both occur during the freezing of the pPBS.

-

𝑑[𝐻 2 𝑂 2 ]/𝑑𝑡 = -𝑑[𝑁𝑂 2 -]/𝑑𝑡 = +𝑑[𝑁𝑂 3 -]/𝑑𝑡 = 𝑘[𝐻 + ][𝐻 2 𝑂 2 ][𝑁𝑂 2 -] (2)
Two of the reactive oxygen and nitrogen species studied here (H2O2 and NO2 -) are reported as the main drivers of the anti-cancer properties of pPBS 9,21 . Thus, the impact of the pPBS storage time and temperature on its cytotoxic capacity was also studied. As a functional assay, clonogenicity or colony formation assay is a complete characterisation of the ability of a single cell to grow into a colony, hence mandatory to determine not only the cell reproductive death after treatment with pPBS, but also the effectiveness of the cytotoxic pPBS at the defined treatment time. Indeed, the assay detects all cells that have retained the capacity for producing a large number of progenies after treatment, which can cause cell reproductive death as a result of damage to chromosomes, apoptosis, etc. 31,48 . Moreover, the cytostatic effect of pPBS stored at different temperatures over different periods has also been considered by analysing small size colonies. Our results revealed that the pPBS retains its cytotoxic capacity on DC-3F adherent cells for at least up to 21 days when it is stored at +4 °C. Contrariwise, its efficacy is drastically reduced over storage time when it is stored at -20 °C. This difference between the two storage temperatures is more profound when considering normal colonies rather than small colonies. Collectively, these results are in good agreement with the data obtained with the chemical analysis of the solutions for the same storage conditions, as degradation of H2O2 and NO2 -is observed at -20 °C but not at +4 °C. Thus, for the pPBS to preserve its cytotoxicity over time, we propose it be stored at +4 °C and, most importantly, storage temperatures below 0 °C should be avoided.

Finally, direct plasma treatment of cells has been reported as being able to permeabilize the cell membrane 49-51 or, controversially, not being able to permeabilize the cell membrane 52 , highlighting the dependence on the cell line. On the other hand, as demonstrated in our previous study 31 , indirect plasma treatment leads to cell membrane permeabilization due to its potential oxidation of the lipids of the cell phospholipid bilayer by the RONS present in the plasma-treated solutions (here pPBS). A key player for the peroxidation of lipids is the concentration of H2O2 39,53 , and for their oxidation is RNS generated in the pPBS (here NO2 -and NO3 -) 22,54 . Also reported in our previous study and documented in the literature 55-57 , different cell types have different responses to oxidative stress, especially cancerous cells and non-malignant cells, undoubtedly resulting in their different behaviors against pPBS treatment. Our results on the cancer cells (DC-3F and LPB) permeabilization did not show any difference between the pPBS stored at +4 °C and -20 °C. For both cell lines, pPBS stored at both temperatures was able to permeabilize the cellular membrane at D0 and after 1 day of storage. On the contrary, after 21 days of storage, only DC-3F cells were permeabilized by pPBS stored at +4 °C or -20 °C. When stored for 21 days, the pPBS was not able to permeabilize LPB cells, even in conditions where the concentration of the RONS studied here is preserved (+4 °C). As discussed before, when pPBS is stored at -20 °C, we observe, on the one hand, massive degradation of NO2 -from the first day of storage (> -85%), while the degradation of H2O2 is, on the other hand, moderate but increasing with storage time (ca. -20-25% at D1 vs ca. -30-35% at D21). As so, while we can state NO2 -does not play an important role in the permeabilization of these cancer cell lines, we also propose that H2O2 could be the main driver of the cellular membrane permeabilization due to pPBS. Indeed, after 21 days of storage, the H2O2 concentration in the pPBS might not be enough anymore for permeabilizing LPB cells. Another explanation could be the presence of another reactive species in pPBS (that is not studied here), which is responsible for the permeabilization of LPB cells and is considerably degraded after 21 days of storage. Different reactive species might be responsible for the cellular membrane permeabilization due to lipid oxidation.

Conclusions

In this study, the efficacy of plasma-treated PBS(Ca 2+ /Mg 2+ ), pPBS, as an anti-cancer agent was investigated in terms of its chemical stability and preserved cytotoxic capacity, when stored at different temperatures over long periods. We found that when pPBS is stored at +4 °C, the concentration of the main reactive species responsible for its anti-cancer capacity, i.e., H2O2 and NO2 -, remains stable for up to 21 days of storage. In parallel, the cytotoxic activity of pPBS is also preserved for the same storage time if stored at +4 °C. On the other hand, we showed that when pPBS is stored at temperatures below 0 °C (-20 °C and -80 °C here) both the concentration of the aforementioned reactive species and the efficacy of pPBS in inducing cancer cells death is degraded rapidly. The freezing of pPBS results in the acidification of the solution, enabling the pH-dependent NO2 -oxidation by H2O2, which results in the degradation of both reactive species. However, it has been found that the freezing rate does not play a role in the acidification process. We conclude that pPBS can preserve their cytotoxic activity, at least for 21 days, if stored at 20 °C or 4 °C, providing a basis for practical application of plasma-treated PBS in cancer therapy. In the future, other storage conditions, such as light exposure and packaging, should be appraised. Finally, we also confirmed that pPBS can permeabilize the cellular membrane, and showed that it retains this ability regardless of the storage temperature. We exclude the role of NO2 - and propose H2O2 to be the main driver of the cellular membrane permeabilization due to pPBS. The pH was evaluated by using bromophenol blue as a pH marker and by comparing the RGB values of the tubes containing pPBS with the RGB values of tubes containing uPBS of well-known pH (Figure 13). The RGB values are the mean value of 3 independent points of the photos of each Eppendorf tube. The data are the mean ± SD of 3 independent experiments for storage at -20 °C. For storage at -80 °C, only one experiment was performed.

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In vivo studies of NTP and its combination with ECT

Those ongoing studies will be presented below in two separate sections (manuscript in preparation).

Animal models of all the in vivo experimentations respected the models and protocol described in section 1.2 of the Results (details will be presented in each experiment).

In vivo investigation into indirect plasma treatment on subcutaneous tumours

Aim of the study

This first in vivo study aimed at exploring the anti-tumour effectiveness of plasma-treated liquids as mono-therapy in mice bearing s.c. tumours, via intratumoural (i.t.) administration of the plasma-treated liquids.

Materials and methods

Unless otherwise specified, all the reagents for biological experiments were purchased from Life Technologies (Courtaboeuf, France).

Animal model

Inbred female immunocompetent C57Bl/6J mice were used in this experimentation. See the description above (section 4.1.) for details of this model and protocol.

Cancer cells culture for sub-cutaneous inoculation

Tumourigenic murine fibrosarcoma LPB cells, a methylcholanthrene-induced C57Bl/6 mouse sarcoma cell line [START_REF] Belehradek | Evolution of cell-mediated antitumor immunity in mice bearing a syngeneic chemically induced tumor. Influence of tumor growth, surgical removal and treatment with irradiated tumor cells[END_REF], were employed to establish subcutaneous tumours in our animal model. LPB cells were sub-cultured for at least two weeks before inoculation. The culture of LBP cells was described previously (Sklias et al., to be submittedsee chapter 3 of the Results) except for some specifications for cells used for inoculation in the animal. Cells were cultured as an adherent monolayer in RPMI 1640 Medium (21875-034)

supplemented with 10% heat-inactivated foetal bovine serum (FBS, F7524), 100 U.mL -1 penicillin and 100 mg.mL -1 streptomycin (15140-122). Adherent cells were spread in this complete RPMI medium at 37°C in a 95% humidity atmosphere containing 5% CO2 (HERAcell

Treatment protocol

When the mean tumour volume of all mice reached ca. 100 mm 3 , mice were randomised into four groups (Table 4) with eight to ten mice per group, depending on the experiments.

Intratumoural administration of PAM (Figure 35) was performed once a day for four days consecutively, at the same moment in the day, through an automatic syringe (controlled flow speed at 50 µL.min -1 ). All the PAM for treatment were freshly prepared on the day of injection.

The volume of administration of PAM was half of the tumour's volume measured before each treatment. Follow-up by tumour size monitoring with a digital calliper was performed three times per week for two weeks after the last treatment. The follow-up was performed until the endpoints.

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The treatment parameters of each group are shown in Table 4. The group tumour evolution is reported until the first animal of each treated group reached an end-point and was thus euthanised.

Groups

Results of in vivo monotherapy with plasma-treated liquids

The results of this in vivo pilot experiment studying PAM as mono-therapy are shown in Figure 36. We, unfortunately, did not observe differences such as tumour growth delays between the treated groups and the control. Those results raise the question of RONS scavengers present in cell culture media, such as pyruvate [START_REF] Kaushik | Responses of solid tumor cells in DMEM to reactive oxygen species generated by non-thermal plasma and chemically induced ROS systems[END_REF][START_REF] Sensenig | Non-thermal plasma induces apoptosis in melanoma cells via production of intracellular reactive oxygen species[END_REF][START_REF] Turrini | Plasma-activated medium as an innovative anticancer strategy: Insight into its cellular and molecular impact on in vitro leukemia cells[END_REF]. The follow-up was performed until the endpoints.

Results of tumour growth delay are displayed in Figure 37. Once again, as reported in the previous in vivo experiment with PAM, no noticeable slowdown of tumour growth delay was observed with mice treated with pPBS compared with sham. There is a slight tumour growth delay of two days with multijet pPBS (group 2) in comparison to sham (group 1). On the contrary, with the "diffuse pPBS" (group 3), there could be a tendency to accelerate the tumour growth, as compared with sham.

Key questions remain on whether PAM and pPBS are not an efficient anti-tumour treatment modality and on the reliability of other in vivo (published) studies showing the anticancer efficiency of those plasma-treated liquids being used alone as monotherapy, even if we are aware that each in vivo experiment has its experimental models and design, thus it is not obvious to compare results between different studies.

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In vivo investigation into the combined treatment with pPBS and ECT

Aim of the study

This study aims at achieving the same level of permeabilisation of tumour cells (and, therefore, the same efficacy of ECT) with less intense and, hence, more "comfortable" electrical pulses.

For this purpose, immunocompetent C57Bl/6J mice bearing s.c. LPB tumours were treated with the combination of pPBS and ECT under conditions that did not cause any toxicity and were already used in clinics (see section 4.1). These are the pPBS and the murine LPB fibrosarcoma cells utilised previously for in vitro studies, as described in our second article (Chung et al., 2020) and in our third article (Sklias et al., to be submitted) (see chapter 2 and 3 of Results).

Treatment parameters

Subcutaneous LPB sarcoma tumours were treated either with i.v. BLM (group 1 Control)

or with ECT (group 2, which received i.v. administration of BLM in the retro-orbital sinus followed by eight square-wave EPs of 100 µs, at 1000 V/cm and 1 Hz) or with pPBS combined with ECT (group 4 and group 5) or with 1-hour-pPBS or 20-minutes-pPBS followed by i.v. BLM (no electric pulse, groups 3 and 6, respectively). The tested parameters in the six different treatment groups are described in Table 5. Note that there were five experimental groups in the first in vivo experimentation (group 1 to group 5), and in the second in vivo experiment, there were six experimental groups.

Groups

Preparation of different pPBS

Sterile PBS (Ca 2+ , Mg 2+ ) 1X was treated continuously with NTP multijet either for twenty minutes (resulting in the "20-minutes-pPBS") or for one hour (resulting in "1-hour-pPBS"), using the protocol established in our previous study (Chung, 2020) (see chapter 2 of the Results). The pPBS were prepared a day before the treatment and stored at +4°C, as described in our previous study (Sklias et al., to be submitted -see chapter 3 of the Results). Note that for the preparation of the 1-hour-pPBS, the compensation of water evaporation (and thus the maintenance of the original osmolarity of the liquid) was performed during the production of the pPBS, i.e., 700 µL of water were added to the plasma-treated PBS every twenty minutes.

EP generator and electrodes

Electroporation was performed using the electric pulse generator Cliniporator™ (IGEA, Carpi, Italy), delivering HV pulses and LV pulses. Eight square-wave EPs of amplitude over a distance ratio of 1000 V/ cm, a duration of 100 µs at 1 Hz, were used. Non-invasive stainlesssteel plate electrodes (P30-8B, IGEA, Carpi, Italy) consisting of two metallic plates 1 mm thick and 4.7 mm apart were employed. Voltage was set up according to the distance between the two plates of the electrodes. Thus 470 V for the plate electrodes, distant 4.7 mm, was used in this study.

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Experimental procedure

Before treatment, mice were anaesthetised with Isoflurane (Isoflurin ® 1000 mg.g -1 , Axience, FR) in an induction chamber. For all groups receiving pPBS, an i. to fill the space between electrodes and the tumours [START_REF] Ivorra | Use of conductive gels for electric field homogenization increases the antitumor efficacy of electroporation therapies[END_REF].

The in vivo treatment protocol of the combined approach is displayed in Figure 38.

Statistical analysis and data handling

Data represents as mean ± SEM values. GraphPad Prism software (version 8.4.0, La Jolla, CA, US) was used for data handling, analysis and graphic representations (ongoing analysis).

Results of in vivo combined treatment with pPBS and ECT

Differences observed in anti-tumour effectiveness were evaluated by tumour growth delay and the probability of tumour cures achievement. The tumour growth delay of the first

RESULTS

-125 -in vivo experiment is shown in Figure 39. The efficacy of the single-and combined treatments was compared with ECT as the gold standard. We did not observe a delay of tumour growth in groups receiving treatment either with pPBS alone (group 3) or with pPBS followed by ECT (group 5), as compared with the ECT (group 2). On the contrary, with the 1-hour-pPBS alone, there could be a tendency to accelerate the tumour growth, as compared with the Control until the occurrence of a given event, here from the one-shot treatment at D0 until the tumour size reaches the ethical limit (1800 mm 3 ). We observed a prolonged survival for mice in groups receiving combined treatments with pPBS and ECT, especially the one treated with the 1-hour-pPBS and ECT, compared with mice receiving single treatment with either pPBS or ECT.

However, there is no significant difference between those two groups on day 34 (group 4) and Following this first in vivo experimentation, we repeated the experiment with six groups (the group 6, i.t. administration of 20-minutes-pPBS followed by i.v. administration of BLM, is the control of group 5).

The tumour growth delay of the second in vivo experiment is shown in Figure 41. There are two types of responses to the treatments: a group of curves presenting all the treatments without µsPEF (groups 1, 3 and 6) and a group presenting the treatments with µsPEFs (groups 2, 4 and 5). We observed that monotherapy with pPBS (i.e., without ECT), either the 20-

RESULTS

-127 -minutes-pPBS (group 6) or the 1-hour-pPBS (group 3), decelerated the tumour growth for only two days, as compared with the control (group 1), thus no noticeable anti-cancer effect.

Interestingly, with the combined treatment employing pPBS followed by ECT, better antitumour effects were observed, tumour growth delay being four days in group 4 (1-hour-pPBS followed by ECT) and seven days in group 5 (20-minutes-pPBS followed by ECT). This displacement of tumour growth curves also reveals the differences in the eventual synergy of ECT and the two pPBS: anti-tumour synergy effectiveness was much more pronounced with the 20-minutes-pPBS combined with ECT than with the 1-hour-pPBS combined with ECT.

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The efficiency of the combined treatments can be observed for example, at 35 days posttreatment, where 88.9% survival was found in the group receiving the "20-min-pPBS + ECT" (group 5), followed by the group 4 that received "1-hour-pPBS + ECT" with 77.2% survival, as compared with 33% survival in mice receiving ECT (group 2). Interestingly, there are two mice in group 4 (combination of 1-hour-pPBS and ECT) with complete tumour regression since day three post-treatment and three mice in group 5 (combination of 20-min-pPBS and ECT) with complete tumour regression since day nine post-treatment. After a hundred days of followup, their tumour volume remained zero (complete regression); those mice were thus considered cured (tumour cure). Indeed, when a mouse has a complete tumour regression (tumour volume is zero) for more than sixty days, the tumour is considered cured.

The cure rate was 22.22% for group 4 and 33.33% for group 5, at a field amplitude for the ECT lower than usual (which results in no cure if applied in the absence of the pPBS). In conclusion, with combined treatments employing pPBS followed by ECT at a µsPEF amplitude

RESULTS

-129 -of 1000 V/cm, a longer tumour growth delay and a higher curability rate of the tumours were obtained compared with ECT alone.

The survival rate based on the individual curve of tumour growth of each mouse is depicted in Figure 43.

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DISCUSSION

This thesis sought to improve electrochemotherapy (ECT) through non-thermal plasma (NTP) medicine, i.e., to explore the combination of electric pulses and NTP.

In the first assessment, we attempted to obtain a stable helium NTP multijet device that could offer not only a large and homogeneous treatment surface, as compared with other NTP devices with single-jet, but also an easier translation from pre-clinical to clinical studies, i.e.,

an NTP system suitable for fundamental research as well as for medical uses. The second assessment consisted of two parallel investigations: (i) the dosimetry of plasma-treated PBS (pPBS) and the study of its different storage conditions for optimal in vitro and in vivo uses, and (ii) the in vitro study on combined treatment with pPBS and µsPEFs on two different cell lines. Finally, we investigated a combination of pPBS and ECT on small animals to elucidate the anti-tumour effect of the combined treatment and the underlying mechanisms of the effects observed. The treatment employed two different pPBS and ECT at low PEF amplitude (lower than that used in conventional ECT in clinics).

In the first article, I contributed by performing the first biological assays of the newly introduced multijet plasma device, both in direct NTP applications and in indirect applications (thus through the preparation of plasma-treated liquids). To get stable and reproducible plasma, a dialogue had to be raised with the engineers that developed the device to ameliorate some issues. Connexion to ground and introduction of a compensating circuit was created, and I tested them. It is perhaps worth noting that these developments had some similarities with impedance matching circuits used for higher frequency (RF) power supplies to minimise reflected power. We highlight that for the examples used in this paper, the compensating course was not used its primary purpose (use in direct NTP applications) but rather for maintaining consistent results between targets with different impedances. We In the second article, we attempted to determine new conditions for ECT, devoid of its side effects mainly due to intense electric pulses. We were interested in reducing the pulsed electric fields (PEFs) of the classical 100 microseconds pulses used in ECT without reducing the permeabilisation of the cell membrane. We investigated a combined treatment of µsPEFs with pPBS to outperform ECT in two different cell lines. We succeeded to enhance the cell membrane permeabilisation, both in terms of the percentage of permeabilised cells and the intracellular content of the used permeabilisation marker YO-PRO-1® Iodide, with reduced µsPEFs (500 V/cm instead of 1100 V/cm if combined with pPBS. This enhancement of the cell membrane permeabilisation level was cell-line dependent. Indeed, multiple research groups discussed the critical influence of both cell type and cancer type on the cell sensitivity to indirect plasma treatment [START_REF] Eline | Influence of Cell Type and Culture Medium on Determining Cancer Selectivity of Cold Atmospheric Plasma Treatment[END_REF]Kim et al., 2016;[START_REF] Tanaka | Plasma-activated medium selectively kills glioblastoma brain tumor cells by down-regulating a survival signalling molecule, AKT kinase[END_REF][START_REF] Wang | Cold Atmospheric Plasma for Selectively Ablating Metastatic Breast Cancer Cells[END_REF]Zucker et al., 2012). Compared to their non-cancerous counterparts, many cancer cell types seem to function with higher levels of endogenous oxidative stress (higher intracellular levels of ROS) in vitro and in vivo, making them more vulnerable to damage by further ROS encroachments produced by exogenous agents [START_REF] Trachootham | Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?[END_REF]. Besides, we have to consider the most common lipid component in animal cell membranes, cholesterol, which is fundamental for determining many structural properties of the plasma membrane and whose content ranges depending on the cell type. Indeed, as cholesterol is mainly responsible for the ordering of the lipids in the phospholipid membrane hence the membrane permeability [START_REF] Meyer | Effect of cholesterol on the structure of a phospholipid bilayer[END_REF][START_REF] Raffy | Control of Lipid Membrane Stability by Cholesterol Content[END_REF], a lower ratio of cholesterol to phospholipids increases the fluidity of the plasma membrane [START_REF] Van Blitterswijk | Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells[END_REF], making the phospholipid bilayer more susceptible to the oxidative stress which arises from the impinging RONS. These observations have also been predicted by molecular dynamic simulation (Delemotte, 2012;[START_REF] Tarek | Membrane electroporation: A molecular dynamics simulation[END_REF], especially for NTP treatment of cancer cells [START_REF] Van Der Paal | Effect of lipid peroxidation on membrane permeability of cancer and normal cells subjected to oxidative stress[END_REF]Yusupov, 2017). Interestingly, cancer cells have a significantly lower cholesterol concentration in their plasma membrane (ca. 2-fold lower) than in the phospholipid bilayer of their non-tumourigenic counterparts [START_REF] Shinitzky | Membrane fluidity in malignancy Adversative and recuperative[END_REF][START_REF] Van Blitterswijk | Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells[END_REF]. Even though to our knowledge, a precise characterisation of the cholesterol level in the membranes of the two studied cell lines has never been investigated, this could, at least in part, explain the different behaviour of the two cell lines to the combined therapy in our DISCUSSION -133 -investigation. Indeed, the origin of the B16 cells is a melanoma explanted from mice, while the DC-3F are spontaneously transformed Chinese hamster lung fibroblasts, the latter being perhaps closer to the normal cells than the former. This could explain the situation where the single pre-and post-treatment of the two studied cell lines with pPBS without any PEF applied has enhanced the percentage of permeabilised cells. This effect is significantly more pronounced in the case of the B16-F10 melanoma cells (significant 2-fold enhancement) than within DC-3F fibroblasts bearing the same treatment condition. Moreover, the increased penetration of RONS in cancer cells explained by the decreased cholesterol fraction of their cell membrane was also observed three years ago by the group PLASMANT in Belgium (Van Der Paal et al., 2017).

