Gianpiero Lazzari

Patrick Couvreur
email: patrick.couvreur@u-psud.fr

Simona Mura
email: simona.mura@u-psud.fr

Valérie Nicolas

Michiya Matsusaki

Mitsuru Akashi

Multicellular tumor spheroids: a relevant 3D model for the in vitro preclinical investigation of polymer nanomedicines

Keywords: Multicellular tumor spheroids, light sheet fluorescent microscopy, confocal laser scanning microscopy, drug penetration, multi-view image fusion, 3D tomography MCTS-on-a-chip, microfluidics, photo-lithography, SU-8 photoresist, microvasculature, multicellular tumor spheroids Multicellular tumor spheroids Pancreatic cancer 3D models Tumor microenvironment Layer-by-layer coating

Application of nanotechnology to medicine, usually termed as nanomedicine, has given a crucial impulse to the design of various drug-loaded nanocarriers driven by the idea to overcome the limits associated to the traditional delivery modalities, in particular in the field of cancer treatment. However, appropriate preclinical evaluation of the real therapeutic potential of nanomedicines suffers from the lack of relevant models, well representative of the human disease and good predictors of the therapeutic response in patients. In this context a great emphasis has been directed toward 3D tumor models aiming to surmount the insufficient predictive power of traditional 2D monolayer cultures of cancer cells. This review focuses on multicellular tumor spheroids (MCTS), which are so far the largest employed 3D tumor model in preclinical studies. After a brief discussion on spheroid construction strategies and analytical/imaging techniques employed in experimental settings, the application of 3D MCTS to the evaluation of nanomedicines displaying various physico-chemical properties is reviewed. Finally, relevant examples of scaffold and microfluidic systems in which MCTS have been included are described.

2-(2-methoxy

Evaluation of intracellular metabolic activity. Reduction of the WST-1 into a water soluble formazan by the NADH dehydrogenase and plasma membrane electron transport. Absorbance measurement at 450 nm. 122, 123 3-(4,5-dimethylthiazol-2yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) a,b Evaluation of intracellular metabolic activity. Reduction of the MTS tetrazolium salt into an water soluble formazan product by the mitochondrial NADPH dehydrogenases. Absorbance measurement at 490 nm. 65, 88 AlamarBlue ® a,b Evaluation of cellular reducing ability. Reduction of resazurin to resorufin. Fluorescence measurement. Ex 540-570 nm/Em 580-610 nm. 110, 146 Acid phosphatase (APH) b Quantification of APH activity. Hydrolysis of p-nitrophenyl phosphate in p-nitrophenol by the APH enzyme. Absorbance measurement at 405 nm. 82, 95, 115, 124 Adenosine triphosphate (ATP) b Measurement of the intracellular ATP content. Oxidation of luciferin by the luciferase enzymes in presence of intracellular ATP and emission of bioluminescence. 66, 67, 120, 125 Lactate dehydrogenase (LDH) a, Evaluation of membrane integrity. Measurement of conversion of lactate into pyruvate via NAD + reduction by the LDH. The resulting NADH catalyzed the reduction of a 53, 136 90, 91

Growth inhibition

Optical microscopy c

Measurement of morphometric parameters (e.g., mean diameter, minimum diameter, maximum diameter, volume, area and circularity).

52

I would first like to thank to Dr Nadia Benkirane-Jessel and Dr Maria de la Fuente Freire who did me the honor of accepting to review this manuscript. I also thank to Dr Anne-Marie Haghiri, Pr Gert Storm and Dr Catherine Le Visage for accepting to be part of my PhD jury.

After three years spent in his research team, I would like to sincerely thank to my PhD director Pr Patrick Couvreur who gave me the opportunity to join a dynamic and international environment of high scientific level. My professional growth would not be the same without his contribution. His great experience and his passion for research taught me to enjoy the success as well as the failures. Thank you for the optimism through which you have supported my work and my personal motivation.

I would like to express my deepest gratitude to my supervisor and PhD co-director Dr Simona Mura, the "engine" of this research project. Through scientific discussions and simple exchanges, she has daily helped me to face small and big issues guiding this work in the right direction. Her dedication to work inspired me and was example of perseverance. Thank you for your constant availability and support; I will never forget when we tried to "save" the experiments with spheroids (after weeks of cell culture work) during the general blackout; we were the only two persons stayed in the university in spite of the evacuation order. Thank you for your professional rigor through which I learned how to do high-quality research. Since life is not only work and sometimes there are sad events, I will be always grateful for what you have done during my unhappy personal moments.

A special thanks to all the people of the Galien Institute, with a special mention to its director, Pr Elias Fattal, who has been always attentive to the student need. Every day he had a smile or an Italian word for me, thank you.

Thank you to Dr Julien Nicolas for his scientific advices and for the nanoparticle picture that I use since my first PowerPoint presentation three years ago. Thank you to Stéphanie Denis for her help in the cell culture lab and for her sincere friendship. Many thanks to Sylvie Zemmour, she always answered to all my administrative questions and "took care" of my reagent orders.

Thank you to Dr Juliette Vergnaud, Dr Nicolas Tsapis, Dr Hervé Hillaireau and Dr Mariana Varna for the precious scientific exchanges.

Dr Anne-Marie Haghiri and Dr Gilgueng Hwang enriched the scientific value of this project by allowing me to work in the nanotechnology laboratory of Marcoussis. I am very thankful for their technical and scientific support in the construction of the microfluidic device.

Spending time in their research team has been for me an occasion of professional and personal growth.

Several peoples from platforms contributed to the success of many experiments and enriched my scientific and technical knowledge. I am very thankful to Velérie Nicolas not only for her precious support with the confocal microscopy but also for her gentle approach and the sincere friendship. Thank you to Delphine Courilleau for bioluminescence measurements and for the almost daily scientific (and not only) discussions on the way to Châtenay Malabry in the 294 bus. Thank you to Meriem Garfa-Traore and Nicolas Goudin for the great help with the light-sheet microscopy. Through their experience, I have learned to exploit the advantages of this innovative microscope and it was also a pleasure to spend time in their friendly laboratory and to have lunch with them on the roof of the Imagine Institute. Thank you to Maryline Favier, Fabiola Ely-Marius and Rachel Onifarasoaniaina of the histology platform for their contribution and their kind availability.

I am grateful to all the PhDs and post-docs of the Galien Institute that made this experience unforgettable! During these three years, I had the chance to meet very friendly colleagues.