In the third article, we investigated the pH and chemical stability of pPBS in terms of the evolution of the concentration of H2O2, NO2 -and NO3 -, the main anti-cancer drivers of the pPBS effects, as well as the cytotoxic and cytostatic effects of the pPBS, and its capability to permeabilise the cell membrane. Two different cell lines were investigated, and the different studied pPBS were stored at different temperatures (ranging from -80°C to +21°C) for different storage times (up to 75 days). One of the most important differences between using plasmatreated liquids and direct plasma application is that any direct effect of UV radiation or electric fields on the biological target can be avoided using plasma-treated liquids. We could thus focus on the generated ROS and RNS in the pPBS used for our in vitro and in vivo investigations, which were precisely analysed in this third article. Understanding the chemical essence of the reactions between RONS and the intracellular molecules would help understand the underlying mechanisms of NTP treatment in cancer therapy. pPBS stored at +20°C (room temperature) or +4°C was revealed to preserve their cytotoxic activity, at least for 21 days.

Also, pPBS can permeabilise the cellular membrane and showed that it retains this ability regardless of the storage temperature. We excluded the role of NO2 -and proposed H2O2 to be the main driver of the cellular membrane permeabilisation due to pPBS.

With those encouraging in vitro results, an eventual combination of pPBS with electrochemotherapy (ECT) as a novel anti-cancer approach appeared to have significant potential and was therefore investigated in small animals. We firstly performed a pilot experiment investigating the anti-cancer efficiency of plasma-treated liquids as single anticancer therapy.

DISCUSSION

-134 -

The mono-therapy with PAM or pPBS intratumourally administered in the LPB tumour model was unsuccessful regarding the tumour growth delay. We firstly suspected the presence of an eventual RONS scavenger in the MEM 1X used to produce the PAM, as reported in the literature (Chance et al., 1979;[START_REF] Kaushik | Responses of solid tumor cells in DMEM to reactive oxygen species generated by non-thermal plasma and chemically induced ROS systems[END_REF][START_REF] Kondeti | Long-lived and short-lived reactive species produced by a cold atmospheric pressure plasma jet for the inactivation of Pseudomonas aeruginosa and Staphylococcus aureus[END_REF][START_REF] Tornin | Pyruvate Plays a Main Role in the Antitumoral Selectivity of Cold Atmospheric Plasma in -163 -Osteosarcoma[END_REF][START_REF] Utsumi | Effect of indirect nonequilibrium atmospheric pressure plasma on antiproliferative activity against chronic chemo-resistant ovarian cancer cells in vitro and in vivo[END_REF][START_REF] Wang | Pyruvate Protects Mitochondria from Oxidative Stress in Human Neuroblastoma SK-N-SH Cells[END_REF]. However, there is no pyruvate present in MEM 1X, according to the medium formulation from the producer's datasheet. There is no scavenger of RONS present in PBS (Ca 2+ /Mg 2+ ) used to produce the pPBS. There could be, on the contrary, other RONS scavengers present in MEM 1X that are not to our knowledge. Therefore, we were very curious about the potential efficiency of the pPBS (instead of the PAM) as anti-tumour mono-therapy and, in addition, about the reliability of other published in vivo studies showing the anticancer efficiency of those plasma-treated liquids. The same lack of efficacy was found with the pPBS.

Another possibility to explain the inefficiency of the i.t. PAM treatment alone in our experiments is the large size of the tumours that we treated (the mean tumour volume of mice in each experimentation was ca. 100 mm 3 ), i.e., tumours that may be considered advanced, hence not easy to be treated, even incurable. The response to treatment could also depend on the tumour type, as demonstrated by various studies. Groselj and colleagues, in their research entitled "Vascularization of the tumours affects the pharmacokinetics of bleomycin and the effectiveness of electrochemotherapy", showed in vitro and in vivo results of intrinsic effectiveness of ECT (conventional one) with BLM in two different cell lines (and thus two different types of tumours), and concluded that carcinoma was significantly more responsive to ECT than melanoma due to differences in tumour vascularisation, as carcinoma tumours had numerous well-distributed, small blood vessels, while melanomas were less vascularised, exhibiting predominantly larger vessels [START_REF] Groselj | Vascularization of the tumours affects the pharmacokinetics of bleomycin and the effectiveness of electrochemotherapy[END_REF]. Nevertheless, ECT is used in the clinics to treat almost all the types of tumour nodules, as all cells are permeabilised by electric pulses of appropriate electric field amplitude and bleomycin is effective on all dividing cells, whatever their origin. In our in vivo experiments combining pPBS and ECT, since we only used one type of tumour, the LPB tumour, we could not compare the treatment effectiveness regarding the tumour microenvironment. the LPB tumours volume for treatment was reduced, being smaller than that of the initial in vivo experiments (ca. 65-71 mm 3 versus ca. 100 mm 3 ). This was also for a homogenous tumour size amongst animals and a better observation of the combined treatment effects. For the second in vivo combined treatment, the control group (administration of BLM only) was constituted with one animal less than the other groups for two reasons: 1) under the experimental conditions followed during the present study, there was repeatedly no antitumour effect of the BLM administered alone [START_REF] García-Sánchez | Successful tumor Electrochemotherapy using Sine Waves[END_REF][START_REF] Miklavčič | The importance of electric field distribution for effective in vivo electroporation of tissues[END_REF]Mir, 1991a); 2) to reduce the number of animals used according to the ethics 3Rs' rule (replacement, reduction and refinement) [START_REF] Workman | Guidelines for the welfare and use of animals in cancer research[END_REF].

For in vivo investigations, often, the effect is not so noticeable. Besides, it is usually obscured by notorious variations of animals' responses. Moreover, in vivo experiments are typically carried out with no more than ten animals per group due to limited resources. In our investigations, all groups possessed from 8 to 10 mice per group, which is considered the correct group size to analyse an eventually relevant effect of the treatment. The estimation for statistically significant identification of the treatment effect was based on the tumourvolume measurements, and the probability of survival was employed.

In our preliminary in vivo studies of PAM and pPBS (section 4.2), we treated tumours from two cancer cell lines (B16-F10 melanoma and LPB fibrosarcoma) syngeneic in C57Bl/6J mice (the treatment was therefore carried out in the same genetic background). The first tumour is characterised by irregular vascularisation and heterogeneous cell organisation, and the second by organised vascularisation and homogeneously arranged cells. This might explain rapid tumour growth in melanoma B16-F10 tumours. Indeed, melanoma exhibits notorious resistance to standard therapies, and the results of adjuvant and especially the palliative treatment of melanoma are more than unsatisfactory.

It is worth mentioning the efficiency of ECT (alone) and the choice of the in vivo models.

Studies on immunocompetent and immunodeficient nude mice demonstrated that the host immune response is crucial for effective ECT with BLM (Mir, 1991a) and cis-Pt (Serša et al., 1997b). The growth delay induced by ECT in tumours was more significant in immunocompetent than in immunodeficient mice. The most important observation was that a tumour could be eradicated by ECT exclusively in animals with a normal immune response.

The importance of the immune system was also demonstrated by other studies in which DISCUSSION -136 -combined use of ECT with various types of immunotherapy resulted in potentiation of ECT effectiveness [START_REF] Calvet | The promising alliance of anti-cancer electrochemotherapy with immunotherapy[END_REF][START_REF] Mir | Electrochemotherapy tumor treatment is improved by interleukin-2 stimulation of the host's defenses[END_REF]Serša, 1997a).

In this in vivo study, we investigated the anticancer effect of two preparations of pPBS, the 20 minutes-pPBS and the one hour-pPBS, in combination with ECT at 1000 V/cm. This amplitude is 300 to 350 V/cm below the classical PEF amplitude in this kind of preclinical trial, and pulses at 1000 V/cm (+ bleomycin) are insufficient to obtain good anti-tumour effects (e.g., no cure). As discussed in state of the art, the electropermeabilisation of cells (the basis of ECT) involves rearrangements of the cell membrane structure by partial oxidation of membrane lipids. Radical reactions cause the oxidation of lipids, and NTP produces radicals (RONS), which are known to pass from the gas phase of the NTP to biological targets exposed to the plasma, here the PBS. A pre-treatment of the tumour with pPBS helped create radicals inside the tumour and in the tumour micro-environment, aimed to favour the electropermeabilisation of the tumour cells at a less intense electric field amplitude (therefore more "comfortable" for the patients in clinics). To respect the 3R rule (Replacement, Reduction and Refinement), the combination of pPBS and µsPEFs at different amplitudes was previously studied in cultured cells in chapters 2 and 3 of the Results (see the second article (Chung, 2020) and the third article (Sklias et al., to be submitted). We increase the rate of wholly cured animals by combining plasma-treated PBS with ECT at low electric field strength.

FURTHER PROSPECTS AND CHALLENGES -137 -

FURTHER PROSPECTS AND CHALLENGES

My thesis aimed to reduce the µsPEFs amplitudes using NTP as a complementary tool.

We succeeded in obtaining exciting results with low µsPEFs amplitudes, promising results for ECT. The in vivo combined treatment with a pre-treatment with pPBS followed by ECT at low µsPEFs has a significant delay in tumour growth and a considerable lifespan of animals compared with those treated with mono-therapy pPBS or ECT.

Theory somehow works, up to now, for in vitro studies investigating indirect NTP (pPBS)

with µsPEFs, with or without chemotherapeutic agents (bleomycin, paclitaxel, dacarbazine) [START_REF] Daeschlein | Enhanced Anticancer Efficacy by Drug Chemotherapy and Cold Atmospheric Plasma Against Melanoma and Glioblastoma Cell Lines In Vitro[END_REF]Wolff, 2020) but those in vitro results show variable anti-cancer responses with still unclear mechanisms of the effects observed. Moreover, the in vitro and in vivo protocols and strategies of the NTP approach, being as monotherapy or in combination with PEFs (or with ECT), are often study-dependant; thus, results are usually not easy to compare (different radicals, different concentrations of the radicals, depending on the NTP production system -also potential caveats with the thickness of the liquid treated by the NTP, in the time between exposure to NTP and application to the cells or animals, not speaking about the conditions for storage if the application is delayed, etc.).

To the best of my knowledge, our in vivo study combining indirect NTP strategy with ECT represents the first in vivo combined therapy in the field and successfully demonstrates a synergistic effect of plasma-treated liquids (here, the plasma-treated PBS) and ECT in vitro and even in vivo (even though, our experiments with mice require further confirmation). In particular, our approach allowed a decrease of the pulsed electric field amplitude used in ECT, i.e., 300 to 350 V/cm lower than that used for practical ECT in clinics, while offering a better treatment result than with the ECT alone. This novel combined approach would pave the way for promising targeted anti-cancer therapies.

In perspective, further confirmation of the last in vivo experimentation will be necessary. The challenges are still ahead for better control of the RONS delivery, understanding the process chain leading to the antitumoural effects, or optimising the combined plasma/(electro)chemotherapy approach.

Indeed, debate still goes on within the plasma science community whether a definition of "dose" should be applicable to perform NTP treatment, as it is available for other physicalbased therapies such as radiation treatment, phototherapy or laser therapy. Moreover, there still is no demonstration on which specific plasma component or plasma parameter would be responsible for a given specific reactive species, thus for a specific biomedical effect or therapeutic outcome. Subsequently, plasma impact on biological experiments and therapeutic applications is usually controlled via treatment time and energy supplied to the device. Because of the different technical setups of NTP devices under investigation or in medical applications, this has to be defined for every device specifically, and a generalisation is not possible. Identifying and defining such a parameter or set of parameters for deviceindependent control of biological plasma effectivity is possibly the biggest challenge in preclinical research in the plasma medicine [START_REF] Adamovich | The 2017 Plasma Roadmap: Low temperature plasma science and technology[END_REF].

Unlike electrochemotherapy, there is, to date, no standard operating procedure for plasma medicine, as it is the newest field of non-thermal plasma technology.

GLOSSARY & KEY TERMS

This glossary gives general definitions of terms used in this manuscript and provides an overview of cancer terms and medical terms related to oncology.

Ablation (also called ablative therapy): treatment that removes or destroys all or part of a body or tissue; can also be used to remove or halt the function of an organ (e.g., removing the ovaries or testicles or taking medicines that cause them to stop making their hormones).

Ablation may be performed by surgery, hormones, drugs, radiofrequency, extreme heat, freezing, irreversible electroporation or other methods.

Abnormal: not normal (a state, condition or behaviour that differs from what is considered normal). An abnormal lesion or growth in or on the body may be benign (not cancer), precancerous or premalignant (likely to become cancer) or malignant (cancer).

Abscess: an enclosed collection of pus in tissues, organs or confined spaces in the body. An abscess is a sign of infection and is usually swollen and inflamed.

Abscopal effect: a hypothesis in the treatment of metastatic cancer whereby the shrinking or disappearance of tumours in parts of the body that were not the direct target of local therapy, such as radiation therapy. It is thought that in the abscopal effect, the immune system is stimulated to fight cancer in the whole body as a result of local treatment. Learning more about the abscopal effect may help develop new cancer treatments, especially for metastatic cancer.

Actinic keratosis, also called solar keratosis (keratoses as plural noun): a rough raised area of skin that can develop after years of sun exposure. They are benign, but a few will develop into squamous cell cancer and can be removed with cold, lasers, chemicals, or creams. See also squamous cell carcinoma.

Acute: severe; sharp; begins quickly.

Adenocarcinoma: a benign growth starting in the glandular (secretory) cells. Glandular cells are found in tissue that lines specific internal organs and makes and releases substances in the body, such as mucus, digestive juices or other fluids. Most breast, pancreas, lung, prostate, and colon cancers are adenocarcinomas.

Adenocarcinoma in situ (also called AIS): a condition in which abnormal cells are found in the glandular tissue that lines specific internal organs, such as the uterus, cervix, lung, pancreas, and colon. Adenocarcinoma in situ, which occurs most often in the cervix, may become cancer and spread to nearby normal tissue.

Adenoma: a tumour that is not cancer and starts in gland-like cells of the epithelial tissue (thin layer of tissue that covers organs, glands, and other structures within the body).

Adenopathy: large or swollen lymph glands.

Adenosarcoma: A tumour that is a mixture of an adenoma (a tumour that starts in the glandlike cells of epithelial tissue) and sarcoma (a tumour that begins in bone, cartilage, fat, muscle,

Apoptosis: an active process of programmed cell death, characterised by visible shrinking cells, cleavage of chromosomal DNA, chromatin condensation, and fragmentation of both the nucleus and the cell. Blebbing of the cell membrane is often seen.

Autophagy: a genetically regulated form of programmed cell death in which the cell digests itself. Being caspase and p53 independent, autophagy is characterised by double-membrane vacuoles in the cytoplasm, which sequesters organelles such as mitochondria and ribosomes.

Autoradiography: the detection of radio-isotopically labelled molecules by exposure to X-ray film.

B cell (also called B lymphocyte): a type of white blood cell that makes antibodies. B cells are part of the immune system and develop from stem cells in the bone marrow.

B-cell lymphoma: a type of cancer that forms in B cells and may be either indolent (slowgrowing) or aggressive. Most B-cell lymphomas are non-Hodgkin lymphomas. There are many different types of B-cell non-Hodgkin lymphomas. These include Burkitt lymphoma, chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma. Prognosis and treatment depend on the type and stage of cancer.

Basal cell: a small, round cell found in the lower part (or base) of the epidermis, the outer layer of the skin.

Basal cell cancer (also called basal cell carcinoma): cancer that begins in the lower part of the epidermis (the outer layer of the skin). It may appear as a small white or flesh-coloured bump that grows slowly and bleeds. Basal cell cancers are usually found in areas of the body exposed to the sun. Basal cell cancers rarely metastasise (spread) to other body parts. They are the most common form of skin cancer.

Bench-to-bedside: a term used to describe how the results of research done in the laboratory are directly used to develop new ways to treat patients.

Benign (also called non-malignant): not cancerous.

Benign tumour: a tumour that is not cancer. It does not invade nearby tissue or spread to other parts of the body but remains confined to its site of origin.

Benign vascular tumour: a type of benign (not cancer) tumour that forms from cells that make blood vessels or lymph vessels. Benign vascular tumours may occur anywhere in the body, and a patient may have several tumours in different parts of the body. They may grow large and sometimes spread to nearby tissue. The most common type of benign vascular tumour is haemangioma, which usually occurs in infants and goes away independently.

Bilateral: affecting both the right and left sides of the body, e.g., bilateral Wilms tumour is cancer in both kidneys.

Bilateral cancer: cancer that occurs in both of a pair of organs, such as both breasts, ovaries, eyes, lungs, kidneys or adrenal glands, at the same time.

Biological therapy (also called biological response modifier therapy (BRM therapy), biotherapy): a type of treatment that uses substances made from living organisms to treat diseases. These substances may occur naturally in the body or be made in the laboratory. Some biological therapies stimulate or suppress the immune system to help the body fight cancer. Other biological therapies attack specific cancer cells, which may help keep them from GLOSSARY & KEY TERMS -170 -growing or killing them. They may also lessen specific side effects caused by some cancer treatments. Biological therapy includes immunotherapy (such as cytokines, cancer treatment vaccines, and some antibodies) and some targeted therapies.

Biomarker (also called molecular marker and signature molecule): a biological molecule found in blood, other body fluids or tissues that signify a normal or abnormal process or a condition or disease. A biomarker may be used to see how well the body responds to a treatment for an illness or condition.

Biopsy: the removal of cells or tissues for examination by a pathologist. The pathologist may study the tissue under a microscope or perform other tests on the cells or tissue. There are many different types of biopsy procedures. The most common types include (1) incisional biopsy, in which only a sample of tissue is removed; (2) excisional biopsy, in which an entire lump or suspicious area is removed; and (3) needle biopsy, in which a sample of tissue or fluid is removed with a needle. The procedure is called a core biopsy when a wide needle is used. When a thin needle is used, the process is called a fine-needle aspiration biopsy.

Biorefinery: a facility that integrates sustainable biomass processing into a spectrum of biobased products and bioenergy. It can also be defined as the optimised use of biomass for materials, chemicals, fuels and energy applications, where use relates to costs, economics, markets, yield, environment, impact, carbon balance and social aspects.

Blood-brain barrier (also called BBB): a network of blood vessels and tissue made up of closely spaced cells that help keep harmful substances from reaching the brain. The BBB lets some substances, such as water, oxygen, carbon dioxide, and general anaesthetics, pass into the brain but keeps out bacteria and other substances, such as many anticancer drugs. The BBB is formed by the tight interactions between the brain endothelium, surrounded by the basal lamina and stabilised by pericytes, glial cells and neurons.

Blood plasma: a "yellowish liquid" component of blood that makes up more than half (about 55%) of its overall content and contains nutrients, glucose, proteins, hormones, minerals, enzymes, and other substances.

Burden: the "load" carried by society expressed in terms of an observable fact -for cancer burden, this would be described in terms of cancer incidence or mortality.

Bypass: a surgical procedure in which the surgeon creates a new pathway for the flow of body fluids.

Cancer: an abnormal (continual and unregulated) proliferation of any of the different kinds of cells in the body.

Cancer cell: a cell that divides and multiplies uncontrollably and has the potential to spread throughout the body, crowding out normal cells and tissues.

Cancer cell line: cancer cells that keep dividing and growing over time, under certain conditions in a laboratory, and are used in research to study cancer biology and test cancer treatments.

Cancer continuum: a concept that describes the spectrum of cancer care from cancer biology and aetiology, prevention, early diagnosis and screening, treatment, survivorship care, palliative care and end of life care.

Cytokines: cell signalling molecules that are secreted in response to external stimuli. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors.

DNA vaccination: a technique for protecting an organism against disease by injecting it with a genetically engineered plasmid containing the DNA sequence encoding the antigen(s) against which an immune response is sought, so cells directly produce the antigen for an immunological response more safely than with viral vectors.

Doubling time (DT): defined as the time required to double the initial tumour volume, DT is a popular end-point of experimental tumour radiobiology (the faster a tumour grows, the smaller DT).

Drug resistance: the reduction in medication effectiveness to cure a disease or condition.

Electrochemotherapy (ECT): reversible electroporation that temporarily increases membrane permeability to enhance the uptake of chemotherapeutic agents in a tumour.

Electrofusion: the use of reversible electroporation to produce a fusion between cells.

Electrogenetransfer (also called electrogenetherapy or EGT): reversible electroporation to introduce plasmid DNA into living tissue cells. See Electrotransfer.

Electropermeabilisation: the generation of defects by pulsed electric fields (PEFs) or the longterm effect of electroporation.

Electrophoresis (from the Greek "ηλεκτροφόρηση" meaning "to bear electrons"): the migration and separation of charged particles (ions) under the influence of a spatially uniform electric field.

Electroporation: the generation of pores in the plasma cell membrane via pulsed electric fields (PEFs). This dynamic phenomenon is reversible or irreversible depending on the induced transmembrane voltage at the plasma membrane.

Electropulsation: exposure of cells to electric pulses, leading to their membranes' structural alteration and increased conductivity and permeability, thus allowing the transport of low-to non-permeant molecules across the membrane, bypassing the physiological limitations. Cell electropulsation comprises cell electroporation and cell electropermeabilisation.

Electrosurgery: application of an HF (radio frequency) alternating polarity, electrical current to biological tissue to cut, coagulate, desiccate or fulgurate tissue.

Electrotransfer: the movement of molecules into cells, by either passive diffusion or electrophoresis, made possible by using electric pulses. From this term, we derive DNA electrotransfer, RNA electrotransfer, PNA electrotransfer, etc.

Enhanced permeability and retention (EPR) effect: the mechanism resulting from pathophysiological processes (e.g., leaky tumour vasculature, poor lymphatic drainage and tumour microenvironment interactions) that leads to the accumulation and retention of nanoparticles or macromolecules in tumours.

Oedema: swelling due to build-up of fluid.

Fibropapilloma: a condition characterised by the presence of benign proliferative neoplasms containing superficial epidermal and subjacent dermal tissue.

Fibrosis: A proliferation of fibroblasts and collagen's deposition in tissues due to a reparative process (e.g., following injury, inflammation, and necrosis) or as part of a reaction to chronic irritants (e.g., pneumoconiosis, chronic lymphedema).

Hodgkin disease: a type of lymphoma where the cells in the lymphatic system abnormally reproduce, eventually making the body less able to fight infection. Steady enlargement of lymph glands, spleen, and other lymphatic tissue occur.

Iatrophysics (from Greek iatrós, "Arzt"; physis, "inanimate nature"): a 17 th and 18 th -century theory and practice of medicine that interpreted all phenomena of health and illness as dependent on the internal physical structure of the body, its external form, and mechanical alterability. With reductionistic simplification, it attempted to apply the findings of the new experimental natural sciences to the realm of life.

IFN-𝛄 (interferon-gamma): cytokine produced by activated T cells and natural killer cells, whose primary action is the activation of macrophages.

Immune system: the system composed of lymph fluid, lymph nodes, the lymphatic system, and white blood cells responsible for protecting the body against infection and disease.

Immunosuppression: a state in which the ability of the body's immune system to respond is decreased. This condition may be present at birth or caused by certain infections (such as human immunodeficiency virus or HIV) or cancer therapies, such as cancer cell killing (cytotoxic) drugs, radiation, and bone marrow transplant.

Immunotherapy: treatments that promote or support the body's immune system response to a disease such as cancer.

Induced transmembrane voltage (induced TMV): an increase in transmembrane voltage resulting from exposure to electric pulses and an increase in a transmembrane electric field.

Irreversible electroporation (IRE): the permanent permeabilisation state of a cell exposed to an electric field that induces the loss of homeostasis and apoptotic-like cell death. IRE is a predominantly nonthermal and adjacent structure-sparring ablation modality that has been proven safe and efficient in treating liver, pancreas, and prostate cancer.

Leukaemia: a blood cancer arising from the precursors of circulating blood cells in the bone marrow, consequently keeping the marrow from making normal red and white blood cells and platelets. There are four main types of leukaemia: acute myelogenous leukaemia (AML), chronic myelogenous leukaemia (CML), acute lymphocytic leukaemia (ALL), and chronic lymphocytic leukaemia (CLL). Leukaemia and lymphomas account for approximately 8% of human malignancies.

Lipids: hydrophobic molecules that function as energy storage molecules, signalling molecules, and the major components of cell membranes.

Locally invasive: a tumour that can invade the tissues surrounding it by sending out "fingers" of cancerous cells into normal tissue. It can cause problems by pressing on nearby tissues and organs, making them unable to work the way they should.

Loco-regional (adj): in medicine, restricted to a localised region of the body.

Lymphoma: cancer that starts in lymphocytes (a type of white blood cell) part of the immune system. Lymphoma cells can build up in lymph nodes and other lymph tissues. Lymphomas are grouped into two categories: Hodgkin lymphoma and non-Hodgkin lymphoma. Leukaemia and lymphomas account for approximately 8% of human malignancies.

Magnetic resonance imaging (MRI): a diagnostic procedure that uses a combination of large magnets, radiofrequencies, and a computer to produce detailed images of organs and structures within the body.

Malignancy (adj malignant) (also called cancer): a term for diseases in which abnormal cells divide without control and can invade nearby tissues. Malignant cells can also spread to other body parts through the blood and lymph systems. There are several main types of malignancy.

Carcinoma is a malignancy that begins in the skin or tissues lining or covering internal organs. Sarcoma is a malignancy that begins in bone, cartilage, fat, muscle, blood vessels or other connective or supportive tissue. Leukaemia is a malignancy that starts in blood-forming tissue, such as the bone marrow, and causes too many abnormal blood cells. Lymphoma and multiple myeloma are malignancies that begin in the cells of the immune system. Central nervous system cancers are malignancies that form in the brain and spinal cord tissues.

Margin: the edge or border of the tissue removed in cancer surgery. The margin is described as negative or clean when the pathologist finds no cancer cells at the edge of the tissue, suggesting that all cancer has been removed. The margin is described as positive or involved when the pathologist finds cancer cells at the edge of the tissue, suggesting that all cancer has not been removed.

Mean (the arithmetic mean, also called the expected value or average): a statistics and probability term, the central value of a discrete set of numbers. The mean is the sum of a set of numbers divided by how many numbers are in the set.

Median: a statistics and probability term, a value separating the higher half from the lower half of a data sample, a population or a probability distribution.

Median overall survival (also called median survival): the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that half of the patients in a group of patients diagnosed with the disease are still alive. In a clinical trial, measuring the median overall survival is one way to see how well a new treatment works.

Metastasis: the spread of cancer cells through the blood or lymphatic system, causing the development of secondary tumours in a part of the body that is far from the original primary cancer.

Metastasis-initiating cells: tumour cells that can survive and proliferate in distant metastatic sites.

Metastatic niche: microenvironment in distant organs that supports the survival and outgrowth of tumour cells.

Mitochondria: cytoplasmic organelles responsible for synthesising most of the ATP in eukaryotic cells by oxidative phosphorylation.

Mitotic cell death: the type of cell death which results from failure to arrest the cell cycle before or during mitosis in response to DNA damage with hallmarks including nuclear fragmentation and multiple micronuclei.

Mortality rate: Proportion of a population who dies from a particular disease in a specified time.

Myeloma: cancer that starts in the plasma cells of bone marrow. In some cases, the myeloma cells collect in one bone and form a single tumour (a plasmacytoma). In other cases, the myeloma cells contain in many bones and form many tumours (multiple myeloma).

Non-Hodgkin lymphoma: a type of lymphoma that causes the cells in the lymphatic system to abnormally reproduce, eventually causing tumours to grow. Non-Hodgkin lymphoma cells can also spread to other organs.

Non-thermal irreversible electroporation (NTIRE): an electroporation-based non-thermal tissue ablation method, leading to cell death either directly through excessive damage inflicted to cells and membranes (necrosis) or via apoptosis Oncogene: a gene capable of inducing one or more characteristics of cancer cells.

Oncogenesis: the maintenance and subsequent evolution of a tumour.

Oncology: a branch of medical science that focuses on disease, symptoms, treatment and other medical aspects of cancer.

Oxidative phosphorylation: the synthesis of ATP from ADP coupled to the energetically favourable transfer of electrons to molecular oxygen as the final acceptor in an electron transport chain.

p53: a transcription factor (encoded by the p53 tumour suppressor gene) that arrests the cell cycle in G1 in response to damaged DNA and is required for apoptosis induced by various stimuli.

Palliative care: an approach that improves the quality of life of patients and their families facing the problem associated with any life-threatening illness through the prevention and relief of suffering using early identification and impeccable assessment and treatment of pain and other issues, physical, psychosocial and spiritual. The primary purpose is to improve the patient's quality of life.

Phospholipid bilayer: the basic structure of biological membranes. The hydrophobic tails of phospholipids are buried in the interior of the membrane, and their polar head groups are exposed to the aqueous solution on either side.

Phospholipid transfer protein: a protein that transports phospholipid molecules between cell membranes.

Phospholipids: the principal components of cell membranes, consisting of two hydrocarbon chains (usually fatty acids) joined to a polar head group containing phosphate.

Plasma membrane: a phospholipid bilayer with associated proteins that surrounds the cell.

Plasmid: a small, circular DNA molecule capable of independent replication in a host cell.

Precancerous lesion: abnormal tissue changes in an early stage of cancer that could progress to invasive cancer if left untreated.

Prediction intervals: range of possible future observations that consider the random variation inherent in the past trends and the future prediction.

Prevalence: the number of people with the disease at a given time point. Cancer prevalence often refers to the number of people living with cancer who require care.

X-ray therapy: a type of radiation therapy that uses high-energy radiation from x-rays to destroy cancer cells and shrink tumours.

Xenobiotics: any substance (synthetic or natural) that is not naturally present in the body of an organism. Xerostomia: dry mouth (occurring when the body is not able to make enough saliva) Title: Enhancement of electrochemotherapy by non-thermal plasma-treated liquids: protocol design and proof of concept Keywords: cancer; electrochemotherapy; non-thermal atmospheric pressure plasmas (NTP); pulsed electric field amplitude; malignant melanoma; reactive oxygen and nitrogen species (RONS)

Xenograft

Abstract:

Electrochemotherapy (ECT) -an established method for the local eradication of cutaneous tumours based on electroporation technology -is widely used in Europe in human and veterinary medicine with very few systemic side effects. Non-thermal plasmas (NTP) are recently gaining momentum for application in oncology based on their rich content of short-lived reactive species as well as long-lived reactive oxygen and nitrogen species (RONS) which can be transferred to liquids being treated with NTP. We sought to combine those technologies to enhance the ECT capabilities and to better understand the contribution of RONS in those anticancer strategies.

As a multidisciplinary study at the crossroads of physics, chemistry, biotechnologies and medicine, this thesis aimed at emphasising the potent beneficial outcome of the combination of ECT and NTP medicine. A novel, stable source of helium plasma multi-jet (PMJ) has been utilised for in vitro investigations on three different cell lines (the DC-3F, B16-F10 and LPB cells), and for in vivo studies on nude and C57Bl/6 mice bearing subcutaneous tumours. We demonstrated that the pH of the plasma-treated PBS (pPBS) and the stability of the three main RONS (H2O2, NO2 -and NO3 -) under various storage conditions were crucial for the storage of plasma-treated liquids while maintaining their anti-cancer capabilities. In combination with ECT at an electric field amplitude lower than the one usually applied, those stable pPBS partially restored the ECT efficacy, delaying tumour growth and hence prolonging mice survival.

  et Thérapeutiques Anticancéreuses) situé à l'Institut Gustave Roussy (Villejuif, France), le GREMI (Groupe de Recherche sur l'Énergétique des Milieux Ionisés, UMR 7344, CNRS / Université d'Orléans, France) et le LPGP (Laboratoire de Physique des Gaz et des Plasmas, CNRS / Université Paris-Saclay, Orsay, France). Pour le projet, le GREMI a conçu et fabriqué un nouveau dispositif de plasmas froids multijet à hélium avec un circuit mis à la terre et compensé pour des utilisations stables en in vitro et in vivo. Une série d'expérimentation in vivo a été réalisée pour tester ces multijets de plasmas froids sur la peau de deux lignées de souris (Nude et C57Bl/6J), appelant le traitement NTP directe. J'ai notamment étudié le NTP indirect, c'est-à-dire des liquides traités aux multijets de plasmas froids et utilisés par la suite comme un traitement anti-cancéreux pour différents modèles in vitro et in vivo. Les expérimentations de NTP indirect étaient notamment réalisées avec le PBS (Ca 2+ , Mg 2+ ) recevant différents temps de traitement par les multijets de plasmas froids, donnant différents « plasma-treated PBS » ou « pPBS ». En combinant ces pPBS avec les µsPEF, nous avons réussi à réduire l'amplitude des impulsions électriques in vitro et in vivo par rapport à celle utilisée dans l'ECT en clinique ou aux NTP en monothérapie, tout en maintenant l'efficacité anti-cancéreux. De plus, en in vivo le traitement combiné les pPBS et ECT a amélioré l'efficacité de l'ECT. La stabilité, les composants et le pH des liquides traités aux plasmas froids dans différentes conditions de stockage ont également été étudiés afin d'établir les meilleures conditions de production et de stockage où ces liquides pourraient être préparés de manière reproductible et stables pour différentes utilisations. Toutes ces études dans le cadre de la nouvelle approche combinant le NTP et les µsPEF ont contribué à compléter la panoplie des thérapies anticancéreuses. Pendant trois ans, ma thèse a donné lieu à la publication de deux articles scientifiques, la soumission d'un troisième article, un article en préparation, et diverses communications scientifiques lors de conférences et ateliers scientifiques nationaux et internationaux listés cidessus.

Figure 1| Figure 2| Figure 3| Figure 4| Figure 5| Figure 8| Figure 9| Figure 10| Figure 11| Figure 13| Figure 14| Figure 15| Figure 16| Figure 17| Figure 18| Figure 19| Figure 20| Figure 21| Figure 23| Figure 24| Figure 25| Figure 26| Figure 27| Figure 28| Figure 29| Figure 30| Figure 31| Figure 36| Figure 37| Figure 38| Figure 39| Figure 42| Figure 43| Figure 44|

 1|2|3|4|5|8|9|10|11|13|14|15|16|17|18|19|20|21|23|24|25|26|27|28|29|30|31|36|37|38|39|42|43|44| Figure 1| Three discoveries in physics that have revolutionised modern medicine ........... -13 -Figure 2| Portrait of Pierre and Marie Curie and a photograph of one of the first radiotherapy units in clinics ...................................................................................................... -15 -Figure 3| The physical principle of electroporation on a cell .............................................. -21 -Figure 4| Schematic of different effects on cells exposed to electric fields, including the consequent thermal effects ................................................................................ -22 -Figure 5| Molecular dynamic simulation of the electropulsation phenomenon in the lipid bilayer .................................................................................................................. -25 -Figure 6| Different methods employed for in vitro detection of plasma membrane electroporation.................................................................................................... -26 -Figure 7| Some electric pulse delivery units ........................................................................ -27 -Figure 8| Different fixed geometry electrode configurations are available for ECT of different nodules with different depths, sizes, and shapes ............................................... -28 -Figure 9| Schematic illustration of non-thermal plasma ..................................................... -29 -Figure 10| Photographs of different NTP devices available for pre-clinical and clinical investigations ...................................................................................................... -35 -Figure 11| Example of wound healing treatment in clinics with NTP ................................. -48 -Figure 12| A photograph of a human spleen before and after thirty seconds of treatment with FE-DBD showing blood coagulates without tissue damage ................................ -49 -Figure 13| The burden of cancer (the WHO cancer report 2020 on global profile)............ -51 -Figure 14| The cancer control continuum ........................................................................... -53 -Figure 15| European code against cancer: 12 ways to reduce cancer risk ......................... -53 -Figure 16| Stage of metastatic progression. ........................................................................ -55 -Figure 17| The interactions of the tumour cell secretory pathway with different hallmarks of cancer .................................................................................................................. -58 -Figure 18| Classification of chemotherapeutic agents and their mechanism of action ..... -63 -Figure 19| Examples of some therapies currently under preclinical or clinical evaluation that might affect the cancer-immunity cycle ............................................................. -69 -Figure 20| Principle of electrochemotherapy...................................................................... -73 -Figure 21| Photos demonstrating a complete response to ECT in a patient with cutaneous metastasis from malignant melanoma ............................................................... -74 -Figure 22| ECT induced vascular lock on a haemorrhagic melanoma metastasis immediately after delivery of the electric pulses ..................................................................... -79 -

Figure 1|

 1| Figure 1|Three discoveries in physics that have revolutionised modern medicine (a) Giovanni Borelli's representation of the body as a mechanism in his De Motu Animalium: how muscles balance the body, and a load carried on the neck. "If the spine of a stevedore is bent and supports a load of 120 pounds carried on the neck, the force exerted by Nature in the intervertebral disks and the extensor muscles of the spine is equal to 25,585 pounds. The force exerted by the muscles alone is not less than 6404 pounds".[START_REF] Marquet | Iatrophysics to biomechanics. From Borelli (1608-1679) to Pauwels (1885-1980[END_REF]. (b) Abbé Nollet's experiment on animals and plants[START_REF] Nollet | Recherches sur les causes particulieres des phénoménes électriques et sur les effets nuibles ou avantageux qu'on peut en attendre[END_REF]. Nollet measured the change in weight of cats and small birds after placing them either near an electrified body or within an electrified cage. Electrified animals lost more weight than the controls[START_REF] Duck | The origins of medical physics[END_REF]. (c) Hand mit Ringen (Hand with Rings): a print of one of the first X-rays by Wilhelm Röntgen of his wife Anna Bertha Ludwig's left hand on November 8 th , 1895. This first-ever X-ray image was presented to Professor Ludwig Zehnder of the Physik Institut, University of Freiburg, on January 1 st , 1896. From Wikimedia Commons, the free media repository.

Figure 2|

 2| Figure 2|Portrait of Pierre and Marie Curie and a photograph of one of the first radiotherapy units in clinics (a) Pierre and Marie Curie, in their humble Paris laboratory in 1899, with Marie Curie proceed to the measurement of a radioactive sample. With the right hand, she lifts progressively the weight applied to the quartz; in the left hand, she holds the chronometer[START_REF] Adloff | THE LABORATORY NOTEBOOKS OF PIERRE AND MARIE CURIE AND THE DISCOVERY OF POLONIUM AND RADIUM[END_REF]. (b) One of the world's first megavoltage radiotherapy units at St Bartholomew's Hospital, London, UK, in 1937[START_REF] Keevil | Physics and medicine: A historical perspective[END_REF].