Alexandre, Anaelle, Arnaud, Céline, "ClaireAlbert", Corentin, Elise, Flavio, Isabella, Johanna, Julie, Julien, Maria, Marie, Mathilde, Quentin, Raul, Roberta, Romain, Sophie, Tanguy, Teresa, thank you to each of you for the beautiful moments spent together. I am particularly grateful to Marion for her sincere friendship. Thank you for being always attentive to me and for your daily "ça va?"; you were at my side in the happy and unhappy moments and I am conscious that your support will continue in the future. Special thanks to Pierre-Louis (Pilou) for our never boring discussions. Thank you for the lessons on the French polite words and for being a very good friend. Thank you to my professional mate, Daniele, with whom I shared this great PhD adventure made of scientific collaborations, seminars, European congress, travels, burgers and beers. The Italian crew could not be the same without Federica, thank you for the funny moments and the typical fondness of an Italian friend.

Thank you to Anna for her support in these last months, probably the most nonsocial period of my PhD.

General introduction

In the last decades, various engineered systems for drug delivery (e.g., nanomedicines) have been developed with the aim to overcome the limits associated to conventional chemotherapy, such as non-specific drug distribution, poor delivery to the target tissue and rapid metabolism.

However, the success of new therapeutic strategies in the clinic is still suboptimal and only a limited number is currently marketed.

A discrepancy between promising preclinical in vitro results and the in vivo performances is often observed in the early stage of drug development and might be ascribed to the lack of capacity of the models commonly used for in vitro studies to faithfully reproduce the pathophysiology of solid tumors. These models mainly consist of cancer cells cultured as flat (two dimensional, 2D) monolayers or assembled to form three dimensional (3D) multicellular tumor spheroids (MCTS).

However, being composed exclusively of one cell type, these models are too simplistic. They do not allow to reproduce the heterogeneous cellular composition, as well as, the complex architecture of the tumor and its surrounding microenvironment. Thus, they fail to replicate the multiple biological barriers that drugs and nanomecidines have to cross in order to reach the target cells.

The aim of this PhD thesis was to overcome these limitations and construct a reliable tool for an appropriate in vitro evaluation of the therapeutic potential of nanomedicines and other chemotherapies. Attention has been focused on the pancreatic ductal adenocarcinoma (PDAC) whose strong fibrotic reaction represents a well-known example of a tumor biological barrier responsible for the limited efficacy of the treatments. The main part of this research work relies on the construction and complete characterization of novel hetero-type MCTS based on a triple co-culture of pancreatic cancer cells, fibroblasts and endothelial cells, and thus capable to integrate the cancerous component and the microenvironment of the tumor. The constructed 3D model has demonstrated the capacity to reproduce in vitro the influence of the microenvironment on the sensitivity of cancer cells to chemotherapy. In addition, by combining the 3D model and the innovative Light Sheet Fluorescence Microscopy (LSFM), we have been able to investigate the penetration of the anticancer drug doxorubicin (in a free form and loaded into nanoparticles (NPs)) in a high informative manner. Then, in order to acquire a better understanding on how nanomedicines and other anticancer chemotherapies interact with the tumor, we sought to combine the hetero-type 3D culture with controlled flow conditions in a microfluidic device. To reach this goal we have designed and fabricated a tailor-made chip suitable to host both a MCTS and a perfusable microvascular network (i.e., MCTS-on-a-chip).

An introduction and three chapters on the experimental work compose this thesis and are presented in the form of articles that have been already published or are currently in preparation.

The Introduction of this thesis is a bibliographic review focused on spheroids as model for the preclinical screening of nanomedicines. After a brief overview on current analytical and imaging techniques applied in the experimental settings, relevant examples of the application of MCTS to the in vitro evaluation of polymer nanomedicines with different physicochemical properties are presented.

The Chapter I describes the step-by-step construction and characterization of a novel heterotype MCTS based on a triple co-culture of cancer cells, fibroblasts and endothelial cells. It is demonstrated how the presence of a microenvironment composed of different cell types affects the sensitivity of the model towards two cytotoxic drugs with a different mechanism of action (i.e., gemcitabine and doxorubicin). The 3D imaging strategy used to assess the drug penetration through the spheroid is detailed in Chapter II in which it is demonstrated that the precise assessment of doxorubicin diffusion in the hetero-type MCTS could be achieved by the fusion of multi-view images collected with the LSFM, while a lower degree of information could be collected through the conventional Confocal Laser Scanning Microscopy (CLSM). Finally, the Chapter III discusses the design and custom-made construction of a microfluidic device which would combine the 3D culture and the controlled flow conditions. In particular, we detail the optimization of the photo-lithography protocol through which we have achieved specific geometrical features that will be crucial to ensure i) a spatial control of all the cellular components and ii) the organization of endothelial cells in luminized perfusable vessels.

Introduction générale

Au cours des dernières décennies, des systèmes de taille nanométrique chargés en principes actifs (nanomédicaments) et des nouvelles stratégies thérapeutiques ont été développés afin de surmonter les limitations liées à la chimiothérapie conventionnelle telles qu'une distribution non spécifique, une mauvaise accumulation dans les tissus cibles ainsi qu'une métabolisation rapide. Cependant, le succès des nouveaux médicaments en clinique reste encore limité et seulement un faible nombre de nanomédicaments est actuellement commercialisé.

Une divergence entre les résultats précliniques in vitro et les performances obtenues in vivo est souvent observée dans la première étape du développement d'un médicament. Cet écart pourrait être attribué au manque de modèles pertinents, représentatifs de la pathologie observée chez l'Homme et qui soient de bons prédicteurs de la réponse thérapeutique chez les patients. En effet, les modèles utilisés aujourd'hui (généralement culture cellulaire en deux dimensions, 2D) ne reproduisent pas la structure complexe de la tumeur in vivo. Ainsi, ils ne permettent pas une évaluation fiable du potentiel thérapeutique réel des médicaments. Dans cette optique, les méthodologies de culture de cellules en trois dimensions (3D) sont extrêmement avantageuses. Ces méthodologies permettent, en effet, la construction de systèmes cellulaires pertinents qui reproduisent in vitro la relation entre les cellules cancéreuses et leur microenvironnement. Parmi ces modèles, l'assemblage de cellules sous forme de sphéroïdes multicellulaires a été largement exploré. Néanmoins, les sphéroïdes décrits jusqu'à présent correspondent à des nodules formés exclusivement de cellules cancéreuses, ce qui constitue une vraie limitation. En effet, ces sphéroïdes ne reproduisent pas l'organisation de la tumeur et l'hétérogénéité du microenvironnement et, par conséquent, ils ne parviennent pas à mimer les multiples barrières biologiques que les médicaments doivent traverser pour atteindre les cellules cibles.

Dans cet esprit, l'objectif de cette thèse de doctorat était de surmonter ces limitations et de construire des modèles pertinents qui reproduisent in vitro la relation entre les cellules cancéreuses et leur microenvironnement afin de i) mieux comprendre les mécanismes de passage des nanomédicaments et ii) mieux prédire l'efficacité des nouveaux traitements.

Au cours de cette thèse nous nous sommes intéressés au cancer du pancréas qui est caractérisé par la présence d'un abondant stroma formant un bloc fibreux (réaction desmoplastique) qui limite la pénétration des médicaments et réduit ainsi leur efficacité. Cette tumeur représente donc un bon exemple de barrière biologique tumorale.

La partie principale de ce travail de recherche repose sur la construction et la caractérisation complète d'un nouveau type de sphéroïde multicellulaire, capable de reproduire in vitro la relation entre les cellules cancéreuses et leur microenvironnement, grâce à la co-culture de cellules cancéreuses pancréatiques, de fibroblastes et de cellules endothéliales. Les études de cytotoxicité in vitro nous ont permis d'investiguer la capacité de ce modèle à reproduire la résistance des cellules cancéreuses aux traitements observés in vivo. Grâce à la Microscopie de Fluorescence à Feuillet de Lumière nous avons pu étudier la pénétration au sein des sphéroïdes de la doxorubicine, soit en forme libre soit encapsulée dans des nanoparticules.

Ensuite, afin de mieux comprendre comment les médicaments et nanomédicaments interagissent avec la tumeur, nous avons cherché à combiner la culture 3D avec des conditions dynamiques contrôlées dans un dispositif microfluidique. Pour atteindre cet objectif, nous avons conçu et fabriqué une puce sur mesure, adaptée pour loger à la fois le sphéroïde et des canaux dans lesquels les cellules endothéliales pourront s'organiser sous forme de vaisseaux.

Une introduction bibliographique et trois chapitres expérimentaux composent cette thèse et sont présentés sous forme d'articles déjà publiés ou en cours de préparation. L'Introduction de cette thèse est une revue bibliographique concertante les sphéroïdes en tant que modèles pour le criblage préclinique des nanomédicaments. Après un bref aperçu des techniques expérimentales d'analyse et d'imagerie appliquées à cette problématique, l'introduction présente des exemples pertinents de l'application des sphéroïdes dans l'évaluation in vitro de nanomédicaments polymères présentant différentes propriétés physicochimiques.

Le Chapitre I décrit la conception et la caractérisation, étape par étape, d'un nouveau modèle 3D de type sphéroïde multicellulaire basé sur une triple co-culture de cellules cancéreuses, de fibroblastes et de cellules endothéliales. Il a été démontré comment la présence d'un microenvironnement complexe affecte la sensibilité des cellules tumorales vis à vis de deux principes actifs cytotoxiques ayant un mécanisme d'action différent (à savoir la gemcitabine et la doxorubicine). Le Chapitre II démontre que l'évaluation précise de la pénétration de la doxorubicine au sein d'un sphéroïde 3D peut être réalisée grâce à la reconstruction d'images multi-vues acquises via la Microscopie de Fluorescence à Feuille de Lumière, alors que seulement un faible degré d'information peut être obtenu par Microscopie Confocale à Balayage Laser conventionnelle. Enfin, dans le Chapitre III, nous discutons de la conception et de la construction d'un dispositif miniaturisé réalisé sur mesure et qui permettra la combinaison de la technologie de la microfluidique et de la culture cellulaire en trois dimensions. Les différentes étapes de l'optimisation du protocole de photolithographie sont détaillées. Elles ont permis d'obtenir des puces avec des critères géométriques bien définis. Ces éléments permettront de garantir i) le contrôle spatial des différents types de cellules ainsi que ii) l'organisation des cellules endothéliales en forme de vaisseaux qui pouvant être perfusés.

Introduction Multicellular tumor spheroids: a relevant 3D model for in vitro preclinical investigation of polymer nanomedicines

Gianpiero Lazzari, Patrick Couvreur, Simona Mura*

Introduction

The efficacy of conventional therapies is often limited by a non-specific cell/tissue distribution of drugs, their rapid metabolization and/or excretion from the body. In this context, nanoscale systems for drug delivery (i.e., nanomedicines) have received in the last decades a tremendous attention because they hold the potential to overcome these limits, providing a solution to medical challenges that urgently require novel therapeutic strategies. [1][2][3] Nanomedicines can indeed improve the therapeutic index of the transported drug by (i) offering protection from degradation, (ii) enabling controlled release and distribution and (iii) increasing bioavailability. In particular, being cancer one of the leading causes of death worldwide, much work has been done in this field with the aim to propose more efficient treatments.

Accordingly, a plethora of well-engineered nanomedicines surface-modified with cell targeting ligands 4 or endowed with stimuli-responsiveness 5 has been designed. However, despite the encouraging results observed in preclinical experimental models as well as in clinical trials, 6,7 the introduction of nanoscale drug delivery systems in the clinical practice is not straightforward, 8,9 and only a limited number of nanomedicine reached the market place.

They include for instance lipid and natural protein-based nanomedicines such as the Doxorubicin(Doxo)-loaded liposomes (i.e., Doxil ® , Myocet ® ) and paclitaxel albumin-bound nanoparticles (NPs) (i.e., Abraxane ® ). Noteworthy is that so far not any nanomedicine based on synthetic polymers has been introduced in the market in spite of the high versatility offered by the macromolecular synthesis and the possibility to opportunely tune the polymers properties (e.g., composition, structure, functionalization, degradability etc…). [10][11][12][13] It has to be noted that reaching the biological targets and ensuring sufficient drug delivery and accumulation is extremely challenging as consequence of the multiple biological barriers that characterize tumors and their microenvironment, which are crossed by nanomedicines at low efficiency or not at all. 14,15 However, such biological transport barriers are generally not taken into account during in vitro preclinical evaluation of nanomedicines, the majority of which is still routinely carried out on two dimensional (2D) monocultures of isolated cancer cells.

Despite their relative ease of handling, these cultures do not show any structural architecture and lack the complex physiology and the microenvironment of the real tumor tissues, which consist of different cell types (e.g., fibroblasts, macrophages, endothelial cells, immune cells) embedded in an extracellular matrix (ECM) mainly composed of fibrous proteins and proteoglicans. [16][17][18] Cells cultured in 2D monolayers display altered gene expression and activation of signaling pathways compared to cells grown in the native tumor tissue. 19 Moreover, growing in a single layer, they do not replicate neither (i) the cell-to-cell and cellto-ECM interactions [20][21][22] nor (ii) the oxygen, nutrients and pH gradients, 23 which play a crucial role in tumor progression, chemoresistance and metastatic spread. 24 Accordingly, there is an urgent need for more relevant models capable to closely mimic the heterogeneity and the microenvironment of the in vivo condition thus allowing a more predictive in vitro evaluation of nanomedicines. In this context, three dimensional (3D) culture models such as multicellular spheroids (MCTS), polymer scaffolds and microfluidic systems have been proposed as an alternative approach to overcome the aforementioned limitations. [25][26][27][28][29][30][31][32] 3D tumor models are indeed capable to recapitulate some key features of the real tumors thus representing a valuable tool for (i) a more accurate preclinical screening of nanomedicines and (ii) the identification of the candidates with the highest chances of success, which would be further evaluated in vivo. According to their predictive capacity, 3D models would enable to limit the number of animals required in preclinical studies allowing to abide by the 3Rs guidelines. 33,34 Although the application of 3D models for systematic assessment of therapeutic efficacy of nanomedicines is still at the beginning, their superiority compared to the 2D models is nowadays clearly acknowledged. This review will provide the reader with an overview of the application of multicellular tumor spheroids, the widest employed 3D tumor model so far, for evaluation of nanomedicines highlighting their usefulness as discriminating tool. In agreement with the scope of the journal, attention has been focused on drug delivery systems made of polymers or in which polymers have been used for surface modifications.