Figure 3|

 3| Figure 3|The physical principle of electroporation on a cell (a) The cell plasma membrane naturally has a native transmembrane potential difference or ΔψO (blue arrows) which value depends on the species and the cell type. Considering the cell as a sphere of a radius r, its plasma membrane as a very thin spherical dielectric bilayer shell, when electric pulses are applied, an induced transmembrane voltage Δψ i is created (orange arrows). The cell permeabilisation appears only on the part of the plasma membrane where the potential difference has been brought above its critical value of ca. 200-300 mV, i.e.,|Δψ O + Δψ i ≈ 200 -300 mV|. The Δψ i effect is position-dependent on the cell surface (dependent on the angular parameter Θ), hence, under an applied electric field, the side of the cell facing the anode (+) is hyperpolarised while the one facing the cathode (-) is depolarised. (b) The localisation and asymmetry of electropermeabilisation can be detected by propidium iodide uptake in a CHO cell submitted to a train of 10 electric pulses, 5 ms, 1 Hz at 700 V/cm. Adapted from[START_REF] Bernhardt | On the generation of potential differences across the membranes of ellipsoidal cells in an alternating electrical field[END_REF][START_REF] Escoffre | Electrochemotherapy: Progress and Prospects[END_REF] Kinosita, 1977a;[START_REF] Teissié | An experimental evaluation of the critical potential difference inducing cell membrane electropermeabilization[END_REF].

Figure 5|

 5| Figure 5|Molecular dynamic simulation of the electropulsation phenomenon in the lipid bilayer (a) Life cycle of an electrically induced pore in the lipid bilayer. For simplicity and clarity, only water molecules (H2O, represented by red and grey spheres) and phosphorus atoms of the polar fatty acids (P, represented by large green spheres) from the lipid headgroups are shown (apolar tails of phospholipids not represented) Stages of pore formation and closure are displayed in their order of appearance but disregarding the differences in their characteristic timescales. The formation of pores begins with the onset of the electric field (E), and closure begins as the field ceases (E = 0). Figure adapted from[START_REF] Kotnik | Membrane Electroporation and Electropermeabilization: Mechanisms and Models[END_REF][START_REF] Levine | Life cycle of an electropore: Field-dependent and fieldindependent steps in pore creation and annihilation[END_REF]. (b) Electrotransfer of a double-stranded siRNA (yellow and green spheres) through an aqueous pore within a model DOPC membrane (headgroups showed as blue and grey spheres and hydrophobic tails as cyan lines) driven by nanosecond electric pulses. When being submitted to a pulse of sufficient field strength, the siRNA is dragged by electrophoresis towards the membrane and several of the siRNA phosphate groups strongly associate with the cholines of the phospholipid headgroups. If the siRNA is close to an electropore (i.e., a field-induced hydrophilic pore), the siRNA can slide electrophoretically along this pore, against the field direction, being finally detached from the membrane phospholipids by electrophoresis. In the simulation, the translocation of a 23 bp siRNA can be completed within less than 10 ns under a transmembrane potential difference of 1.6 V. For clarity, water molecules are omitted, and the lipid headgroups initially in interaction with the siRNA strand are depicted in red to highlight their lateral diffusion.(Breton et al., 2012b;[START_REF] Mir | Electroporation-based gene therapy: Recent evolution in the mechanism description and technology developments[END_REF]

Figure 6|

 6| Figure 6|Different methods employed for in vitro detection of plasma membrane electroporation Abbreviations: TIRF -total internal reflection fluorescence microscopy, DEP -dielectrophoresis, SHG -second harmonic generation, FTIR -Fourier transform infrared spectroscopy. Graphic adapted from[START_REF] Napotnik | In vitro electroporation detection methods -An overview[END_REF].

The

  Cliniporator™ was produced by IGEA®, an Italian spin-off company of the University of Modena, in 2006 during the ESOPE project (European Standard Operating Procedures of Electrochemotherapy). The NanoKnife 3.0 Generator is a product of AngioDynamics, Inc. (New York, US), employing IRE technology for surgical ablation of soft tissue (US) and cell membrane electroporation (CE). The computer-controlled Cliniporator™ allows the setting and controlling pulse parameters on a screen and monitoring and storage of delivered current and voltage

Figure 7|

 7| Figure 7| Some electric pulse delivery units (a and b) The Cliniporator™ (IGEA® Clinical Biophysics, Carpi, Italy). (c) The NanoKnife 3.0 System (AngioDynamics, Inc., New York, US).

Figure 8|

 8| Figure 8| Different fixed geometry electrode configurations are available for ECT of different nodules with different depths, sizes, and shapes (a and b) Adjustable (a) linear and (b) hexagonal configuration needle electrodes, 10 to 30mm in length for treatment of lesions up to 3 cm deep (recommended voltage 400 V). (c) Hexagonal needle electrodes, 10 to 40mm in length (recommended voltage 730 V). (d) Plate electrodes, 30mm length (recommended voltage 960 V). (e and f) Finger electrodes with (e) perpendicular or (f) axial needles, 5 to 10mm in length (recommended voltage 400 V) for the treatment of small lesions in anatomical cavities (up to 1 cm deep). Figure adapted from "Electrodes and Accessories" of the IGEA® Clinical Biophysics (Carpi, Italy) and (Miklavčič et al., 2014).

Figure 9|

 9| Figure 9|Schematic illustration of non-thermal plasma (a) The four fundamental states of matter. The triangular tails represent the thermal motion strength of particles. Scheme adapted from(Yan et al., 2017). (b) Different components of non-thermal plasma (also called cold plasma) (scheme from www.terraplasma.com/en/cold-plasma/).

  kV direct current (DC) (positive and negative); 10-250 µA; dry or wet; O2, N2, Ar, He at 10 L.min -1

Figure 10|

 10| Figure 10|Photographs of different NTP devices available for pre-clinical and clinical investigations (a) The floating-electrode dielectric barrier discharges (FE-DBD), electrically safe for treatment where the target is the counter electrode, here a finger[START_REF] Fridman | Blood coagulation and living tissue sterilization by floatingelectrode dielectric barrier discharge in air[END_REF]. (b) The CE-certified argon plasma jet kINPen® MED developed by the Institute of Plasma Physics (INP) Greifswald and manufactured at Neoplas tools GmbH (Greifswald, Germany)(Isbary et al., 2013a). (c) Some NTP multijet devices developed by the GREMI (Orléans, France): a hand-held NTP multijet (left panel), and an NTP jet array consisting of two branches that deliver 13 and 21 secondary jets in ambient air over a grounded target. The full assembly is connected to a single plasma gun reactor flushed with a 2 slm helium flow rate[START_REF] Maho | Atmospheric Pressure Multijet Plasma Sources For Cancer Treatments[END_REF][START_REF] Robert | New insights on the propagation of pulsed atmospheric plasma streams: From single jet to multi jet arrays[END_REF].

Fig. 1

 1 Fig. 1 Electrically safe non-thermal e-plasma for treatment of living tissue without causing damage

(

  updated statistics from WHO Coronavirus Disease (COVID-19) Dashboard), the US. Food and Drug Administration (FDA) has announced to clear the Cliniporator™ platform for use in the upcoming Phase 1 trial investigating CORVax12, a DNA-encodable vaccine candidate for COVID-19. CORVax12 of the US. biotechnology OncoSec Medical Incorporated combines TAVO™ (tavokinogene telseplasmid), a DNA plasmid-based IL-12, with the National Institute of Health (NIH)'s SARS-CoV-2 virus "spike" protein. Furthermore, OncoSec announced that it had licensed the exclusive rights to the Cliniporator™ electroporation or gene electrotransfer platform from IGEA Clinical Biophysics, a license encompassing a broad field for gene delivery in oncology. The unique approach with CORVax12 delivered by the Cliniporator™ brings new hope to better protection against COVID-19 and other virus diseases.

Figure 11|

 11| Figure 11| Example of wound healing treatment in clinics with NTP Visualisation of (a to c) a 61-y-o patient with venous ulcers receiving a daily therapy of 2 min MicroPlaSter®: (a) wounds before plasma treatment, (b) after 7 and (c) after 11 treatments. At the beginning of NTP treatment, Klebsiella oxytoca and Enterobacter cloacae were detectable, after the 11 th treatment (23 days later) swabs were sterile. Adapted from (Heinlin, 2010) and (d) the MicroPlaSter® plasma torch (Microwave 2.46 GHz, 86 W; Ar 2.2 slm; distance to torch 2 cm) used in the treatment of a chronic infected wound (Isbary, 2010).

Figure 12| A

 12| Figure 12| A photograph of a human spleen before and after thirty seconds of treatment with FE-DBD showing blood coagulates without tissue damage

Figure 13|

 13| Figure 13| The burden of cancer (the WHO cancer report 2020 on global profile) (a) Estimated global burden of cancer and (b) distribution of cases and death by the leading 10 cancer types for both sexes in 2018 (World Health Organization, 2020).

  , clearly sets a global priority on cancer prevention. Primary prevention of cancer consists of modifying factors that increase the risk of cancer (Figure 15). Early detection aims to identify cancer or precancerous lesions as early as possible by one of two distinct approaches: early diagnosis or screening. The main diagnostic steps are assessing the anatomical extent of the disease and the cancer type and subtype. Standard cancer treatments are systemic surgery, therapy (e.g., chemo-, endocrine and targeted therapy), radiotherapy, nuclear medicine, bone-marrow transplantation and cancer cell therapy. Survivorship care is the provision of services after completion of cancer treatment and includes surveillance to identify diseases and their families. It is part of the management strategy for a cancer diagnosis. STATE OF THE ART Biophysical-based treatments in cancer therapy -53 -

Figure 14|

 14| Figure 14|The cancer control continuum Adapted from (World Health Organization, 2020).

Figure 15|

 15| Figure 15| European code against cancer: 12 ways to reduce cancer risk With technical support from International Agency for Research on Cancer (IARC) and the European Code against Cancer, the approach used in the European Union is a simple, clear, comprehensive, evidence-based set of 12 ways to reduce cancer risk (World Health Organization, 2020).

Figure 16

 16 Figure 16 highlights different stages of metastatic progression.

Figure 16|

 16| Figure 16| Stage of metastatic progression.

  Current conceptualisation identified the eight hallmarks of cancer or what are arguably necessary conditions to manifest malignant disease are (Hanahan et al., 2017): • Sustaining proliferative signalling: In physiological conditions, proliferation is regulated by growth factors produced in an autocrine, endocrine or paracrine STATE OF THE ART Biophysical-based treatments in cancer therapy -57 -fashion. Unlike noncancerous cells, cancer cells are endowed with extensive proliferation capacity by making their growth factors; • Evading growth suppressors: Unlike noncancerous cells, cancer cells may keep growing in the presence of inhibitory signals from the surrounding environment;

Figure 17

 17 Figure 17 displays the interactions of the tumour cell secretory pathway with different hallmarks of cancer, rationalising the complexities and variability at all levels of consideration of this neoplastic disease with yet unknown mechanisms.

Figure 17|

 17| Figure 17| The interactions of the tumour cell secretory pathway with different hallmarks of cancer Arrows indicate either positive (blue) or negative (orange) regulatory mechanisms linking the secretory pathway (SP) to the hallmarks of cancer. The question mark indicates an absence of information available in the literature on the relation between the SP and replicative immortality.Figure adapted from[START_REF] Dejeans | Addicted to secrete -novel concepts and targets in cancer therapy[END_REF][START_REF] Hanahan | Hallmarks of cancer: The next generation[END_REF].

Figure 18|

 18| Figure 18| Classification of chemotherapeutic agents and their mechanism of action Source: https://www.amboss.com/us/knowledge/Chemotherapeutic_agents

  Immunotherapy based strategies: ✓ Immune checkpoint inhibitors ✓ Immune cytokine-based strategies ✓ Tumour superantigen targeting and superantigen ligand targeting • Enzyme or small molecules strategies: ✓ Proteolysis targeting chimaera ✓ Antibody-drug conjugates ✓ Antibody-directed enzyme prodrug therapyTo design an appropriate targeted treatment protocol, almost all molecules are administered orally or intravenously, with molecular tests carried out before the treatment to determine the presence or absence of specific biomarkers in patients' tumours. To date, while being actively investigated for development, these targeted therapies have the US FDA approval for more than fifteen types of cancer, including the most common types, such as the cancers of STATE OF THE ART Biophysical-based treatments in cancer therapy -65 -breast, lung, prostate, colon, liver, kidney, bladder, and the colorectal carcinomas, the leukaemias and the lymphomas.

  transduction pathways in cancer cells are deregulated so that every cancer case itself poses new challenges for medicine. Signal transduction inhibitors, as their name indicate, block the activities of molecules that participate in signal transduction, the course by which a cell responds to signals from its environment. The signal is transmitted within the cell through biochemical reactions that ultimately produce the appropriate response(s). In some cancers, the malignant cells are stimulated to divide endlessly without being prompted by external growth factors -inappropriate signalling that signal transduction inhibitors can STATE OF THE ART Biophysical-based treatments in cancer therapy -66 -perturb or even suppress. Particular examples of FDA approved mAbs are the anti-human epidermal growth factor receptor 2 (HER2) antibody trastuzumab (Herceptin®) against breast and stomach cancer and the anti-CD20 antibody Rituximab (Mabthera®, Rixathon®, Truxima®) used for a variety of B-cell malignancies.

Figure 19|

 19| Figure 19| Examples of some therapies currently under preclinical or clinical evaluation that might affect the cancerimmunity cycle Abbreviations: CARs: chimeric antigen receptors; CTLA4: cytotoxic Tlympocyte antigen-4; GM-CSF: granulocyte macrophage colonystimulating factor; IDO: indoleamine 2,3-dioxygenase; IFN: interferon; IL: interleukin; PD-L1: programmed deathligand 1; VEGF: vascular endothelial growth factor. Although not illustrated, intratumoral T regulatory cells, macrophages, and myeloid-derived suppressor cells are key sources of many of these inhibitory factors (Chen, 2013).

Figure 23|

 23| Figure 23| Percutaneous image-guided ECT of spine metastases as a local palliative care option Study case of a 59-year-old woman with breast carcinoma who underwent percutaneous image-guided ECT of spine metastases. (A) A blastic tumour of the L2 vertebra (white arrow) was observed during pre-procedure CT tomography reconstructed in the sagittal plane. (B) Pre-procedural axial post-contrast T1-weighted MRI confirms the epidural extension of the tumour in the L2 vertebra. (C) Preprocedural sagittal post-contrast T1weighted MRI shows the metastases located on L1, L2, and L3 vertebra (arrow). (D) Following the individualised treatment plan and under the imaging guidance under cone-beam CT, six needles were inserted to cover the whole tumour. (E) Coronal reconstruction of the individualised treatment plan, allowing the production of a sufficiently intensive electric field encompassing the tumours. (F) Enhancement decreased on the sagittal post-contrast T1-weighted MRI performed the day after ECT (dashed arrow). (Cornelis et al., 2019).

Figure 24|

 24| Figure 24| Invasive electrodes developed for the treatment of deep-seated tumours (a) Individual needle electrodes for the treatment of bone or visceral lesions. (b) Retractable electrodes developed for the treatment of brain: Rendering of the electrode in the fully retracted state (left panel), electrode in the fully extended state. Recommended voltage is 1000 V for the fully extended electrodes (middle panel), diagram of the insertion into the brain (right panel). Figures a and b adapted from the "Electrodes and Accessories" of the IGEA® Clinical Biophysics (Carpi, Italy) and from (Linnert, 2012; Probst, 2018). (c) NanoKnife Single Electrode Activation Probe (15 or 25 cm insertion depth) from the NanoKnife 3.0 System (AngioDynamics, NY, USA).

Figure 25|

 25| Figure 25| The outcome of a carcinoma in a cat treated with ECT (A) White cat (whose race is highly prone to sun-induced malignancies) at presentation with a palpebral squamous cell carcinoma (SCC); (B) Patient after one session of ECT: the tumour mass is reduced and appears less vascularised; (C) Patient after two sessions of ECT, showing complete resolution of the palpebral neoplasm. Photos from (Spugnini et al., 2019) cited the treatment performed by D. Santos dos Anjos, DVM, MSc, PhD student (Unesp-Jaboticabal), Brazil, Mexico.

  Figure 26.

Figure 26|

 26| Figure 26| Timeline of clinical electrochemotherapy Figure adapted from (Campana, 2019a).

Figure 27|

 27| Figure 27| Photograph of the progress of a palliative care employing NTP treatment

  To date, there are four scientifically based plasma medical devices available on the market for clinical utilisation, with European Conformity (CE) certification as medical devices class IIa according to the European Council Directive 93/42/EEC[START_REF] Metelmann | Comprehensive clinical plasma medicine: Cold physical plasma for medical application[END_REF][START_REF] Privat-Maldonado | ROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy[END_REF]:• kINPen MED® (developedby the Institute of Plasma Physics (INP) and manufactured at Neoplas tools GmbH (Greifswald, DE)): the first CE-marked NTP jet for the treatment of chronic wounds as well as pathogen-induced diseases of the skin, skin appendages, extremities and body. This Argon plasma jet is generated by an RF powered (1 MHz) maximum power of 8 W (110/230 V, 50/60 Hz). • PlasmaDerm® (CINOGY GmbH, Duderstadt, DE) and PlasmaCare® (terraplasma medical GmbH, Garching, Germany): atmospheric air-driven DBD based on NTP technology which received CE approval for (chronic) wound-healing disorders such as acute wounds, venous and arterial ulcers, pressure sores, and the diabetic foot STATE OF THE ART Biophysical-based treatments in cancer therapy -87 -syndrome. The novel CE-marked PlasmaCare® is a hand-held, and battery-driven plasma device.

  STATE OF THE ART Biophysical-based treatments in cancer therapy -88 -(see the complete review of Trachootham et al., 2009 describing the cancer redox biology, which includes cell proliferation and angiogenesis).

Figure 28|

 28| Figure 28| Cancer redox biology: a biological basis for therapeutic selectivity (a) The vicious cycle of ROS stress in cancer development and progression. (b) The double-edged sword ROS.[START_REF] Trachootham | Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?[END_REF]

Figure 3 |

 3 Figure 3 | The vicious cycle of ROS stress in cancer.Reactive oxygen species (ROS) that are generated from extracellular or intracellular sources can cause DNA damage, which may activate wild-type p53 in normal cells and trigger stress responses and DNA repair to eliminate the ROS-mediated damage to genetic materials. However, in cancer cells with defective p53, ROS-mediated DNA damage would accumulate owing to compromised DNA repair function. In cancer cells with wild-type p53, ROS-mediated DNA mutations or deletion may cause a loss of p53 function, resulting in a defect in DNA repair leading to an accumulation of gene mutations and deletions. This would promote genomic instability leading to activation of oncogenes, aberrant metabolic stress, mitochondrial dysfunction and a decrease in antioxidants. All these events can further increase ROS levels, leading to more DNA damage and genetic instability. Such a vicious cycle can effectively amplify oxidative stress and promote genomic instability and cancer development.

  ROS generation in cancer cells as a means to kill these cells remains to be explored. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) seems to exert its cytotoxic effect against cancer cells, at least in part, by elevating the p67phox subunit of NOX 112 .

  PLASMANT (Antwerp, Belgium), lead by Dr A. Bogaerts, has performed several MD simulations to understand and explain the effect of NTP treatment on cancer cells. Firstly, Yan et al. proposed a mechanism for the possible selectivity of ROS towards cancer cells based on aquaporins (AQPs), i.e., the only in vitro verified H2O2 channels present at the cell membrane of cancer cells

Figure 30

 30 Figure30highlights recent understanding of molecular mechanisms involved in the effectiveness of NTP in cancer cells could be resumed in eight major steps[START_REF] Semmler | Molecular Mechanisms of the Efficacy of Cold Atmospheric Pressure Plasma (CAP) in Cancer Treatment[END_REF]:(1) While minimal amounts of RONS can diffuse through the cell plasma membrane (PM), an increase expression of different aquaporins (AQ) in cancer cells facilitates the intracellular transition of RONS;(2) In parallel, lipid peroxidation of the PM by free radicals leads to pore formation that further facilitates the diffusion of RONS into the cell. As discussed above, this effect may be enhanced in cancer cells due to reduced concentration of cholesterol in the PM;(3) Increased intracellular RONS interfere with calcium signalling (e.g., through interaction with inositol trisphosphate receptor [IP3-RR] and ryanoid receptor[RR]) resulting in increased calcium influx into cytosol; (4) Besides, RONS induced endoplasmic reticulum (ER) stress leads to a calcium influx into mitochondria, reducing the membrane potential (ΔψO), thus inducing mitochondria-dependent apoptosis; (5) NTP induced DNA double-strand breaks (DSB) -an effect that may not directly due to NTP but rather as a consequence of NTP induced apoptosis.DNA damage response includes activation of ATM, H2AX, p53, and p73; (6) Increased intracellular levels of RONS overwhelm the cell antioxidant system, hence reduce its protective capacity against oxidative stress; (7) The reduction of cell adhesion, migration, and invasion after NTP treatment may be explained by the reduction of integrins expression;(8) Notwithstanding the reported necrosis, apoptosis, and senescence induced processes as consequences of NTP treatment, little is known about their underlying pathways triggered by NTP treatment.