3D multicellular tumor spheroids

Multicellular tumor spheroids are scaffold-free spherical self-assembled aggregates of cancer cells displaying an intermediate complexity between 2D in vitro cell cultures (i.e., cell monolayers) and in vivo solid tumors with which they share important similarities. Since their first introduction in the early 1970s by Sutherland and coworkers, 35,36 the application of multicellular tumor spheroids in drug discovery has grown exponentially, offering the possibility to screen a large variety of different molecules [37][38][39] and they are currently considered a suitable 3D model for drug evaluation in the oncology field. 37,40 The physiological communication and the signaling established between cells growing in close contact make possible to reproduce in spheroids key aspects of the tumor and its microenvironment such as: (i) different cell proliferative rates, (ii) specific gene expression, (iii) deposition of ECM components, (iv) cell-to-cell and cell-to-microenvironment interactions and (v) drug resistance. [40][41][42] Still, a limitation of MCTS relies on the fact that they mimic only the avascular region of in vivo tumor tissues thus leaving out relevant aspects of real tumorigenesis such as the surrounding vasculature, the immune system components as well as the fluid dynamics. 38,41 Large spheroids (~400-500 µm diameter) display an internal layered cell distribution analogous to that observed in solid tumors. This is the result of mass transport limitations, which interfere with the diffusion of oxygen, nutrients and metabolic wastes through the spheroid creating specific gradients. 43,44 Accordingly, thanks to the easier access to oxygen and nutrients, highly proliferating cells are located in the external layer of spheroids and correspond to tumor cells close to capillaries in vivo. Conversely, quiescent cells characterize the middle layer, because cell metabolism decreases progressively with the increasing distance from spheroid periphery. Finally, a critical situation characterizes the spheroid core in which the oxygen depletion (i.e., hypoxia), the nutrient shortage and the metabolic waste accumulation result in cell necrosis (Fig. 1). 40,[43][44][45] The organization in cell layers and the presence of diffusive gradients, acting as microenvironmental stresses, force the inner cells to a specific metabolic adaptation, 43 responsible of the observed impaired therapeutic efficacy of various anticancer drugs or drug-loaded nanocarriers. 40,46 For instance, cells in the hypoxic region are resistant to drugs which promote cellular death through reactive oxygen species, while the existence of necrotic and quiescent cells reduces the therapeutic efficacy of drugs active against the proliferating ones. 40 Another important feature of MCTS is the presence of a network of structural (i.e., collagen and elastin) and adhesive (i.e., fibronectin and laminin) ECM proteins embedded in a gel of glycosaminoglycans and proteoglycans. 40,42 This microenvironment acts as a regulating factor influencing cell proliferation, differentiation and tumor growth. 41,43 In addition, it closely mimics the physical barriers found in real solid tumors, which obstruct the free penetration of drugs thorough the whole mass. 40 Undoubtedly, the presence of this tumor microenvironment represents an advantage compared to the conventional monolayer cultures although it has to be noted that ECM components found in MCTS present a different cellular origin compared to those present in the in vivo tumor tissues. In the former, they are secreted by the same cancer cells forming the spheroids while in the latter by the tumor-associated fibroblasts (TAF). 42 To face this issue, spheroids composed of cancer cells and components of the supportive stroma have been recently proposed. 47,48 These hetero-type multicellular spheroids better reproduce the cellular heterogeneity of the tumor tissues and are expected to allow a more reliable evaluation of cancer cells-microenvironment interactions and their impact on the therapeutic outcomes. 43, 47- 49 Fig. 1 Schematic representation of cell organization and gradients in tumor spheroids.

Adapted with permission from ref. 44. Copyright 2008 Wiley-VCH.

3D MCTS culture methods

Different techniques for spheroid construction are currently available and many of them have been optimized for large-scale production under highly reproducible conditions. 40 Principally they involve the use of cell attachment-resistant surfaces or physical forces to promote cell-tocell interactions and support the 3D spheroid formation (Fig. 2). These techniques will be briefly discussed in the following paragraphs while for a more detailed and extensive description the interested reader could refer to recently published reviews. 38,41,45,50,51

Liquid overlay methods

This technique mainly relies on the use of non-adherent 96-well plates, in which a coating with poly-2-hydroxyethyl methacrylate (poly-HEMA) [52][53][54][55][56][57][58] or agarose prevents cell attachment. This method allows to prepare both mono and hetero-type spheroids, whose size and morphology can be finely tuned by varying the number of cells seeded in each well. 48,50,51,86 The large number of produced spheroids and the ease of handling make this approach applicable to different kind of high-throughput investigations. 51,86 For instance, cells could be seeded using a bulk liquid dispenser or a liquid handling robot, which also offers the possibility of scaling-up the spheroid fabrication. 37 Compared to the agitation-based approaches, the liquid overlay methods require lower volume of media, and testing materials (e.g., drugs). 50,87 However, a major drawback is the time-consuming plate-coating step.

Although pre-coated low cell adhesion plates are commercially available (e.g., Ultra-low Attachment Surface from Corning ® ; PrimeSurface low adhesion culture plate from Sumitomo Bakelite Co.; Lipidure ® -coated plates developed by NOF Corporation), their use increases the overall costs. 38

Hanging drop methods

This technique exploits the surface tension to hang small cell suspension droplets (20-50 µL) on the underside of a tissue culture dish lid. 44,51 Then, gravity drives cells accumulation at the tip of the drop (liquid-air interface) and induces their rapid aggregation into a single spheroid. 44,50 By changing the cell density, control of spheroid size has been achieved with a high reproducibility for numerous cell lines 38 both in mono and co-cultures. 47 However, the volume of the seeding suspension is generally limited and does not provide nutrients enough for a long term culture. 44,50 Therefore, once formed, spheroids need to be transferred on 96well plates or embedded in polymer matrices for further studies. [88][89][90][91][92][93][94][95] Not only such transfer can affect the integrity of spheroids but clearly makes this technique extremely labor intensive and time-consuming thus preventing large scale application. 44,50,51 Commercially available platforms (InSphero GravityPlus™ technology and PERFECTA3D™ hanging drop plates of 3D Biomatrix) can reduce the workload and enable to carry out experiments in a high-throughput manner, although with a significant increase of the expenses.