Figure 30|

 30| Figure 30|Overview of the current understanding of molecular mechanisms involved in the efficacy of nonthermal plasmas in cancer cells ATM: Ataxia-telangiectasia-mutated protein, H2AX: a histone H2A variant, MAM: mitochondria-associated ER membranes, NTP: non-thermal plasma. Scheme adapted from[START_REF] Semmler | Molecular Mechanisms of the Efficacy of Cold Atmospheric Pressure Plasma (CAP) in Cancer Treatment[END_REF]

Figure 31 :

 31 Figure 31:

Figure 31|

 31| Figure 31|Two basic strategies in plasma oncology (using a plasma jet source) (a) A direct NTP treatment of cancer cells in vitro or subcutaneous tumours in vivo. (b) An indirect NTP approach with the treatment of liquids will be used in vitro on cells or administered in tumours in vivo. Schematic illustration adapted from(Yan, 2017)

(

  pre-print in August 2019 and published in May 2020), introduced below (article #1).

  Manuscript receivedMay 23, 2019; revised July 26, 2019; accepted August 14, 2019. Date of publication August 21, 2019; date of current version May 1, 2020. This work was supported in part by the GdR 2025 HAPPYBIO and in part by the PLASCANCER Project (INCa-PlanCancer-n • 17CP087-00) aimed at investigating potential synergistic effects between plasma and pulsed electric field. (Corresponding author: A

Fig. 1 .

 1 Fig.1. Graphical representation of the proposed method to make in vitro and in vivo models look more similar to the reference substrate for plasma medical studies and the human body.

  Authorized licensed use limited to: Universita degli Studi di Bologna. Downloaded on November 08,2020 at 12:52:25 UTC from IEEE Xplore. Restrictions apply.

Fig. 2 .

 2 Fig. 2. Schematic summary of the investigated in vitro conditions for the PG impinging on a 24-multiwell plate filled with salted water.

Fig. 3 .

 3 Fig. 3. (a) Ground connection for the 24-multiwell plate. (b) NTP treatment on animal model on a grounded plate. (c) NTP treatment on a human fingertip. (d) Compensation circuit electrical scheme. (e) Photograph of the compensation circuit with dimensions.

Fig. 4 .

 4 Fig. 4. Voltage (V) and current (I) waveforms during direct treatment of water in 24-multiwell plates at different electrical potentials (FP or GR) and on different supports (dielectric or conductive). Gap distance 10 mm.

  Authorized licensed use limited to: Universita degli Studi di Bologna. Downloaded on November 08,2020 at 12:52:25 UTC from IEEE Xplore. Restrictions apply.

Fig. 5 .

 5 Fig. 5. PG (0.5 slm, 10 kV, 1 kHz, and 15 mm gap) emission intensity (expressed in arbitrary unit) for selected spectral lines and for different electrical conditions of an in vitro target (FP and GR) and a grounded human body. Acquisition were collected at different distances from the surface of the target.

  Authorized licensed use limited to: Universita degli Studi di Bologna. Downloaded on November 08,2020 at 12:52:25 UTC from IEEE Xplore. Restrictions apply.

Fig. 7 .

 7 Fig. 7. Voltage (V) and current (I) waveforms during direct treatment of human body and healthy mice with or without compensation circuit. Gap distance 15 mm.

Fig. 8 .

 8 Fig. 8. Voltage (V) and current (I) waveforms during direct treatment of human body and mice with grafted tumour and with or without compensation circuit. Gap distance 15 mm.

  experimentation. All animal experiments were performed in strict compliance with the ethical guidelines issued by the European Committee (Directive 2010/63/EU). Animals were handled in strict accordance with good animal practice. The study and experimental protocol (#2017_061_11278), reviewed by the Comité d'Éthique pour l'Expérimentation Animale CEEA 26, were approved by the registered ethics committee in animal experimentation of the Ministry under the identification code APAFIS#13409-2018020711382163.

Figure 33|

 33| Figure 33|In vivo NTP direct treatments on nude mice at 2 kHz, 11 kV for 3 minutes continuously Mice were treated with two different modes of plasma exposure: multijet mode (dplasma-skin = 5 mm, upper panel) and diffuse mode (dplasma-skin = 2 mm, lower panel), with at least two mice per condition. The potential skin effects due to thermal heating during plasma-surface interaction were monitored using an IR camera (FLIR i7) during the treatment and up to a minute post-treatment and by photographs of the treatment surface on mice skin at 30 seconds post-treatment and after one day and four days. The purple dashed circles indicate the skin zones exposed to the plasma.

34 .

 34 times 1 minute (1 or 2 minutes apart), once a day 3) Possibly 2 (or 3) minutes, once a dayThe NTP multijet source will be used to develop a new medical device for advanced Before in vivo studies on animals, we established in vitro protocols of a combination treatment with indirect plasma medicine and pulsed electric fields (PEFs) on two different cell lines (DC-3F Chinese hamster lung fibroblasts and B16-F10 murine melanoma). These in vitro studies aimed at outperforming in vitro cell electropermeabilisation, the basis of ECT, at an amplitude lower than that employed in conventional ECT (i.e., lower than 1300 V/cm).We used a novel helium plasma setup based on a Plasma Gun device for the aforementioned purposes. With the set-up developed and described in section 1 of the Results, I prepared plasma-treated liquids using PBS 1X (Ca 2+ , Mg 2+ ) for two different exposure times to the NTP multijet (so-called plasma-treated PBS or pPBS), which is used afterwards for in vitro and in vivo studies. A schematic illustration of the in vitro experimental set-ups and procedure of the combined treatment with pPBS and µsPEFs is displayed in FigureAfter preliminary results testing several conditions (data not shown), an established treatment protocol combining pPBS and µsPEFs was proposed. It significantly enhanced the cell permeabilisation level under very low electric pulse amplitudes. The design of these in vitro studies and their results with discussion and perspectives have been published in the journal Cancers (Basel) in January 2020, which is introduced below. A critical discussion of the paper with some hypotheses explaining the observed results is figured in the Discussion section. This paper paved the way for in vivo studies of plasma-treated liquids combined with ECT and new perspectives for their pre-clinical and clinical applications.

Figure 34|

 34| Figure 34|Schematic illustration of the in vitro experimental set-ups and procedure of the combined treatment with pPBS and µsPEFs A day before the treatment, cells were seeded and cultured as a monolayer on a defined area of 2 cm 2 of Ø 35 mm Petri dishes. On the day of the treatment, after being gently washed twice with PBS 1X (Ca 2+ , Mg 2+ ), cells were incubated at RT in pPBS in the presence of the YO-PRO®-1 Iodide (an indicator of membrane permeabilisation) and this pre-treatment time varied in different protocols. Afterwards, electric pulses were delivered to the cells (8 square-wave pulses of 100 µs, 1 Hz, amplitude ranging from 500 to 1400 V/cm) via two stainless steel parallel plate electrodes connected to the Cliniporator™. Cells were post-treated with a new treated liquid, always in the presence of YO-PRO®-1 Iodide, for a fixed incubation time of 10 minutes. After the post-treatment, the cell layer was harvested in TripLE Express solution, and the cell membrane permeabilisation was analysed by flow cytometry. Graphical abstract adapted from(Chung et al., 2020).

Figure 1 .

 1 Figure 1. Assessment of the electrical characteristics of the non-thermal plasma (NTP) multi-jet source during 20 min. treatment of PBS + /+ at +7 kV (a,c) and + 11 kV (b,d) pulses peak values. Current signals, averaged from 128 single recordings (a,b) and the evolution of the maximum current (c,d) were measured during the treatment process.

Figure 2 .

 2 Figure 2. Chemical analysis of the plasma-treated PBS (pPBS) prepared with the NTP multi-jet source using (a) NTP setup 1 and (b) NTP setup 2. (a) The evolution of the concentration of H2O2 in +7 kV-and +11 kV-pPBS as a function of the treatment time with the plasma, (b) Dosimetry of H2O2, NO2 -, and NO3 - levels in PBS + /+ treated during 20 min. at +11 kV. Data are presented as mean values ± SD of independent duplicates ((b): 1, 5, and 15 min.) or triplicates (all other points).

Figure 1 . 21 Figure 1 .

 1211 Figure 1. Assessment of the electrical characteristics of the non-thermal plasma (NTP) multi-jet source during 20 min. treatment of PBS + / + at +7 kV (a,c) and + 11 kV (b,d) pulses peak values. Current signals, averaged from 128 single recordings (a,b) and the evolution of the maximum current (c,d) were measured during the treatment process.

Figure 2 .

 2 Figure 2. Chemical analysis of the plasma-treated PBS (pPBS) prepared with the NTP multi-jet source using (a) NTP setup 1 and (b) NTP setup 2. (a) The evolution of the concentration of H2O2 in +7 kV-and +11 kV-pPBS as a function of the treatment time with the plasma, (b) Dosimetry of H2O2, NO2 -, and NO3 - levels in PBS + /+ treated during 20 min. at +11 kV. Data are presented as mean values ± SD of independent duplicates ((b): 1, 5, and 15 min.) or triplicates (all other points).

Figure 2 .

 2 Figure 2. Chemical analysis of the plasma-treated PBS (pPBS) prepared with the NTP multi-jet source using (a) NTP setup 1 and (b) NTP setup 2. (a) The evolution of the concentration of H 2 O 2 in +7 kVand +11 kV-pPBS as a function of the treatment time with the plasma, (b) Dosimetry of H 2 O 2 , NO 2 , and NO 3 levels in PBS + / + treated during 20 min. at +11 kV. Data are presented as mean values ± SD of independent duplicates ((b): 1, 5, and 15 min.) or triplicates (all other points).

Figure 3 .

 3 Figure 3. Relative concentration of H2O2, NO2 -and NO3 -in pPBS treated with NTP setup 2 at +11 kV during 20 min. and stored afterwards at +4 °C for up to 14 days. The reference (100%) is the concentration measured after plasma treatment at day 0. Data are presented as mean values ± SD of independent quadruplicates.

Figure 3 .

 3 Figure 3. Relative concentration of H 2 O 2 , NO 2 -and NO 3 -in pPBS treated with NTP setup 2 at +11 kV during 20 min. and stored afterwards at +4 C for up to 14 days. The reference (100%) is the concentration measured after plasma treatment at day 0. Data are presented as mean values ± SD of independent quadruplicates.

Figure 4 .

 4 Figure 4. Schematic illustration of the protocols applied in the combined treatments, with µsPEF at 0, 500, 600, 1100 or 1400 V/cm.

Figure 4 .

 4 Figure 4. Schematic illustration of the protocols applied in the combined treatments, with µsPEF at 0, 500, 600, 1100 or 1400 V/cm. Cancers 2020, 1, x 7 of 21

Figure 5 .

 5 Figure 5. Effects of the combined treatment on DC-3F fibroblasts at 0, 600, and 1100 V/cm. (a) Percentage of electropermeabilised cells and (b) intracellular fluorescence of YO-PRO ® -1 iodide entering the cells as a function of the 7 combined protocols applied. The data are presented as mean (for a) and median (for b) values ± SD of independent triplicates. Statistical differences were analysed while using One-way ANOVA followed by Bonferroni's multiple comparison test. * p < 0.05, ** p < 0.01, and **** p < 0.0001 significant differences. 2.3. Investigations of the Effects of the Combined Treatment on B16-F10 Murine Melanoma Cells 2.3.1. Comparison of the Effect of µsPEF at 600 V/cm versus 1100 V/cm on B16-F10 Cells

Figure 5 .

 5 Figure 5. E↵ects of the combined treatment on DC-3F fibroblasts at 0, 600, and 1100 V/cm. (a) Percentage of electropermeabilised cells and (b) intracellular fluorescence of YO-PRO ® -1 iodide entering the cells as a function of the 7 combined protocols applied. The data are presented as mean (for a) and median (for b) values ± SD of independent triplicates. Statistical di↵erences were analysed while using One-way ANOVA followed by Bonferroni's multiple comparison test. * p < 0.05, ** p < 0.01, and **** p < 0.0001 significant di↵erences.

2. 3 .

 3 Investigations of the E↵ects of the Combined Treatment on B16-F10 Murine Melanoma Cells 2.3.1. Comparison of the E↵ect of µsPEF at 600 V/cm versus 1100 V/cm on B16-F10 Cells

Figure 6 .

 6 Figure 6. Effects of the combined treatment on malignant B16-F10 melanoma cells using µsPEF at 0, 600, and 1100 V/cm. (a) Percentage of electropermeabilised cells and (b) intracellular fluorescence of YO-PRO ® -1 iodide entering the cells as a function of the seven combined protocols applied. Data are presented as mean (for a) and median (for b) values ± SD of independent triplicates. Statistical differences were analysed while using One-way ANOVA followed by Bonferroni's multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 significant differences. 2.3.2. Comparing the Effect of 500 V/cm versus 1400 V/cm µsPEF on B16-F10 Murine Melanoma Cells

Figure 6 .

 6 Figure 6. E↵ects of the combined treatment on malignant B16-F10 melanoma cells using µsPEF at 0, 600, and 1100 V/cm. (a) Percentage of electropermeabilised cells and (b) intracellular fluorescence of YO-PRO ® -1 iodide entering the cells as a function of the seven combined protocols applied. Data are presented as mean (for a) and median (for b) values ± SD of independent triplicates. Statistical di↵erences were analysed while using One-way ANOVA followed by Bonferroni's multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 significant di↵erences. 2.3.2. Comparing the E↵ect of 500 V/cm versus 1400 V/cm µsPEF on B16-F10 Murine Melanoma Cells

Figure 7 .

 7 Figure 7. Effects of the combined treatment on adherent malignant B16-F10 melanoma cells using µsPEF at 0, 500, and 1400 V/cm. (a) Percentage of electropermeabilised cells and (b) intracellular fluorescence of YO-PRO ® -1 iodide entering the cells as a function of the seven combined protocols applied. Data are presented as mean (for a) and median (for b) values ± SD of independent triplicates. Statistical differences were analysed using One-way ANOVA followed by Bonferroni's multiple comparison test. * p < 0.05, ** p < 0.01, ***, p < 0.001, and **** p < 0.0001 significant differences.

Figure 7 .

 7 Figure 7. E↵ects of the combined treatment on adherent malignant B16-F10 melanoma cells using µsPEF at 0, 500, and 1400 V/cm. (a) Percentage of electropermeabilised cells and (b) intracellular fluorescence of YO-PRO ® -1 iodide entering the cells as a function of the seven combined protocols applied. Data are presented as mean (for a) and median (for b) values ± SD of independent triplicates. Statistical di↵erences were analysed using One-way ANOVA followed by Bonferroni's multiple comparison test. * p < 0.05, ** p < 0.01, ***, p < 0.001, and **** p < 0.0001 significant di↵erences.

Figure 8 .

 8 Figure 8. Flow cytometry analysis of a few interesting conditions of the combined treatment in adherent B16-F10 murine melanoma cells to compare effects of protocol 6 (e-h) versus the control (a-d) with µsPEFs of 0 (a,e), 500 (b,f), 600 (c,g), and 1100 (d,h) V/cm. The peak of count as a function of the green fluorescence (FL1-A) shifts towards higher values of the fluorescence per cell, indicating the enhancement of the permeabilisation of the cell membrane.

Figure 8 .

 8 Figure 8. Flow cytometry analysis of a few interesting conditions of the combined treatment in adherent B16-F10 murine melanoma cells to compare e↵ects of protocol 6 (e-h) versus the control (a-d) with µsPEFs of 0 (a,e), 500 (b,f), 600 (c,g), and 1100 (d,h) V/cm. The peak of count as a function of the green fluorescence (FL1-A) shifts towards higher values of the fluorescence per cell, indicating the enhancement of the permeabilisation of the cell membrane.

4. 2 .

 2 Plasma-Treated PBS + / + Preparation Using NTP Multi-Jet Setups 4.2.1. Specifications of the NTP Multi-Jet Setup

Figure 9 .

 9 Figure 9. Schematic representation (a) and photo (b) of the NTP device with a multi-jet nozzle.

Figure 9 .

 9 Figure 9. Schematic representation (a) and photo (b) of the NTP device with a multi-jet nozzle.

Figure 10 .

 10 Figure 10. Schematic illustration of the electropulsation setup used in the combined treatment.Figure 10. Schematic illustration of the electropulsation setup used in the combined treatment.

Figure 10 .

 10 Figure 10. Schematic illustration of the electropulsation setup used in the combined treatment.Figure 10. Schematic illustration of the electropulsation setup used in the combined treatment.

Figure A1 .

 A1 Figure A1. Cytotoxicity of pPBS to adherent DC-3F cells. Survival curves of DC-3F cells that were plated immediately after being incubated with pPBS between 1 and 60 min. are presented as the number of viable colonies formed five days after the treatment. Results from three independent experiments (each in triplicate) are shown as mean values ± SD for each parameter set.

Figure A2 .

 A2 Figure A2. Enhanced electropermeabilisation of adherent DC-3F cells with pPBS treatment. Cells were pre-treated with pPBS for 10 min. and exposed to 8 pulses of 100 µs and 1100 V/cm at a 1 Hz repetition rate. The cell membrane permeabilisation was monitored by the fluorescence of the YO-PRO ® -1 iodide taken up by the cells. Data are presented as median values ± SD of independent triplicates. Statistical analysis used the One-way ANOVA followed by Bonferroni's multiple comparison test. **** p < 0.0001.

Figure A2 .

 A2 Figure A2. Enhanced electropermeabilisation of adherent DC-3F cells with pPBS treatment. Cells were pre-treated with pPBS for 10 min. and exposed to 8 pulses of 100 µs and 1100 V/cm at a 1 Hz repetition rate. The cell membrane permeabilisation was monitored by the fluorescence of the YO-PRO ® -1 iodide taken up by the cells. Data are presented as median values ± SD of independent triplicates. Statistical analysis used the One-way ANOVA followed by Bonferroni's multiple comparison test. **** p < 0.0001.

Figure 1 .

 1 Figure 1. a) Conceptual view of the experimental setup and the two reactors used in this study i) for the plasma production and ii) for the liquid treatment. b) Electrical characteristics of the non-thermal plasma multi-jet source: high voltage measured on the power supply (black dots) and current measured on the compensation circuit (blue line). c) Conceptual view of the experimental procedure employed for the treatment, storage and analysis of the pPBS samples.

Figure 2 .

 2 Figure 2. The concentration of H2O2, NO2 -and NO3 -in pPBS measured on the day of the plasma treatment (D0), within 2 hours after it. The data shown are the mean ± SEM of 34 independent experiments for H2O2 and 30 independent experiments for NO2 -and NO3 -.

Figure 3 .

 3 Figure 3. The concentration of H2O2 in the pPBS as a function of the storage time, when stored at four different temperatures: +20, +4, -20 and -80 °C. The concentration of H2O2 in % is calculated concerning the initial concentration measured on the day of plasma treatment (D0), within 2 hours from it. For up to 21 days of storage (D21), the data are the mean ± SD of 9, 9, 18 and 9 independent experiments for +20, +4, -20 and -80 °C, respectively. For 75 days of storage (D75), the data are the mean ± SD of 3 independent experiments for all the storage temperatures.

Figure 4 .

 4 Figure 4. H2O2, NO2 -and NO3 -absolute concentrations as a function of the plasma treatment time, measured on the day of the plasma treatment (D0), within 2 hours from it. The data for the H2O2 is the mean ± SEM of 18 or 10 or 5 independent experiments for 20 or 10 or 5, 2.5 and 1 minutes of plasma treatment, respectively. For NO2 -and NO3 -, the data are the mean ± SEM of 6 independent experiments.