Agitation-based approaches

Spinner flasks and rotational culture systems (i.e., NASA Rotary Cell Culture System) are bioreactors in which cell aggregation and spheroid formation are mediated by a continuous spinning that keeps in motion a cell suspension supporting cell-to-cell interactions and avoiding their attachment to the container wall. 38 The movement of the medium provides a controlled environment and maintains the transport of nutrients and waste allowing (i) longterm culture and (ii) large-scale production. However, although by tuning cell-seeding density, spinning rate and culture time it is possibly to influence the average spheroid diameter, 44 the overall control on the number of cells per spheroid and their size is extremely poor. 38,50 Therefore, manual selection of similarly sized spheroids and their transfer in different supports are mandatory before any further assay (e.g., cytotoxicity, penetration, etc…). [96][97][98] To be noted that only cell lines that can withstand to high shear stress can be cultured in spinner flasks. 38,50 For less resistant cells types, the NASA Rotary Cell Culture System characterized by lower shear forces, thanks to simultaneous rotation of the culture chamber together with its content, should be instead preferred. 38,44 However, both approaches require specialized equipment and large amount of culture media, which therefore limit their large application. 38

Patterned surfaces and microfluidic devices

Patterned surfaces in combination or not with microfluidics devices have been recently developed and offer a new range of advanced strategies for spheroid construction. 37,38,[99][100][101][102] For instance, spheroids with defined size and composition have been generated using arrays of microwells fabricated with the micromolding or the photolithography technique. 37,44,45,103,104 Low attachment surfaces have been obtained using non-adherent materials such as the poly(dimethyl siloxane) (PDMS) 46,105,106 or applying a coating of agarose [107][108][109][110] or Pluronic ® F-127. 48 Compatible with traditional well plate formats and microscopes, such microwell plates are suitable for high-throughput screening. 111 Similarly, formation of cellular aggregates in a controlled environment and with high simplification of the liquid handling procedures has been obtained with microfluidic systems containing various micro-sized chambers and channels. 45,50,102 Requiring only limited amounts of cells, media and reagents they are extremely attractive and convenient for drug screening applications, however the impossibility to retrieve and extensively characterize the formed spheroids is a general drawback of these approaches. 38,111 Further level of complexity is achieved with microfluidic devices displaying (i) distinct compartments loaded with different cell types (e.g., epithelial cells, endothelial cells and fibroblast), (ii) collagen gel inserts and (iii) variously shaped channels, which ensure the cell-to-cell chemical communication thus mimicking the complex in vivo-like organizations. [111][112][113] Nevertheless up to now the complexity and the costs of the equipment required for their use have hindered their wide application in preclinical investigation of nanomedicines.

End-points assessment in 3D MCTS

Thanks to the capacity to recreate key features of the real tumors, 3D MCTS have been largely used for the assessment of the efficacy of various therapeutic strategies. In this context, assays and detection methods specific for the 3D cultures are highly required to carry out an accurate and predictive evaluation. Being their detailed description out of the scope of this review, the most common techniques are only summarized in the next paragraphs, while a comprehensive presentation can be found in recently published articles. 37,[39][40][41]45 Optical microscopy is the simplest and widely used technique for the visual assessment of morphological changes in spheroids. 52, 55-58, 60-65, 70, 73, 74, 77, 84, 89-93, 95, 107, 110, 114-126 Following exposure to various treatments, the measure of the variation of morphometric parameters (e.g., mean diameter, minimum diameter, maximum diameter, volume, area and circularity) of spheroids from bright field images is used for a quantitative analysis of the extent of growth inhibition. 37,87 After interruption of the treatment, the spheroid growth delay might be calculated in comparison to untreated samples as the time lag required to reach the quintuple spheroid volume of day 4 (5 × V d4 ; assuming V d4 as the starting spheroid volume at the onset of treatment). 87 The assays currently used in 2D monolayer cultures as extensively validated methods to assess the drug cytotoxicity mainly rely on the measurement of (i) cell membrane integrity (i.e., LDH assay) 127 or (ii) intracellular metabolic activity (e.g., MTT, WTS-1, AlamarBlue ® assays). 128 Some of them have been adapted to spheroids and are currently commercially available. Among them, the quantification of the Acid Phosphatase (APH) activity and the intracellular adenosine triphosphate (ATP) level are well-suited assays that do not require preliminary dissociation of spheroids into single-cell suspensions. 37,45 The APH assay is a simple and inexpensive technique based on the quantification of cytosolic acid phosphatase activity in viable cells through the measurement of the absorption of the p-nitrophenol, obtained by APH hydrolyzation of p-nitrophenyl phosphate, at 405 nm using any standard plate detection reader. 37,129 The APH assay has been validated to measure spheroid viability and cytotoxic effects of both free drugs 87,129 and drug-loaded nanocarriers. 82,95,115,124 The ATP assay exploits the oxidation of the luciferin, the substrate of the firefly luciferase enzymes, in presence of O 2 , Mg 2+ and ATP. This reaction brings the luciferin to an electronically excited state, which then decays with the emission of a photon of light (i.e., bioluminescence). Thus, when substrate and enzyme are added to cell cultures the light output, which is directly proportional to the intracellular ATP content (marker of metabolically active cells), allows to quantify the number of viable cells. Ready-to-use assay kits, which exploit the high sensitivity and the low background of the bioluminescence signal 45 (e.g., CellTiter-Glo ® 3D Cell Viability Assay (Promega); Molecular Probes ® ATP Determination Kit (Invitrogen,)) are currently available. The CellTiter-Glo ® 3D Cell Viability Assay represents a time-effective and well standardized assay since the addition of a single reagent directly to the 3D cell culture results into cell lysis and simultaneous generation of the bioluminescence. 130 This assay is especially formulated with robust lytic capacity to overcome 3D MCTS obstacles such as tight cell-cell junctions and the presence of the extracellular matrix. 130 The relatively simple workflow and data analysis make this ATP assay scalable to highthroughput screenings of drug-loaded nanocarrier efficacy. 125 Qualitative assessment of proliferating or dead/necrotic cells can be performed by Confocal Laser Scanning Microscopy (CLSM) imaging following spheroid incubation with various live/dead reagents. 46,118,131 Preliminary spheroid dissociation into single cells is instead required for a quantitative measurement by Fluorescence-Activated Cell Sorting (FACS). 70 Staining cell dispersions of dissociated spheroids with fluorescently labeled Annexin V (AnnexinV-FITC) and propidium iodide (PI) has also been largely used to detect early apoptotic and dead/necrotic cells, respectively. 96,[132][133][134] Apoptosis can be detected also on spheroid cross-sections by using the Terminal deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling (TUNEL) method. The TdT recognition of the blunt ends of double stranded DNA breaks, which characterizes the late stages of apoptosis, 135 catalyzes the addition of biotinylated dUTPs then visualized using streptavidin-conjugated detection agents (i.e., peroxidase or fluorescent markers). 56,136 Monitoring of fluorescent drugs and dye-labeled nanocarriers by CLSM has been largely applied for evaluating their uptake and penetration in individual spheroids. 63,66,69,76,95,105,133 Unfortunately, compared to conventional 2D cultures, the 3D structure represents a technical challenge for conventional instrumentation. Indeed, the increase of spheroid size and thickness result in loss of image quality due to scattering, reflection and absorbance of light. 37,41 The layered structure of spheroids limits the inner scanning depth and, as consequence, the penetration ability of different systems can be compared only on a portion of the spheroid generally corresponding to a depth of 80-100 µm from the spheroid surface. 63,76,95,131,137 Although more time consuming, a better information on the real depth of penetration can be provided by acquisition of fluorescent images from serial sections obtained from fixed spheroids. 46, 71, 88, 96-98, 138, 139 Compared to traditional CLSM, significant improvements in the evaluation of the penetration in living tumor spheroids can be obtained with Two-Photon (TPM) or Multi-Photon microscopy (MPM) in which the energies of two (or more) photons are combined to promote the transition of a fluorescent marker to an excited state. 140 The use of excitation wavelength in the NIR region (700-1000 nm) offers the possibility to increase the laser penetration into 3D MCTS (up to 500-800 µm according to the instrumentation) with low phototoxic effects. 50,140 These techniques have been already applied to visualize with high resolution the diffusion through spheroids of various polymer-based nanocarriers. 70,81,85,120,137,141 Imaging cellular processes and drug delivery in spheroids with an even superior 3D isotropic resolution and limited photodamage are the promises of Light-Sheet Fluorescent Microscopy (LSFM) methods such as the Selective Plane Illumination Microscopy (SPIM) 47,142,143 already applied to the imaging of large biological samples. 144,145 However so far they have not found application yet in the preclinical investigation of polymer nanomedicines. Imaging of nanocarrier diffusion ability. Pinhole-equipped microscope to remove out-of-focus light and increase optical resolution. Light penetration depth limited to 100-150 µm from spheroid surface.