Figure 5 .

 5 Figure 5. The concentration of H2O2 in the pPBS as a function of the plasma treatment time, when stored at a) +4 °C and b) -20 °C for 1 day (D1), 7 days (D7), 14 days (D14) and 21 days (D21).The concentration of H2O2 in % is calculated concerning the initial concentration of H2O2 measured on the day of the plasma treatment (D0), within 2 hours from it. For the 20 minutes plasma treatment time, the data are the mean ± SD of 9 (for +4 °C) and 17 to 18 independent experiments (for -20 °C), while for 10 minutes plasma treatment time, of 7 to 10 independent experiments (for both storage temperatures). For all the other plasma treatment times (1, 2.5 and 5 minutes), the data are the mean ± SD of 2 to 5 independent experiments (for both storage temperatures).

Figure 6 .

 6 Figure 6. The concentration of a) NO2 -and b) NO3 -in the pPBS as a function of the storage time, when stored at four different temperatures: +20, +4, -20 and -80 °C. The concentration in % is calculated with the initial concentration of a) NO2 -or b) NO3 -measured on the day of the plasma treatment (D0), within 2 hours from it. For NO2 -, the data are the mean +-SD of 8 and 3 independent experiments for up to 21 (D21) and 75 days of storage (D75), respectively. For NO3 -, the data are the mean +-SD of 6 and 2 independent experiments for up to 21 (D21) and 75 days of storage (D75), respectively.

Figure 7 .

 7 Figure 7. The concentration of a) H2O2 and b) NO2 -in mimicking solutions of H2O2, NO2 -and NO3 -in PBS(Ca 2+ /Mg 2+ ) of pH 7 .1, as a function of the storage time, when stored at four different temperatures: +20, +4, -20 and -80 °C. The blue and red points correspond to mimicking solutions containing 2*[NO2 -] and 2*[H2O2], respectively, stored at -20 °C (squares) or -80 °C (down triangles). The concentration in % is calculated with the initial concentration of a) H2O2 or b) NO2 -measured on the day of preparation of the solutions (D0). The data are the mean ± SD of 4 independent experiments for all experimental conditions, except for mimicking solutions containing 2*[NO2 -] and 2*[H2O2] (2 independent experiments) and mimicking solutions containing 1*[NO2 -] and 1*[H2O2] and stored for 75 days (D75) (3 independent experiments).

Figure 8 .

 8 Figure 8. The concentration of a) H2O2 and b) NO2 -in pPBS (black symbols; same data as in Figures3 and 6) and in mimicking solutions of H2O2, NO2 -and NO3 -in PBS(Ca 2+ /Mg 2+ ) of pH = 6.1 (blue symbols), as a function of the storage time, when stored at -20 °C (squares) and -80 °C (down triangles). The concentration in % is calculated with the initial concentration of a) H2O2 or b) NO2 -measured on the day of preparation of the solutions (D0). For the mimicking solutions stored at -20 °C and -80 °C, the data are the mean ± SD of 3 and 2 independent experiments, respectively.

Figure 9 .

 9 Figure 9. The concentration of a) NO2 -and b) NO3 -in pPBS containing or not 5 mM or 50 mM of pyruvate, as a function of the storage time, when stored at -20 °C. The concentration in % is calculated with the initial concentrations of NO2 -and NO3 -, measured on the day of the plasma treatment (D0), within 2 hours from it. The data are the mean ± SD of 2, 3 and 8 independent experiments for the pPBS + 50 mM Pyruvate, pPBS + 5 mM Pyruvate and the pPBS alone, respectively.

Figure 11 )

 11 , or normally frozen when just stored at -20 °C (see full circles in Figure10) or -80 °C (see open squares in Figure11). As depicted in Figures10 and 11, the freezing rate of the solution has almost no impact on the degradation of H2O2 and NO2 -. It seems that the evolution over the storage time of the reactive species concentration at -20 °C and -80 °C depends only on the final storage temperature of the solution.

Figure 10 .

 10 Figure 10. The concentration of a) H2O2, b) NO2 -and c) NO3 -in pPBS as a function of the storage time, when stored at -20 °C, but frozen at two different freezing rates: standard as a consequence of storage at -20 °C or fast by liquid nitrogen (LN2) before immediate storage at -20 °C. The concentration in % is calculated with the initial concentration of a) H2O2, b) NO2 -and c) NO3 -measured on the day of the plasma treatment (D0), within 2 hours from it. The data are the mean ± SD of 5 independent experiments.

Figure 11 .

 11 Figure 11. The concentration of a) H2O2, b) NO2 -and c) NO3 -in pPBS as a function of the storage time, when stored at -80 °C, but frozen at two different freezing rates: standard as a consequence of storage at -80 °C or slow by using isopropanol-filled containers when storing at -80 °C. The concentration in % is calculated with the initial concentration of a) H2O2, b) NO2 -and c) NO3 -measured on the day of the plasma treatment (D0), within 2 hours from it. The data are the mean ± SD of 5 independent experiments.

Figure 12 .

 12 Figure 12. pH values of pPBS as a function of the storage time at -20 °C and -80 °C. The pH was evaluated by using bromophenol blue as a pH indicator and the polynomial function relating the sum of R+G-B values and the pH value determined in Figure SF10 b). The RGB values used are the mean values of a large area of the photographs of the Eppendorf tubes containing pPBS and bromophenol blue after storage for 30 minutes, 1, 3 and 6 hours at -20 °C and -80 °C (Figure SF8). The data are the mean ± SD of 5 and 3 independent experiments for storage at -20 °C and at -80 °C, respectively.

Figure 13 .

 13 Figure 13. The concentration of H2O2, NO2 -and NO3 -6 hours after the preparation of mimicking solutions of PBS(Ca 2+ /Mg 2+ ) with different pH values, maintained at room temperature. The concentration in % is calculated with the concentration of H2O2, NO2 -or NO3 -measured in the solution with pH = 7 6 hours after preparation. The data are the mean ± SD of 6 independent experiments.

Figure 14 .

 14 Figure 14. Cytotoxicity of pPBS previously-stored under different storage conditions to adherent DC-3F cells. After being incubated in pPBS for 10 min, DC-3F cells were plated immediately. The cell viability rate is presented as the number of colonies formed from viable cells five days after the treatment. Results from three independent experiments (each in quadruplicate) are shown as mean values ± SD for each parameter set. Statistical significance levels are presented as p-values of < 0.05 (*), < 0.01 (**), < 0.001 (***) and < 0.0001 (****). If no statistical significance is indicated, then p-values > 0.05 (NS).

Figure 15 .

 15 Figure 15. Effect of pPBS, previously stored at different storage conditions, on cell membrane permeabilization of adherent DC-3F and LPB cells. The ability of pPBS to permeabilize the cell membrane after 1 hour of treatment (incubation in pPBS) was analyzed by flow cytometry and expressed as an increase of the intracellular fluorescence of YO-PRO ® -1 iodide in permeabilized cells. The data from 3 independent experiments (each in at least 4 independent replicates) are shown as mean values ± SD for each parameter set. If no statistical significance is indicated, then p-values > 0.05 (NS).

Figure SF2 .

 SF2 Figure SF2. RONS alone pH = 7.1 Concentration over storage time of a) H2O2, b) NO2 -and c) NO3 -in ad-hoc solutions at pH = 7.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only a) H2O2 or b) NO2 -or c) NO3 -. The data are the mean ± SD of 2 independent experiments.

Figure SF3 .

 SF3 Figure SF3. RONS alone pH = 6.1 Concentration over storage time of a) H2O2 , b) NO2 -and c) NO3 -in ad-hoc solutions of pH = 6.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only a) H2O2 or b) NO2 -or c) NO3 -. The data are the mean ± SD of 2 independent experiments.

Figure SF4 .

 SF4 Figure SF4. NO2 -+ NO3 -pH = 7.1 Concentration over storage time of a) NO2 -and b) NO3 -in ad-hoc solutions of pH = 7.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only NO2 -and NO3 -.The data are the mean ± SD of 2 independent experiments.

Figure SF5 .

 SF5 Figure SF5. NO2 -+ NO3 -pH = 6.1 Concentration over storage time of a) NO2 -and b) NO3 -in ad-hoc solutions of pH = 6.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only NO2 -and NO3 -.The data are the mean ± SD of 2 independent experiments.

Figure

  Figure SF6. H2O2 + NO3 -pH = 7.1 Concentration over storage time of a) H2O2 and b) NO3 -in ad-hoc solutions of pH = 7.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only H2O2 and NO3 -.The data are the mean ± SD of 2 independent experiments.

Figure SF7 .

 SF7 Figure SF7. H2O2 + NO3 -pH = 6.1 Concentration over storage time of a) H2O2 and b) NO3 -in ad-hoc solutions of pH = 6.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only H2O2 and NO3 -.The data are the mean ± SD of 2 independent experiments.

Figure SF8 .

 SF8 Figure SF8. H2O2 + NO2 -pH = 7.1 Concentration over storage time of a) H2O2, b) NO2 -and c) NO3 -in ad-hoc solutions of pH = 7.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only H2O2 and NO2 -. The data are the mean ± SD of 2 independent experiments, except for D21 for NO2 -and NO3 -(only 1 experiment).

Figure

  Figure SF9. H2O2 + NO2 -pH = 6.1 Concentration over storage time of a) H2O2, b) NO2 -and c) NO3 -in ad-hoc solutions of pH = 6.1 stored at +4, +20, -20 and -80 °C. Pure ad-hoc solutions were prepared containing only H2O2 and NO2 -. The data are the mean ± SD of 2 independent experiments.

Figure SF10 .

 SF10 Figure SF10. Concentration over storage time at -20 °C of H2O2 in pPBS containing 5 mM of sodium pyruvate. The H2O2 initial concentration, before the addition of pyruvate, was 4 mM (>90% reduction of H2O2 due to pyruvate). The data are the mean ± SD of 3 independent experiments.

Figure SF11 .

 SF11 Figure SF11. Concentration of a) NO2 -and b) NO3 -in ad-hoc solutions of pH = 3 in the presence of different H2O2 concentrations. The blue and red vertical lines correspond to the minimum and maximum values of H2O2 concentration measured in pPBS in the presence of 5 mM pyruvate (Figue SF7). Only one experiment was performed.

Figure SF12 .

 SF12 Figure SF12. pH values and photos of Eppendorf tubes as a function of the storage time at a) -20 °C and b) -80 °C.

  , Thermo Scientific, France) and passaged upon confluency (every two days at a 1:10 dilution or every three days at a 1:30 dilution) using TrypLE™ Express Enzyme solution (12604-013), considering only viable cells. The trypan blue exclusion dye method assessed short-term cell viability for routine sub-culturing (Trypan Blue Solution, T8154) with a Countess™ II FL Automated Cell Counter (Invitrogen, FR). Cells were regularly checked for the absence of mycoplasma contamination via polymerase chain reaction (PCR) (data not shown).On the day of inoculation, LPB cells were detached using TrypLE™ Express Enzyme solution and resuspended in sterile PBS 1X (14190-094) at a final density of 5 ×10 6 lived cells/mL. Soon after that, and under general anaesthesia, each mouse was inoculated with 5 ×10 5 cells/100 µL on the right flank. When the LPB tumour volume of mice reached a defined average volume (detailed below in each experiment), mice were randomised and included in the different experimental groups.Preparation of plasma-treated medium (or plasma-activated medium, termed PAM)Minimum Essential Medium (MEM) 1X was used to produce plasma-activated medium (PAM). MEM 1X was exposed to helium NTP at two different modes, the multijet mode (dplasmaliquid surface = 5 mm) or the diffuse mode (dplasma-liquid surface = 2 mm), with the amplitude of HV pulses being set at 20 kV, 2 kHz, He flow rate at one standard litre per minute, for 10-minutes plasma treatment continuously. Similar to the controlled distance of the plasma exposure on mice skin, the distance between the NTP multijet output and the liquid surface was maintained thanks to customised 3D-printed spacers (data not shown).

Figure 35| 6 .

 35|6 Figure 35| Illustration of the in vivo procedure for indirect plasma treatment using PAM Female C57Bl/6J mice bearing s.c. LPB tumours were treated with (a) PAM or (b) PBS as a negative control. The i.t. administration of the treatment was monitored through an automatic syringe with a controlled flow speed (50 µL.min -1 ). The administered volume was half of the tumour volume, measured just before the i.t. injection.

Figure 36|

 36| Figure 36|The tumour growth delay in C57Bl/6J mice treated with multijet PAM or diffuse PAM The number of mice was n = 10 in each experimental group. Data represents as mean ± SD values. Mice were treated with i.t. administration of treated liquids (multijet PAM or diffuse PAM) or with MEM 1X, compared with the negative control being treated with PBS). The treatment was performed once a day for four days consecutively (D0 to D3). The tumour evolution is shown until day 14 after the first treatment.

Figure 37|

 37| Figure 37|The tumour growth delay in C57Bl/6 mice treated with multijet pPBS or diffuse pPBS by i.t. administrationThe number of mice was n = 8 in each experimental group. Data represented as mean ± SEM values. Mice were daily i.t. injected with pPBS (or with PBS 1X for group 1) for fourteen days consecutively (except D7 without treatment). The tumour evolution is shown until the first animal of each group reached an end-point.

  t. administration of pPBS, either the 20-minutes-pPBS or the 1-hour-pPBS, was performed 6 minutes before the administration of bleomycin. The administered volume was half of the tumour volume, individually measured during the randomisation. Bleomycin (Roger Bellon SA, Neuilly-Sur-Seine, France) was i.v. administered in the retro-orbital sinus 4 minutes before the delivery of the EPs, at a dose of 10 μg per 100 µL per animal. For mice receiving EPs (groups 2, 4 and 5), their tumour was placed between the electrodes. To ensure a correct electric field distribution around the tumour volume, conductive gel (Asept Uni'Gel US, Aspet Inmed, France) was used

Figure 38|

 38| Figure 38| Illustration of the protocol and experimental strategy of the combined treatment with pPBS and ECT in C57Bl/6J mice bearing s.c. LPB tumour

(group 1 )

 1 . If compared to the Control group, the combined treatment employing 1-hour-pPBS followed by ECT (group 4) delayed the tumour growth for six days, and ECT alone decelerated the tumour growth by four days.

Figure 39|

 39| Figure 39|The tumour growth delay in female C57Bl/6J mice treated with pPBS combined with ECT in the first experimentThe number of mice was n = 9 in each experimental group. Data represents as mean ± SEM values. The tumour evolution is shown until the first animal of each group reached an end-point.

  group 2) post-treatment. Contrariwise, one mouse in group 4 (combination of 1-hour-pPBS and ECT) had a complete tumour regression after 60 days (thus considered a tumour cure). This mouse was monitored until day 100 post-treatment with no sign of tumour recurrence, i.e., we obtained 11.11% complete regression for this group N°4. All other treatments showed the life span of the last surviving mouse of 53 days (for group 3: combined 1-hour-pPBS with BLM), 67 days (for group 2: ECT) and 78 days (for group 5: combined 20min-pPBS with BLM) after treatment.

Figure 40|

 40| Figure 40|The probability of survival (Kaplan-Meier estimator) of female C57Bl/6J mice treated with pPBS combined with ECT in the first experimentThe number of mice was n = 9 in each experimental group. Survival times were measured from the one-shot treatment until the occurrence of an end-point. The observation time ended at day 100 post-treatment for mice with complete tumour regression (here, the mouse N°49 of group 4), considered a tumour cure.

Figure 41|

 41| Figure 41|The tumour growth delay in female C57Bl/6J mice treated with pPBS combined with ECT in the second experimentThe number of mice was n = 9 in each experimental group except for the control group (n = 8). Data represents as mean ± SEM values. The tumour evolution is shown until the first animal of each group reached an endpoint.

Figure 42|

 42| Figure 42|The probability of survival (Kaplan-Meier estimator) of female C57Bl/6J mice treated with pPBS combined with ECT in the first experimentThe number of mice was n = 9 in each experimental group except for the control group (n = 8). Survival times were measured from the one-shot treatment until the occurrence of an end-point. The observation time was ended at day 100 post-treatment for mice with complete tumour regression (here, two mice in group 4 and three mice in group 5), considered a tumour cure.

Figure 43|

 43| Figure 43| The tumour growth delay of each female C57Bl/6J mouse treated with pPBS combined with ECT in the second in vivo experimentation The number of mice was n = 9 in each experimental group except for the control group (n = 8). Treatments without application of µsPEFs are shown in left panel (graphs a, c and e corresponding to the groups 1 (control), 6 (i.t. 20-minutes-pPBS + BLM) and 3 (i.t. 1-hour-pPBS + BLM), respectively), being the control of treatments with application of µsPEFs shown in the right panel (graphs b, d and e corresponding to the groups 2 (ECT), 5((i.t. 20-minutes-pPBS + ECT)) and 4 ((i.t. 1-hour-pPBS + ECT)), respectively). The individual tumour evolution was shown until the animal reached an end-point.

  proposed to use the International Standard IEC 60601-1 electrical equivalent circuit for the human body. This circuit is applicable at frequencies below 10 MHz, where electrostatic effects dominate. The compensation circuit was designed to match the model of the human equivalent circuit when placed in series with the chosen target. This work has contributed to plasma medicine research to obtain more directly relevant results to human targets. Besides, DISCUSSION -132 -a compensation circuit should help minimise purely electrical differences between nonhuman targets.

  Thus, for investigating the effectiveness of the combined treatment with pPBS and ECT, we modified the protocol and treatment design: (i) Double the treatment time of PBS 1X by the NTP multijet, being 20 minutes of treatment (20-minutes-pPBS) instead of 10 minutes of exposure to the plasma multijet, to augment the RONS concentration present in the pPBS as DISCUSSION -135 -demonstrated in chapter 3 of Results section (article #3 of Sklias et al., to be submitted); (ii)

FURTHER

  It would also be essential to scrutinise the mechanisms (what is not yet understood) and applications of pPBS and pPBS combined with ECT at different parameters. It would be worth investigating the proposed combined treatment with pPBS and ECT in other tumours (e.g., melanoma, which are less "rigid" than the studied fibrosarcoma LPB tumours, hence probably favour a more homogeneous distribution of pPBS or in other in vitro models such as spheroids.

:•

  The transplant of an organ, tissue or cells to an individual of another species. • Cell line xenograft models: established human tumour cell lines transplanted into immune-deficient host • Patient-derived xenograft (PDX) models: Human tumour explants grown in immunedeficient host Syngeneic models: established mouse tumour cell lines transplanted into immunecompetent hosts (allograft) • Genetically engineered mouse models (GEMMs): Genetic modification that permits spontaneous or induced tumour development Xenogram (also called xeroradiograph): a body picture recorded on paper rather than film.

TABLE OF FIGURES

 OF

TABLE OF TABLES

 OF

Table 1|

 1| Typical classification of plasma with some applications ....................................... -31 -

	Table 2| Different electrical discharge methods for atmospheric pressure NTP generation. ... -
	33 -

Table 3|

 3| Summary of the in vitro and in vivo cellular mechanisms and effects of plasma-treated liquids from studies of the past ten years ........................................................... -99 -Table 4| In vivo parameters of the mono-therapy using PAM in female C57Bl/6 bearing s.c. LPB tumours (n = 10) ......................................................................................... -119 -Table 5| In vivo studied parameters of the combined treatment with pPBS and ECT in female C57Bl/6J mice bearing s.c. murine LPB fibrosarcoma ....................................... -122 -

Table 1

 1 (this table contains some applications of plasmas that are presented in more detail in the next chapter 2.2).

	Classification	Temperature (K)	Electron density (m -3 )	Examples of discharge type	Applications
	High-
	temperature (equilibrium)	Te ≈ Tion ≈ Tgas = 10 6 -10 8	ne ≥ 10 20	Laser fusion Tokamak	Fusion plasma for energy
	plasma
				Plasma torch, arc
	Thermal (quasi-equilibrium) plasma	T e ≈ T ion ≈ T n ≈ Tgas ≤ 2 × 10 4	ne ≥ 10 20	plasma, Radio-frequency (RF) inductively coupled discharges,	Radiation, welding and cutting, waste treatment, material processing, etc.
				Microwave plasma
	Low-temperature (or non-thermal or non-equilibrium) plasma	Te ≥ Tion ≥ Tn ≈ Tgas = 300-1000	ne ≈ 10 10	Glow or corona discharges, dielectric barrier discharge (DBD), micro-hollow cathode discharge	Ozoniser, volatile organic compound treatment, surface modifications (coating, etching, activation, cleaning, nitration, etc.),

Table 1| Typical classification of plasma with some applications

Table 2|

 2| Different electrical discharge methods for atmospheric pressure NTP generation.