Use of 3D MCTS to screen different physico-chemical properties of polymer nanocarriers

Distinguishing characteristics of polymer nanocarriers, such as chemical composition, size, shape and surface properties might strongly affect their capacity to diffuse into tumors and therefore have a profound impact on their anticancer efficacy. 158 Accordingly, these parameters must be taken into account during the design and evaluation of any novel nanomedicine. In this context the 3D MCTS models, thanks to their similarity in morphology and biological microenvironment to solid tumors, have already been used as robust tool for easy polymer nanoparticle screening, 70,97 and to accurately predict the in vivo behavior of the nanocarriers as function of their specific physicochemical properties. 139 The most relevant results will be discussed in the following sessions.

The size effect

The influence of size on the penetration profile of nanoscale systems has been clearly highlighted and, according to the general trend observed in vitro with 3D MCTS and further confirmed in vivo, it is evident that the diffusion capacity is inversely correlated to the particle size. 56,72,97,98,139,148 For instance, nanogels obtained by chelating ligands-modified hyaluronic acid were able of deeper penetration into MKN74 gastric cancer spheroids compared to the polymer in the linear form and their small size (21 nm) ensured a uniform distribution over the entire 3D mass. 106 Similarly, following release from poly(ethylene glycol) (PEG)-poly(d,l-lactide) (PLA) nanoparticles, in which they have been loaded, small polyamidoamine (PAMAM) dendrimers (G2, 2 nm) penetrated efficiently into the core of KB papilloma cells spheroids (diameter ~ 50 µm) compared to their larger counterparts (G4 (5 nm) and G7 (7 nm)) whose florescent signal was detected only in the peripheral rim. 72 Noteworthy is that the size plays a major role in the capacity of tumor penetration even in case of surface functionalization with specific ligands. By using a murine breast cancer MCTS model (4T1 cells) it was observed that RGD functionalization endowed Pluronic F127coated paclitaxel (Ptx) nanocrystals with better penetration ability compared to the free drug (Taxol) and to the non-functionalized carriers. However, for nanocrystals displaying similar functionalization, a smaller size (10 nm vs 70 nm) conferred a greater advantage in terms of depth of penetration (Fig. 3a). 56 It has to be noted that not any difference was observed when the particles were evaluated on a 2D culture model, thus highlighting the usefulness of the 3D MCTS to discern the crucial role of the size. TUNEL staining of spheroid's cross-sections revealed that the deeper penetration was associated to a more extensive cell apoptosis, which caused spheroid growth inhibition and their further collapse following prolonged drug exposure (Fig. 3b,c). 56 In spite of these results, the identification in vitro of the optimal size of a nanocarrier to achieve the highest drug delivery in vivo is extremely complex because although MCTS closely mimic the tumor tissues they lack of predictive value in terms of pharmacokinetics and biodistribution.

Accordingly, evaluation of nanomedicines in animal tumor models still remains a mandatory step to assess these parameters. It has been indeed shown that the requirements for tumor penetration and tumor retention are often in contradiction to each other. Thus, despite unfavorable for penetration in the tumor mass, due to their considerable diffusional hindrance, 159 nanocarriers with a size of around 100 nm, are the most advantageous in improving pharmacokinetics and extravasation. 160 On the other hand, smaller nanocarriers show much better penetration in the tumor interstitial space. 139,148 However, for extremely small particles ( ≤ 5.5 nm), rapid clearance from tumors and short halflife have been observed. 161 To face this issue, the ideal drug delivery system should be able to shrink and adapt its size in response to the encountered microenvironment. Interestingly, sizeswitchable stimuli-responsive nanoparticles able to overcome multiple tumor barriers have been developed by various groups. 58,76,119,133,134,138,141 For instance, pH sensitive nanoparticles were formulated by the molecular assembly of platinum (PtIV)-prodrug conjugated polyamidoamine (PAMAM) dendrimers with two amphiphilic polymers contain either ionizable pH-responsive amide bonds (i.e., polycaprolactone-2-propionic-3-methylmaleic anhydride (PCL-CDM), PCL-CDM-PAMAM/Pt nanoparticles) 134 or tertiary amine groups (i.e., poly(ethylene glycol)-b-poly(2azepane ethyl methacrylate) (PEG-b-PAEMA) PEG-b-PAEMA-PAMAM/Pt) (Fig. 4a). 133 Size variation as function of pH has been evaluated on pancreatic cancer multicellular spheroids (BxPC3 cells). At physiological pH the clustered nanoparticles (i.e., pH-sensitive cluster nanobombs (SCNs/Pt)) displayed a size of around 100 nm, while the pH drop (from 7.4 to 6.5-7) in the tumor extracellular space triggered an instantaneous disassembly of these pH sensitive nanoparticles in small Pt-PAMAM prodrugs (≈5 nm) able to penetrate deeply and uniformly into the spheroid mass (Fig. 4b). Then, once internalized, the intracellular redox environment led to the release of the active molecule (i.e., Pt(II) species) resulting in significant cell apoptosis. As expected a higher cell viability was observed when spheroids were incubated with pH-insensitive nanoparticles (i.e., ICNs), which demonstrated limited capacity of penetration and drug delivery as a consequence of their stable size (Fig. 4b). Whether such advantage was maintained in vivo was then assessed after intravenous injection of both nanoparticles in an experimental model of pancreatic cancer (BxPC3 cancer cells) characterized by an important desmoplastic reaction and limited permeability. 162 Tumor accumulation studies confirmed a higher capacity of penetration of pH-sensitive nanoparticles, which diffused in the tumor interstitium, after vessel extravasation, for several hundreds of nanometers. On the contrary, the pH-insensitive NPs accumulated in the tumor vessels with little penetration in the tumor mass (Fig. 4c) leading to an inefficient drug delivery. In agreement with the in vitro results, an extensive apoptosis was detected in tumor sections of mice treated with the pH-sensitive nanoparticles, thus confirming the predictive potential of the 3D multicellular spheroids. Copyright 2016 American Chemical Society.

The shape effect

3D tumor spheroids have found application also for the early screening of nanocarriers with different shape (i.e., spherical vs elongated such as filaments, rod-like or worm-like vectors). However, whether this parameter may positively or negatively affect the nanocarriers penetration, the cellular uptake and the therapeutic efficacy still remains matter of debate. 85,120,147 For instance, when worm-like poly(ethylene oxide-b-ε-caprolactone) (PEO-PCL) photosensitizer-loaded micelles were compared to spherical ones on 3D models of colon (HCT-116 cells) and head and neck cancer (FaDu cells), surprisingly not any advantage in terms of photodynamic therapy efficiency was observed in contradiction with the previously reported superior uptake of so-called "filomicelles". 163 Nevertheless, the different size of the studied systems as well as other parameters such as the nanocarrier length, rigidity and surface properties might explain such differences and would need to be further explored. 120 The influence of length was studied by Stenzel and coworkers, which investigated rod-shaped poly(1-O-methacryloyl-β-D-fructopyranose)-b-poly-(methyl methacrylate) (poly(1-O-MAFru)-b-PMMA)-based micelles, clearly showing that, among the various fructose-coated rod-like micelles, the shortest ones displayed the highest capacity of penetration in a 3D MCTS of breast cancer cells (MCF-7). 147 Similarly, in order to better highlight the role of these parameters, poly(ethylene glycol) diacrylate (PEGDA)-based anionic nanohydrogels were synthetized by Jet and Flash Imprint Lithography in form of disk-shaped nanocylinders and cuboidal nanorods of two different sizes (low and high aspect ratio; aspect ratio (H/D) = height/diameter). 85 The resulting nanohydrogels displayed negative charge of ~ -55 mV, which should limit the interactions with cell membranes and serum proteins and promote their penetration into HEK293 spheroids (human embryonic kidney cells). Fluorescent intensity analysis of Two-Photon microscopy images revealed a two folds higher accumulation near the spheroid outer half of the disk-shaped nanocylinders with the lowest aspect ratio (H/D ~ 0.3, 325 nm diameter and 100 nm height) (Fig. 5f) compared to both nanocylinders with higher aspect ratio (H/D ~0.45) (Fig. 5e) and nanorods (Fig. 5g,h). Such preferential penetration might be attributed to their larger surface contact area, which promoted the interaction with the cells and the diffusion (either passive or active) across the 3D tumor mass.

The charge effect

As observed for shape and size, also the surface charge of the nanocarriers might also affect tissue penetration and efficient drug delivery. 72,82,164,165 For instance, the penetrating capacities of PAMAM dendrimers were discriminated according to their size (see 2.3.1.), but also a charge effect was clearly observed showing that only the amino-modified (positively-charged) ones were capable of significant accumulation in the outer layers of breast cancer (MCF7 cells) tumor spheroids while negatively and neutral ones were completely excluded (Fig. 6a). 72 Noteworthy is that small G2-NH2 dendrimers penetrated deeper than the larger G7-NH2 ones (214 ± 36 µm vs 81 ± 8.31 µm), but surface accumulation was directly proportional to the dendrimer generation (G7 > G2) and therefore to the surface charge density (i.e., number of NH2 groups) (Fig. 6b). This behavior was correlated to the different strength of dendrimers-cells interaction: while it was negligible for the G2 dendrimers leading to low accumulation and fast penetration by paracellular diffusion, the firmer interaction of G7 ones led to a strong uptake, which however translated led to a low diffusion via a transcellular mechanism. The key role of a positive surface charge in the accumulation into multicellular spheroids has been observed also by comparing gold nanorods (AuNRs) (55 nm length x 14 nm diameter) coated either with (i) cetyltrimethylammonium bromide (CTAB) and poly(diallyldimethylammonium chloride) (PDDAC), or (ii) polystyrene sulfonate (PSS) displaying a surface charge of +40-50 mV or -25 mV, respectively. 82 When incubated with MCF-7 breast cancer MCTS, the coating with a cationic polymer ensured the highest gold accumulation, which was expected to induce strong photothermal cytotoxicity after NIR irradiation as it was observed in 2D monolayer cultures. However, despite their tendency to be largely retained, irradiation of these positively charged AuNRs led to a 40% lower hyperthermia efficacy compared to the negatively charged PSS-coated AuNRs. Such unexpected behavior was related to the different penetration capacity of the different AuNRs.

Indeed, cationic polymer-coated AuNRs highly accumulated in the spheroids but only in the outer region and simple surface adsorption was also observed. Moreover the interaction with negativelycharged serum proteins led to an increase of their size preventing diffusion. On the contrary, the negative charge of PSS-coated AuNRs allowed a more homogeneous distribution in the spheroid core, which resulted in higher viability loss and destruction of the inner compact spheroid structure.

The role of the chemical composition

3D multicellular spheroids have been used to discern whether the chemical composition of the polymer building blocks could affect the drug delivery efficacy of the resulting nanoparticles. 60,91,96,152,153,155 Thus, curcumin-loaded biodegradable and non-biodegradable nanoparticles prepared by self-assembly of bovine serum albumin (BSA) conjugated with poly-(ε-caprolactone) (PCL) or poly(methyl methacrylate) (PMMA) were investigated on LNCaP prostate cancer MCTS in comparison to conventional 2D cultures. 91 The latter did not reveal appreciable differences between the two types of nanoparticles, which were both rapidly taken up by cells and induced similar cytotoxicity. On the contrary, the MTCS allowed to detect the faster and deeper accumulation of BSA-PMMA NPs, which occurred via a rapid sequence of endo and exocytosis events. However, such high penetration rate hindered a sufficient intracellular drug release thus leading to a lower cytotoxicity compared to the BSA-PCL NP, whose biodegradability ensured, on the contrary, high intracellular drug concentrations (Fig. 7a).