GLOBAL Total # cancer cases (2018) Total # cancer deaths (2018) 18,078,957 9,555,027 25.0%

a PAF, cancer deaths b PAF, cancer cases c PAF,

melanoma cases TRENDS Probability of premature death from cancer per year INVESTMENT CASE (2019) At this income level, investing in a package of essential services and scaling-up coverage will: Costs per year Projected lives saved per year UV (2012) c Occupational risk (2017) a PAFs (population attributable fractions) BURDEN OF CANCER 29.7% Total population (2019) 13.0% 3-4%

7,676,965,500 15,179,108 Premature deaths from NCDs (2016) Cancer as % of NCD premature deaths (2016) 4-5% Tobacco (2017) a Alcohol (2016) a Infections (2012) b Obesity (2012) b Cancer Global Profile 2020 1.0% 2-8% Estimated past and future trends in total cases per year (breast and lung)

	Lung	11.6%	18.4%
	Breast	11.6%	6.6%
	Colorectum	10.2%	9.2%
	Prostate	7.1%	3.8%
	Stomach	5.7%	8.2%
	Liver	4.7%	8.2%
	Oesophagus	3.2%	5.3%
	Cervix uteri	3.2%	3.3%
	Thyroid	3.1%	0.4%
	Bladder	3.0%	2.1%

Most common cancer cases (2018) Incidence Mortality 0 200,000 400,000 600,000 800,000 1,000,000 1,200,000

	8.3%
	8.2% 8.1% 8.0% 7.9% 7.8% 7.7% 7.6% 7.5% 7.3% 7.2% 7.1% 7.1% 7.0%
	2020	2022	2024	2026	2028	2030

4.61% 2.0% 7.0% 12.0% 17.0% 2000 2005 2010 2015 2020 2025 2030 Past trends Projected linear trend SDG target 1,655,589 1,810,532 2,069,792 2,076,106 2,778,850 3,299,640

	3,500,000
	3,000,000
	2,500,000
	2,000,000
	1,500,000
	1,000,000	6.45% 6.30% 6.15% 5.99% 5.84% 5.69% 5.53% 5.38% 5.22% 5.07% 4.92% 4.76%
	500,000
	-

Breast cancer Lung cancer 2012 2018 2040 $0.00 $0.50 $1.00 $1.50 $2.00 $2.50 $3.00 $3.50 $4.00 $4.50

						STATE OF THE ART
						Biophysical-based treatments in cancer therapy
	genes but genetic damages occurring during one's lifetime. Genetic damage may result from
	both internal and external factors.
	US$ (per capita)
	2020	2022	2024	2026	2028	2030
						-52 -

  redox adaptation response, leading to an upregulation of antioxidant capacity and a shift of redox dynamics with high ROS generation and elimination to maintain the ROS levels below the toxic threshold. As such, cancer cells would be more dependent on the antioxidant system and more vulnerable to further oxidative stress induced by exogenous ROS-generating agents or compounds that inhibit the antioxidant system.

		A further increase of
		ROS stress in cancer
		cells (red bar)
		using exogenous
		ROS-modulating
		agents is likely to
		cause elevation
		of ROS above the
		threshold level,
		leading to cell death.
		This might constitute
		a biochemical basis
		to design therapeutic
		strategies to
		selectively kill
		cancer cells using
		ROS-mediated
		mechanisms.		1
				disulphide), f
				inhibitor, wi
				Specific inhib
				SOD, catalas
				also been ide
				of drug devel
				inhibitors in
				as 2-methoxy
				such as ATN
				HMOX1 inclu
				zinc protopor
				Overcoming
		Endogenous and exogenous		adaptation. E with intrinsi
		ROS stimuli		by exogenou
				kill the malig
				experimental
		ROS	No adaptation	the ROS-mod activity in cli
				especially tho
				highly adapte
	Genomic instabilit y	Redox adaptation Transcriptional factors	Cell death	lated antioxi only enables ROS stress, b
				to many antic
				of exogenous
	Cell proliferation Senescence evasion Angiogenesis Metastasis	Cell death Survival factors DNA repair	ROS-scavenging systems	and increased multidrug re
		NATURE REVIEWS | DRUG DISCOVERY
		Cell survival	Altered drug metabolism
		Cancer	Drug
		development	resistance
		Nature Reviews | Drug Discovery

a

Table 3|

 3| PTL, promoting an easier transition to clinical practice. Table3depicts the studies, models, cellular mechanisms, and effects of plasma-treated liquids reported in some major in vitro and in vivo studies of the past ten years, either as monotherapy or in combination with molecules, cytotoxic drugs, or other therapies. Summary of the in vitro and in vivo cellular mechanisms and effects of plasma-treated liquids from studies of the past ten years

				STATE OF THE ART STATE OF THE ART STATE OF THE ART STATE OF THE ART STATE OF THE ART
			Biophysical-based treatments in cancer therapy Biophysical-based treatments in cancer therapy Biophysical-based treatments in cancer therapy Biophysical-based treatments in cancer therapy Biophysical-based treatments in cancer therapy
	in vitro; spheroids Human colorectal carcinoma cells in vitro; adherent cells Human ovarian carcinomas (ES2 in vitro; adherent cells & spheroids in vitro: Murine MC38 colon and tumour-suppressor); PTL: plasma-treated liquid; TRPM2: transient receptor potential cation channel, subfamily M, He plasma jet; DMEM ↘ spheroid growth DNA damage (peripheral (Judée et al., 2016) Ar plasma jet; ↘ cancer cell motility ↘ MMP-9 (Nakamura et al., 2017) kINPen® plasma jet; Cell cycle arrest; Actin network reorganisation; (Freund et al., 2019) member 2; ttt: treatment; w/ or w/o: with or without.
	Study; models in vitro; suspended cells in vivo; i.p. PAM 15min post-inoculation (repeat 3 days) in vivo; i.p. PAM at 48h post-xenografts in vitro; adherent cells in vitro; spheroids Green fluorescent Cell lines HCT 116 Human cervical carcinoma HeLa and Chinese hamster CHO-K1 and SKOV-3) and foetal lung fibroblasts WI-38 PDA6606 pancreatic cancer cells in vivo: CT26 human GBM cell lines (U87, U251 and LN229) colorectal cancer cells in vitro; adherent cells U251SP human glioblastoma in vitro; adherent cells in vivo; i.t. PAM at 24h post-inoculation NOS2 and NOS3 (chronic resistance to paclitaxel and cisplatin, respectively) in vitro; adherent cells Human melanoma (A375, A2058), lung adenocarcinoma protein (GFP) transduced human colorectal carcinoma cells HCT 116-GFP epithelial (A549) and osteosarcoma (MG63, SAOS-2, HOS) cell lines In vitro; Mouse melanoma (B16), human in vitro; adherent Human suspended cells; combined ttt w/ melanoma (A375) and glioblastoma cells glioblastoma U251SP cells BLM, paclitaxel or dacarbazin (A172) derived in vitro; adherent cells Human carcinoma (lung epithelial A549, liver HepG2 and breast MCF7), and neuroblastoma SH-SY5Y in vitro; adherent cells Human skin fibroblasts (NHSF), human colon cancer (HCT116) and human melanoma (Lu1205) cell lines in vitro; spheroids Human head and neck carcinomas (FaDu) in vitro; adherent Human immune-cells suppressive PSC (hPSC21, hPSC128 and RLT-PSC) and pancreatic cancer PCC (MIA-Paca-2, PANC-1, BxPC3, Capan-2) in vitro; adherent cells in vivo; i.t. PAM at 24h post-xenograft Human pancreatic cancer cell lines (PANC-1, in vitro; adherent cells in vivo; subcutaneous tumour (i.t. PAM at 24h post-Human glioblastoma U251SP, mammary epithelial MCF10A, cervical cancer SiHa and in vitro; adherent Human cells pancreatic cancer in vivo; i.t. PAM at 24h post-xenograft cell lines Capan-2 in vitro; adherent Endometrial and cells cancer cell lines BxPC-2 AMEC, HEC50, ISHIKAWA, and inoculation, repeated ttt) ovarian cancer in vitro; adherent Human RL95 SK-OV-3 cells cells endometrial Capan-2, BxPC-3 and MIA PaCa-2) in vitro; adherent cells Human ovarian carcinomas (OVCAR-3 NOS2) in vitro; adherent cells human glioblastoma (U251SP) and mammary epithelial (MCF10A) cells at D1 to 4, then D8 post post-xenografts cancer cells cells glioblastoma human gastric in vitro; adherent U251SP human (GCIY-EGFP) cells squamous cell in vitro; adherent cells in vivo; i.p. PAM SC-2-NU, AGS, and enhanced GFP-tagged GCIY phosphamide) tumour (at 7 days w/o i.p. cyclo-i.t. PAM w/ or post inoculation: melanoma line SCC15 subcutaneous metastatic in vitro; adherent Human oral in vivo; B16-F10 murine TOV21G, ES-2 and RPCI-WM1 in vitro; suspended cells Human lymphoplasmacyti c lymphoma (LPL) MWCL-1 and in vivo; i.p. PAM at D0, 1, 2, 6, 7, 8 post-xenografts carcinoma HEC-1 and gastric cancer GCIY cells in vitro; spheroids Human colorectal 3 cells carcinoma SKOV-ovarian HCT 116 and	Plasma device; Plasma-treated liquids stored at -80°C & +4°C HV DBD; DMEM (direct ttt) and PBS and FBS (indirect RPMI PBS kINPen®; PBS; catalase ttt) He plasma Ar plasma jet; DMEM Ar plasma jet; RPMI-1640 He plasma jet; DMEM stored at jet; PBS; combined ttt with PEFs +4°C DBD PlasmaDerm Ar plasma ® jet; DMEM and PBS RLS Ar plasma jet; DMEM stored at -80°C He plasma micro-jet; PBS (Ca 2+ /Mg 2+ ) He plasma jet; DMEM kINPen® plasma jet; PBS Ar plasma jet; RPMI-1640 Ar plasma jet; RLS Ar plasma jet; RLS Ar plasma jet; RPMI-1640 Ar plasma jet; Ar plasma jet; DMEM w/ or w/o FBS and P/S DMEM jet; Ar plasma Ar plasma jet; RPMI DMEM jet; DMEM jet; Air plasma He plasma Ar plasma jet; RPMI-1640 He plasma jet; IMDM He plasma DMEM NaCl 0.9% PBS, jet;	Cellular effects and molecular mechanisms spheroid cells) ↘ cell re-adhesion and cell growth; PBS more cytotoxic than DMEM ↗ mutagenic potential of FBS ↘ tumour burden ↘ metastatic nodules by 60% ↗ actin protrusion dynamics ↗ CRT, HSP70 and HMGB1 ↗ ICD Anti-cancer capacity of pPBS: H2O2 is a more ↘ tumour burden ↗ i.t. macrophages and T lymphocytes important than NO2 -; in long-term exposure pPBS more stable than other Early DNA damage; Caspase-dependant apoptosis; Caspase 3/7 cleavage; ↘ phosphorylation kinases pathways PI3K/AKT/mTOR ↘ PDK1 ↘ cell viability ↘ NOS2 cell -inoculated tumour by 66% Caspase-dependent apoptosis; Activate intracellular ROS generation and mitochondrial ROS accumulation; PTL; not same anticancer Caspases 3/7 activation; effect w/ PBS supplemented with H2O2 and/or NO2 -or w/ pPBS Alteration of cell junctions and cytoskeleton; ↘ spheroid growth ↘ cell viability (most Excessive mitochondrial fragmentation and clustering; ↗ Drp1 ↘ cell viability prominent with the combined ↘ phosphorylation of AKT; DMEM induces more plasma-treated PBS and BLM) intracellular ROS than LRS Caspase-dependent apoptosis; ↗ TRMP2 ↗ mitochondrial stress Zn 2+ -dependant cell death ↗ Zn 2+ , ↘ NAD + , ↘ ATP H2O2 was not toxic (effect cell-type dependent); Synergism of H2O2 and NO2 -(but not NO3 -): ↘ cell viability; He plasma jet interaction with Detachment of peripheral spheroid cells since the first day after 1 ttt, followed by a ↘ cell viability Co-culture with DC: ↗TNF-α and IFN-γ rapid spheroids' regrowth ↘ TGF-β (more than the Ctrl); PCCs: ↗ ICD (expressed and ambient air was required to ↘ cell growth after 4 released damage-associated generate NO2 -and NO3 -successive ttt molecular patterns); Caspase-dependent apoptosis; Caspase 3/7 cleavage; ↘ tumour volume Only lactate exhibited anti-Caspase-dependant apoptosis PSCs: ↘ immunosuppressive tumour microenvironment. DNA fragmentation tumour effects through activation by NTP ↘ cell proliferation ↗ autophagic cell death & late apoptotic cells (time and ↘ tumour burden concentration-dependant); ↘ metastatic nodules ↘ phosphorylation of mTOR ↘ cell viability (better with and AKT ↘ cell proliferation PAM + cis-Pt than only PAM or ↘ tumour volume phenotype) Apoptotic cell death for U251SP but not for MCF10A; Antitumour effect was not due and NO2 -(that was incubation-time dependent) ↘ pentose phosphate pathway ↗ glycolysis Metabolomic analysis solely to the synergism of H2O2 p53 pathway, hypoxia pathway ↘ cell adhesion and migration PAM + drug = ↗ cell death ↘ tumour burden strategy; more efficiency than indirect Direct NTP treatment was Transcriptomic analysis ↗ BAX/BCL2, apoptosis sensitive (mesenchymal ↘ cell viability ↘ tumour growth TOV21G and ES-2 are more Cell shrinking, blebbing, and detachment; and in vivo ↘ cell proliferation ↗ cellular differentiation ↗ human PRDM1 α ↘ tumourigenic population cis-Pt) ↗ cell death Anticancer effects both in vitro ↘ ALDH Caspase 3 cleavage; ↘ spheroid growth	(Boehm et al., 2016) (Van Boxem et al., 2017) (Griseti et al., References (Tanaka et al., 2011., Tanaka et al., 2012) 2019) (Saito et al., 2016) (Daeschlein et al., 2018) (Tanaka et al., 2019) (Utsumi et al., 2013) (Adachi et al., 2015; Hara et al., (Girard, 2016) (Van Loenhout et (Chauvin et al., al., 2019) 2018) 2015., Hara et al., 2017) (Hattori et al., 2015) (Tanaka et al., (Sato et al., 2016) 2018) (Yoshikawa et al., 2020) (Ikeda et al., 2018) 2019) (Kurake et al., (Kurake et al., 2016) (Takeda et al., 2017) 2018) (Shi et al., 2017) (Saadati et al., (Utsumi et al., 2016) (Wada et al., 2017) (Griseti et al., 2020)
		-96 --97 --98 --99 --100 -

generate Legend: ↗ activation or enhancement; ↘ inhibition or reduction; ALDH: aldehyde dehydrogenase; AKT/PI3K/mTOR pathway: cell cycle progression, prevention of apoptosis pathway; ATP: adenosine triphosphate; Ar: argon; BAX/BCL2: apoptotic regulators; CRT: calreticulin; DBD: dielectric barrier discharge; DCs: dendritic cells ; DMEM: Dulbecco's Modified Eagle's Medium; Drp1: dynamin-related protein 1; GFP: green fluorescent protein; He: helium; HMGB1: high-mobility group protein 1 and amphoterin; HSP70: 70kDa heat shock proteins; HV: high voltage; ICD: immunogenic cell death; IMDM: Iscove's Modified Dulbecco's Medium; i.p.: intraperitoneal; i.t.: intratumoural; MMP-9: matrix metallopeptidase 9 (extracellular matrix degradation); NAD + : nicotinamide adenine dinucleotide; PAM: plasma-activated medium; PEF: pulsed electric field; P/S: penicillin/streptomycin; RLS: Ringer's lactate solution; RPMI: Roswell Park Memorial Institute medium; PDK1: 3-phosphoinositide-dependent protein kinase-1; PRDM1 α: PR domain zinc finger protein 1 (a repressor of beta-interferon (β-IFN) gene expression and a

TABLE I CONCENTRATIONS

 I OF H 2 O 2 , NO -3 , AND NO -2 GENERATED IN 1 mL OF WATER IN 24-MULTIWELL PLATES FOR DIFFERENT TARGET ELECTRICAL CONDITIONS. TREATMENT TIME 5 MIN. GAP DISTANCE 10 mm

TABLE II CONCENTRATIONS

 II OF H 2 O 2 , NO -3 , AND NO -2 GENERATED IN WATER IN A 24-MULTIWELL PLATE (3 mL) AND IN THE CUSTOM WELL (9 mL) WITH AND WITHOUT THE COMPENSATION CIRCUIT. GAP DISTANCE 10 mm

Table 1 .

 1 pH and conductivity ( ) of the control (sham) and the pPBS at +7 kV and +11 kV for a PBS + / + treatment time of 20 min. Data are presented as mean values ± SD of independent quadruplicates.

		Control (sham)	+7 kV pPBS	+11 kV pPBS
		Mean	SD	Mean	SD	Mean	SD
	pH	7.18	0.05	6.68	0.02	6.24	0.10
	(S/m)	10.7	0.08	11.27	0.21	13.08	0.27

Table 1 .

 1 pH and conductivity (σ) of the control (sham) and the pPBS at +7 kV and +11 kV for a PBS + /+ treatment time of 20 min. Data are presented as mean values ± SD of independent quadruplicates.

		Control (sham) +7 kV pPBS +11 kV pPBS
		M e a n	S D	M e a n SD Mean SD
	pH	7.18	0.05	6.68 0.02	6.24	0.10
	σ (S/m)	10.7	0.08	11.27 0.21 13.08 0.27

Table 4|

 4| In vivo parameters of the mono-therapy using PAM in female C57Bl/6 bearing s.c. LPB tumours(n = 10)

replace MEM 1X with PBS 1X (Ca 2+ , Mg 2+ )

  When the s.c. LPB tumour volume of mice reached an average of 80 mm 3 , mice were randomised and included in 3 different groups with 8 mice per group. The treatments were daily i.t. administrated to mice for fourteen days consecutively (except D7).

	which was studied previously in in vitro tests (see chapters 1 and 2 of the Results). Indeed, as
	a buffered and isotonic biological solution (pH 7.20), PBS 1X (Ca 2+ , Mg 2+ ) is suitable for almost
	all living biological targets. Again, experiments were performed with female C57Bl/6J mice
	bearing s.c. LPB tumours, respecting the protocol established previously. The protocol of
	preparation of pPBS remained the same as that of PAM, either with a "multijet mode" (being
	group 2 "multijet pPBS") or with a "diffuse mode" (being group 3 "diffuse pPBS"). The

To investigate this question, we decided to parameters of the NTP device used to treat PBS remained unchanged, except the amplitude of the high voltage pulses being set at 11 kV (parameter at which our plasma generator was the more stable -data and observation not shown). Untreated PBS 1X (Ca 2+ , Mg 2+ ) was used RESULTS -121 -as vehicle control (group 1).

  The LPB cell culture for sub-cutaneous inoculation and the inoculation were performed with respect to established protocol, as described in section 4.2.2. above. At around 12 days post-inoculation, when the average tumour volume reached ca. 70 mm 3 mice were randomised and included in the different experimental groups, with nine mice per group (except for the second in vivo experimentation where the Control group contained eight mice).

		RESULTS
		4.2.2 Materials and methods
	Animal model
	Again, experiments were performed with female C57Bl/6J mice bearing s.c. LPB tumours,
	respecting the protocol established previously (see section 4.2.2.).
	Cancer cells culture for sub-cutaneous inoculation
		Treatment parameters
	1	Control (i.v. administration of BLM)
	2	ECT (i.v. BLM followed by 8 square-wave EPs of 100 µs, at 1000 V/cm and 1 Hz)
	3	i.t. 1-hour-pPBS followed by i.v. BLM
	4	i.t. 1-hour-pPBS followed by ECT
	5	i.t. 20-minutes -pPBS followed by ECT
	6	i.t. 20-minutes-pPBS followed by i.v. BLM
		-123 -

Table 5| In vivo studied parameters of the combined treatment with pPBS and ECT in female C57Bl/6J mice bearing s.c. murine LPB fibrosarcoma

  Amélioration de l'efficacité de l'électrochimiothérapie par association avec des liquides traités par des plasmas froids : mise au point des protocoles expérimentaux et preuve de concept Mots clés : cancer ; électro-chimiothérapie ; plasmas froids ; amplitude du champ électrique ; mélanome ; espèces réactives de l'oxygène et de l'azote (RONS) L'électrochimiothérapie (ECT) -une méthode établie pour l'éradication locale des tumeurs cutanées basée sur la technologie d'électroporation -est largement utilisée en Europe en médecine humaine et vétérinaire avec très peu d'effets secondaires systémiques. Les plasmas froids (NTP) ont récemment pris de l'ampleur pour leur application en cancérologie grâce à leur riche source en espèces réactives à courte durée de vie ainsi qu'en espèces réactives de l'oxygène et de l'azote à longue durée de vie (RONS) qui peuvent être transférées aux liquides traités par du NTP. Nous cherchions à combiner ces technologies afin d'améliorer les capacités de l'ECT et de mieux comprendre la contribution des RONS dans ces stratégies anticancéreuses.En tant qu'étude multidisciplinaire utilisant à la fois de la physique, de la chimie, des biotechnologies et de la médecine, cette thèse visait à mettre en évidence les effets bénéfiques de la combinaison de l'ECT et du plasma froid. Une nouvelle source de plasma d'hélium multi-jet (PMJ) a été utilisée pour des études in vitro sur trois lignées cellulaires différentes (les cellules DC-3F, B16-F10 et LPB), et pour des études in vivo sur les souris nude et C57Bl/6 porteuses de tumeurs sous-cutanées. Nous avons démontré que le pH et la température de stockage du PBS traité par le PMJ (pPBS), ayant une influence importante sur la stabilité des trois principaux RONS (H2O2, NO2 -et NO3 -), étaient cruciaux pour conserver la capacité anticancéreuse du pPBS. Le traitement combinant du pPBS et une ECT à une amplitude de champs électrique plus faible que celle utilisée habituellement a permis de restaurer l'efficacité de l'ECT, ralentissant la croissance des tumeurs chez la souris et, en conséquence, améliorant le taux de survie des animaux traités.

	Résumé :

Université Paris-Saclay

Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre :

"Blood" consists of two major parts: blood cells and blood plasma. Blood cells are red blood cells, platelets, and white blood cells (lymphocytes, monocytes, neutrophils, eosinophils, and basophils). Blood plasma consists of water, ions (calcium, magnesium, sodium, etc.), large organic molecules (amino acids, glucose, lipids, nitrogenous waste, and proteins, like albumins, globulins, clotting factors and fibrinogen), and some trace elements and vitamins. Herein we use "blood" as whole blood, and "blood plasma" as defined.

Authorized licensed use limited to: Universita degli Studi di Bologna. Downloaded on November 08,2020 at 12:52:25 UTC from IEEE Xplore. Restrictions apply.

GLOSSARY & KEY TERMS

ACKNOWLEDGEMENTS ix

ACKNOWLEDGEMENTS

Ayant parcouru un long chemin avant d'arriver à mon doctorat, je souhaite que toutes personnes mentionnées ci-après aient la certitude de ma profonde gratitude pour avoir partagé ma vie professionnelle et personnelle.

ACKNOWLEDGEMENTS xi

ou un au revoir à 20h du soir en passant demander aux étudiants si tout allait bien, ça fait partie d'une vie agréable au labo).

• Leslie, ma chère, je me rends compte de la chance que l'on a (et que j'ai) de t'avoir dans

ACKNOWLEDGEMENTS xii

Acknowledgments: The authors thank T. García-Sánchez, J-R. Bertrand (Gustave Roussy) and G. Bauville (LPGP) for technical support.

Acknowledgements:

The authors thank P. Jeanney and M. Fleury (LPGP) for technical support, and P.-M. Girard (Institut Curie) for technical support and valuable discussions.

supported by a consortium of three laboratories in the framework of the ANR (Agence Nationale de la Recherche) project cô. nuôi nấng chị em con. Cảm ơn mẹ đã hy sinh rất nhiều cho gia đình mình, đã sẵn sàng hy sinh mạng sống của mình để cho con có chị, có em.

Funding: We acknowledge the financial support from the PLASCANCER project (INCa-PlanCancer N°17CP087-00), the LabEx LaSIPS project PHeCell3D, the GDR 2025 HAPPYBIO, the CNRS, the Université Paris-Saclay and the Institut Gustave Roussy.

Supplementary Materials:

The following are available online at http://www.mdpi.com/2072-6694/12/1/219/s1, Video S1: NTP multi-jet generated at +7 kV peak voltage; Video S2: NTP multi-jet generated at +11 kV peak voltage. Funding: We acknowledge the financial support from the PLASCANCER project (INCa-PlanCancer N 17CP087-00) and the GDR 2025 HAPPYBIO.

Conflicts of Interest:

The authors declare no conflict of interest.

Appendix A.

Treatment with PBS + / + Increases Cell Electropermeabilisation without A↵ecting the Cell Viability: Proof of Concept.

Appendix A.1. Assessment of the Plasma-Treated PBS + / + Cytotoxicity Appendix A. 1.1. Materials and Methods The cytotoxicity of pPBS was determined via a clonogenic survival assay that overcomes some of the limitations of other in vitro assays, such as apoptosis measurements, by monitoring all types of cell death, even the mitotic cell death. This colony forming technique is a robust in vitro cell survival test based on the ability of each single viable cell to grow into a colony (defined to consist of at least 50 cells) after being treated by specific agents. 300 µL of adherent DC-3F cells at a density of 1.60 ⇥ 10 5 cells•mL 1 were seeded per well in the wells of a 48-well plate (Nunclon ® Delta Surface, Thermo Fisher Scientific, DK, 150687) for an overnight cell culture at 37 C in a humidified, 5% CO 2 incubator. In the day of experiment when the cell layer reached ca. 70 to 80% confluency, the medium above the cells was removed and the cell layer was washed twice with PBS + / + . After being prepared (plasma setup not grounded neither compensated, plasma parameters: +7 kV, 2 kHz, Helium 1 slm, distance 5 mm between the plasma outlet and the liquid layer in well), 300 µL of fresh pPBS was added to the cell layer. After di↵erent incubation times in pPBS, pPBS was removed from the cell layer; cells were dissociated by 100 µL Adherent DC-3F cells were pre-treated with either sham or pPBS for 10 min., before being exposed to a pulsed electric field (8 pulses, 100 µs, 1100 V/cm, 1 Hz) and post-treated for 10 min. with sham. Cell permeabilisation was evaluated by flow cytometry immediately after the treatments (Figure A2). In the condition where no PEF was applied (0 V/cm), a slight though not significant increase of YO-PRO ® -1 uptake was observed in cells pre-treated with pPBS compared to those pre-treated with Sham. The exposure of the cells to the EPs increases the intracellular fluorescence intensity of YO-PRO ® -1 Iodide (present in all the treatment liquids), indicating membrane permeabilisation (Figure A2). This effect is significantly greater in cells pre-treated with pPBS than in control cells pre-treated with sham (1.68-fold enhancement).

The treatment of DC-3F cells with pPBS combined with an EP at 1100 V/cm intensity has thus given Adherent DC-3F cells were pre-treated with either sham or pPBS for 10 min., before being exposed to a pulsed electric field (8 pulses, 100 µs, 1100 V/cm, 1 Hz) and post-treated for 10 min. with sham. Cell permeabilisation was evaluated by flow cytometry immediately after the treatments (Figure A2). In the condition where no PEF was applied (0 V/cm), a slight though not significant increase of YO-PRO ® -1 uptake was observed in cells pre-treated with pPBS compared to those pre-treated with Sham. The exposure of the cells to the EPs increases the intracellular fluorescence intensity

Abstract

To be considered as efficient anti-cancer agents, plasma-treated solutions (PTS) should maintain their anti-cancer efficacy over long periods. This study investigates the chemical stability of plasma-treated PBS(Ca 2+ /Mg 2+ ), or pPBS, as a function of storage time and temperature, in terms of the evolution of the concentration of H2O2, NO2 -and NO3 -, the main drivers of the PTS anti-cancer effects, as well as the pPBS cytotoxic and cytostatic effects, and its cell membrane permeabilization capability, on two different cell lines (non-malignant DC-3F and tumorigenic fibrosarcoma LPB). pPBS was stored for up to 75 days at 4 different temperatures: +20, +4, -20 and -80 °C. The aforementioned long-lived reactive species remained stable for 21 days of storage at +20 or +4 °C. Contrariwise, significant degradation of H2O2 and NO2 -was observed at -20 and -80 °C, even after only 1 day of storage. It is shown that the considerable acidification occurring during freezing (from pH 6 to 2-3) favors the conversion of NO2 - into NO3 -through peroxidation by H2O2. However, the freezing rate is inconsequential. In good agreement with the chemical dosimetry of pPBS, the cytotoxic and cytostatic capacities of pPBS were also preserved when stored at +4 °C (for at least 21 days) but not at -20 °C. The cell membrane permeabilization was cell-dependent and linked to the H2O2 content of the pPBS, thus also dependent on the pPBS storage conditions. In conclusion, the simple and stable storage at room temperature makes the practical application of pPBS in cancer therapy easy and accessible to all people.

Author Contributions: conceptualization: K.S., T.-H.C, A.S., E.R., L.M.M., J.S.S.; methodology: K.S., T.-H.C., A.S., J.-M.P., E.R., L.M.M., J.S.S.; data acquisition: K.S., T.-H.C.; data validation: K.S., T.-H.C., F.M.A., L.M.M., J.S.S.; data analysis and visualization: K.S., T.-H.C., J.S.S.; data interpretation: K.S., T.-H.C., A.S., J.-M.P., E.R., L.M.M., J.S.S.; resources: E.R., L.M.M., J.S.S.; supervision: L.M.M., J.S.S.; funding acquisition: C.D., J.-M.P., E.R., L.M.M., J.S.S.; writing-original draft preparation: K.S., J.S.S.; writing-review and editing: K.S., T.-H.C., L.M.M., J.S.S.. All authors discussed the results, reviewed the manuscript, and have read and agreed to the published version of the manuscript. blood vessels or other connective or supportive tissue). An example of an adenosarcoma is Wilms tumour.

Supplementary Information

Adenosis: a disease or abnormal change in a gland. Breast adenosis is a benign condition in which the lobules are more prominent than usual.

Adenosquamous carcinoma: a type of cancer that contains squamous cells (thin, flat cells that line specific organs) and gland-like cells.

Adjunct agent: a drug or substance used in cancer therapy in addition to the primary treatment.

Adjunct therapy (also called adjunctive therapy): another treatment used together with the primary treatment. Its purpose is to assist the primary treatment.

Adjuvant therapy: additional cancer treatment given after the primary treatment to lower the risk that cancer will come back. Adjuvant therapy may include chemotherapy, radiation therapy, hormone therapy, targeted therapy or biological therapy.

Advanced cancer: cancer that is unlikely to be cured or controlled with treatment. Cancer may have spread from where it first started to nearby tissue, lymph nodes, or distant body parts. Treatment may be given to help shrink the tumour, slow the growth of cancer cells or relieve symptoms.

Adverse effect (also called an adverse event): an unexpected medical problem during treatment with a drug or other therapy. Adverse effects may be mild, moderate or severe and may be caused by something other than the drug or treatment.

Adult-type fibrosarcoma: a type of cancer that forms in fibrous (connective) tissue. It can occur anywhere in the body. Adult-type fibrosarcoma may spread to nearby tissue or other body parts, such as the lungs or bones. It may also recur (come back) after treatment. Adulttype fibrosarcoma usually occurs in young and middle-aged adults but also children. It is a type of soft tissue sarcoma.

Agonist: a substance or drug that binds to a receptor inside a cell or on its surface and causes the same action as the substance that normally binds to the receptor.

Allograft: see Xenograft

Alopecia: hair loss.

Anaesthesia: A loss of feeling or awareness caused by drugs or other substances. Anaesthesia keeps patients from feeling pain during surgery or other procedures. Local anaesthesia is a loss of sensation in a tiny body area. Regional anaesthesia is a loss of sensation in a part of the body, such as an arm or leg. General anaesthesia is a loss of sensation and a complete loss of awareness that feels like profound sleep.

Angiogenesis: blood vessel formation. Tumour angiogenesis is the growth of new blood vessels that tumours need to develop, a process caused by the release of chemicals by the tumour and by nearby host cells.

Angiogenesis inhibitors: are used to prevent the formation of blood vessels, thereby depleting the cancer cells of oxygen and nutrients.

Cancer immunoediting: an extrinsic tumour suppressor mechanism that engages only after the cellular transformation has occurred, and intrinsic tumour suppressor mechanisms have failed.

Cancerology: a branch of basic research in medical science interested in molecular changes in cells and tissues that lead to cancer.

Carcinogen: an agent (chemical, physical or viral) that promotes carcinogenesis but does not cause cancer at all times, under all circumstances.

Carcinogenesis: the initiation of a tumour. Carcinoma: cancer found in cells that make up epithelial -tissue that covers or lines the surfaces of internal organs, glands, and body structures. Carcinoma usually forms a solid tumour. Carcinomas include approximately 80% to 90% of human cancers.

Carcinomatosis (also called carcinosis): a condition in which cancer is spread extensively throughout the body or, in some cases, to a relatively large region of the body.

carcinomatous lymphangitis

Catalase (CAT,1.11.1.6): an antioxidant enzyme present in all aerobic organisms that decompose hydrogen peroxide (H2O2) into water and oxygen in an energy-efficient manner in the cells exposed to environmental stress.

Cauterisation (or cautery): a medical practice of burning a part of the patient body (destroying some tissue) to remove or close off a part of it in an attempt to mitigate bleeding and damage, remove an undesired growth or minimise other potential medical harm, such as infections when antibiotics are unavailable.

Chemoprevention: the ability to use drugs or lifestyle modifications to prevent cancer from developing.

Chemotherapeutic agents: antineoplastic agents used to directly or indirectly inhibit the uncontrolled growth and proliferation of cancer cells and are classified according to their mechanism of action and include alkylating agents, and antimetabolites, topoisomerase inhibitors, antibiotics, mitotic inhibitors, and protein kinase inhibitors.

Chemotherapy: the use of any chemotherapeutic agent (drug) to treat any disease. For cancer treatment, chemotherapy is also called "chemo" and refers to drugs to kill cancer cells that grow and divide rapidly.

Circulating tumour cells: rare cells shed by solid tumours into the systemic circulation at an estimated frequency of 1:500.000-1:1.000.000 circulating cells.

Clinical trial: research studies performed on people to evaluate a medical, surgical or behavioural intervention. Usually advanced through four phases, they are the primary way researchers find out if a new treatment (a new drug or diet or medical device) is safe and effective in people by evaluating their treatment, side effects, and survival.

Complementary therapy: therapies used in addition to standard treatment.

Computed tomography scan (also called a CT or CAT scan): a diagnostic imaging procedure that uses a combination of X-rays and computer technology to produce horizontal or axial images (often called slices) of the body, showing details of any part of the body, including the bones, muscles, fat, and organs. CT scans are more detailed than general X-rays.

Prevention: eliminating or minimising exposure to the causes of cancer, and includes reducing individual susceptibility to the effects of such reasons.

Primary prevention: Elimination or reduction of exposure to recognised risk factors in susceptible populations to prevent disease. Primary prevention of cancer comprises a broad spectrum of strategies, from legislation and policies to minimise or eliminate exposure to carcinogens to promoting healthy behaviour and health sector programmes.

Primary tumour (also called original or primary cancer): a tumour grows at the anatomical site where tumour progression began and proceeded to yield a cancerous mass. See secondary tumour.

Programmed cell death: a standard physiological form of cell death characterised by apoptosis.

Proto-oncogene: a normal gene in the cell that can be converted into an oncogene.

Pulsed electric field (PEF) treatment: see electropulsation.

Radical: an atom or group of atoms with at least one unpaired electron, a feature that makes a radical very chemically reactive.

Reactive oxygen species (ROS): include oxygen ions, free radicals and peroxides, both inorganic and organic. They are generally highly reactive due to unpaired valence shell electrons. ROS form a natural by-product of normal oxygen metabolism and are essential in cell signalling. However, ROS levels can increase dramatically during environmental stress, resulting in significant damage to cell structures.

Redox status: balance of the reduced state versus the oxidised form of a biochemical system, influenced by the level of reactive oxygen and nitrogen species (ROS and RNS) relative to the capacity of antioxidant systems to eliminate ROS and RNS.

Reversible electroporation: the temporary permeabilisation state of a cell exposed to an electric field that disturbs the phospholipid bilayer, allowing molecules to reach the cell interior through the cell plasma membrane. After the end of the electric pulse, the transport of molecules ceases, the plasma membrane reseals, and the cells remain viable.

Sarcoma: cancer that starts in connective tissue cells, including blood and lymph vessels, cartilage, fat, muscle, tendon, and bone cells. For example, osteosarcoma is the most common type of cancer that starts in the bone; chondrosarcoma is cancer that begins in cartilage cells.

Secondary prevention: (of cancer) involves the use of examinations and tests to detect cancer as early as possible before signs and symptoms would cause a patient to seek care. It has previously been used synonymously with screening.

A secondary tumour (also called secondary cancer): cancer that has spread (metastasised) from the place where it first started (the primary tumour) to another part of the body. Secondary tumours are the same type of cancer as the original (primary) cancer. See primary tumour.

Standard deviation (SD): measures the amount of variability or dispersion from the individual data values to the mean.

Standard ECT: fixed electrode-geometry electrochemotherapy.

GLOSSARY & KEY TERMS -177 -

The standard error (SE) of a statistic: the approximate standard deviation of a statistical sample population that measures the accuracy of a sample distribution representing a population using SD. In statistics, a sample mean deviates from the actual mean of a population-this deviation is the standard error of the mean (SEM).

The standard error of the mean (SEM): the measure of how far the sample mean of the data is likely to be from the actual population mean. The SEM is always smaller than the SD.

Thermo-neutral point (TNP): a discrete ambient temperature below which energy expenditure increases and above which body temperature increases. Humans do not have a TNP. The mouse TNP is ca. 29°C in the light phase and ca. 33°C in the dark phase.

Thermo-neutral zone (TNZ): the ambient temperatures where metabolic rate is at a minimum. The TNZ is critical for understanding the thermal biology differences between individuals.

Transfection: introducing a foreign genetic material (DNA, RNA) into eukaryotic cells, done by viruses, electroporation, microinjection, liposomes, etc.

Transforming growth factor β (TGF β): polypeptide growth factor that generally inhibits animal cell proliferation.

Tumour: any abnormal proliferation of cells.

Tumour necrosis factor (TNF): a polypeptide growth factor that induces programmed cell death.

Tumour promotor: a compound that leads to tumour development by stimulating cell proliferation.

Tumour suppressor gene: a gene whose inactivation leads to tumour development.

Tumour virus: a virus capable of causing cancer in animals or humans.

Ultrasound (also called sonography): a diagnostic imaging technique that uses high-frequency sound waves and a computer to create images of blood vessels, tissues, and organs. Ultrasounds are used to view internal organs as they function and to assess blood flow through various vessels.

Unilateral: affecting one side of the body (e.g., unilateral kidney cancer occurs in one kidney only.)

Variable-geometry ECT: long-needle variable electrode-geometry electrochemotherapy.

Vascular disruption effect of ECT: a cascade of tumour cell death surrounding obstructed tumour vessels, resulting from the drug used in the electroporation of endothelial cells and contributing to the overall effectiveness of ECT Vascular lock: transient reduction of perfusion, at a greater extent in tumours than in normal tissues that occurs after application of electric pulses to tissues X-ray: a type of radiation (electromagnetic energy beams) that can pass through the body and onto film, making pictures of areas inside the body. X-rays may be used in a diagnostic test (in low dose) and treatment (in high amount) of cancer and other diseases.

X-ray crystallography: a method in which the diffraction pattern of X rays is used to determine the arrangement of individual atoms within a molecule.