Non-biodegradable BSA-PMMA NPs induced a growth inhibition comparable to the biodegradable NPs, only when tested at 5 times higher drug concentration (Fig. 7b). Other examples of anticancer drug-loaded biodegradable nanocarriers evaluated on 3D spheroids included milk protein nanoparticles 153 and micelles composed of either (i) pseudo block copolymers formed by the assembly of β-cyclodextrin terminated multi armed poly(N-vinylpyrrolidone) (PVP) and adamantane functionalized polycaprolactone (PCL) 155 or (ii) poly (ethylene oxide)-poly [(R)-3hydroxybutyrate]-poly (ethylene oxide) triblock copolymers. 96 The capacity of penetration of these nanosystems in 3D models was consistent with further in vivo experiments thus supporting the predictive capacity of the tumor spheroids. 96,153,155

The role of the crosslinking

The capacity of amphiphilic block copolymers to self-assemble in water as micelles has been largely applied for efficient drug delivery. 90,120,124,166,167 Micelles display a typical core-shell structure and a variety of crosslinking strategies has been proposed to reach a higher stability. For instance the irreversible crosslinking of poly(ethylene glycol methyl ether acrylate)-bpoly(carboxyethyl acrylate) (POEGMEA-b-PCEA) micelles with 1,8-diaminooctane 90,124 protected against disassembly leading to better cellular uptake when compared to uncrosslinked counterparts. However, such crosslink induced a reduction of the drug release, leading to micelles with lower cytotoxicity compared to the free drugs when evaluated on 2D monolayer cell cultures of human prostate carcinoma (LNCaP cells). 124 Analogous results have been obtained when glycol dimethacrylate (EGDMA) crosslinked poly(ethyleneoxide-b-3-caprolactone) (PEO-PCL) have been investigated on HCT-116 (human colorectal carcinoma) and FaDu cells (human squamous cell carcinoma). 120 On the contrary, the aforementioned micelles were characterized by superior antitumor activities than the free drug in different MCTS made with HCT-116, FaDu or LNCaP cells, 120,124 clearly demonstrating the limited predictive potential of 2D cultures in the assessment of the real value of nanocarriers as drug delivery systems.

Whether the crosslink could affect the mechanism and depth of penetration as well as the cytotoxicity of drug-loaded micelles has been recently investigated by the group of Stenzel using pancreatic (AsPc-1 cells) multicellular tumor spheroids. 90 Hence, 1,8-diaminooctane-crosslinked poly(N-(2-hydroxypropyl) methacrylamide-co-methacrylicacid)-block-poly(methyl methacrylate) (P(HPMAco-MAA)-b-PMMA) micelles (CKM) were compared to their uncrosslinked version (UCM) (Fig. 8a). Results revealed that CKM were capable of moving through the cell layers via a transcellular process and delivered higher doxorubicin amounts to the spheroid core which resulted in greater cytotoxicity compared to the UCM (Fig. 8b,c,d). The latter quickly disassembled after penetration into the cells of the outer layers, releasing the loaded drug and causing cell death. As a result, no further micelle transcytosis could occur. The lower efficacy of the free drug (evaluated in terms of DNA content and inhibition of spheroid growth) correlated with its limited diffusion. 90 Nevertheless, the crosslinking is not always the best strategy to improve drug cytotoxicity. Thus, the evaluation of the effect of the reversible disulfide core-crosslinker cystamine in micelles formed by poly(ethyleneglycol methyl ether acrylate)-b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA)

The role of targeting ligands

Functionalization of polymer nanocarrier surface with various ligands has been largely exploited as strategy for selective cell targeting. 4 As discussed in the following sections, MCTS are currently widely used in order to assess the effectiveness of such modifications and evaluate the tumoral behavior of targeted nanoparticles.

Transferrin-targeted nanocarriers

Overexpression of transferrin receptor (TfR) has been detected in different types of rapidly proliferating tumors. 168 Accordingly, opportune surface functionalization with transferrin (Tf) has been applied to efficiently deliver drugs to cancer cells. Such modification clearly enhanced the cellular uptake and penetration depth of polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) micelles 67,68 and poly(amidoamine) (PAMAM) dendrimers 61 in MCTS models of ovarian carcinoma 67,68 and glioma 61 comparatively to the non-functionalized counterparts. The more efficient delivery of the loaded drugs (paclitaxel (Ptx)) 67,68 or doxorubicin (Doxo) 61 resulted in a significant inhibition of cell proliferation confirmed by reduction of spheroid volume and metabolic activity (ATP content). A 6-fold reduction of the IC 50 value (8.92 µM vs 1.35 µM) was measured on the 3D spheroids incubated with Tf-functionalized Ptx-loaded PEG-PE micelles (as compared to non-targeted ones) but these values were higher than those observed in 2D monolayers. These results reflect a reduction of efficacy due to the existence in the MCTS of physical barriers to diffusion and a different cells sensitivity due to the 3D spatial organization. 67 Reflectance confocal microscopy and synchrotron X-ray fluorescence microscopy (XFM) have been used to monitor in quantitative and qualitative manner, the capacity of penetration into 3D breast cancer spheroids (MCF-7 cells) of transferrin-decorated polymer-modified gold nanoparticles (100 nm). 105 Images revealed that although functionalization with human Tf increased the amount of internalized NPs compared to controls (i.e., bovine transferrin functionalized particles and naked ones), after 48 h the penetration of functionalized NPs was however limited the a depth of 50 µm thus representing a real issue for further therapeutic applications due to their incapacity to diffuse in the tumor core. 105

Carbohydrates-targeted nanocarriers

The natural affinity of dextran for highly glycosylated surfaces has been exploited for achieving targeted delivery of ald-dex-Doxo nanoparticles (i.e., aldehyde-dextran polymer conjugated to doxorubicin via a pH sensitive bond) to SK-N-BE(2) cells by the interaction with the glycocalyx at their surface. 126 Investigation of these NPs in 3D neuroblastoma spheroids (∼400-500 µm) enabled to discern their potential as drug delivery systems, while it was not evident in 2D conditions where the free drug displayed superior cytotoxicity. Such difference disappeared in the spheroid model in which a drastic reduction of the free Doxo efficacy was observed while that of the ald-dex-Doxo nanoparticles remained unvaried and resulted in a more efficient reduction of tumor outgrowth.

Such difference was related to the different capacity to overcome the encountered biological barriers: after 24 h the free Doxo penetrated up to 50 µm in the spheroids while nanoparticles were detected at that depth after 1h only and diffused in the whole mass at 4 h. Interestingly, such capacity was exclusive of ald-dex-Doxo nanoparticle and was not observed when cells were incubated with dextran based NPs in which the drug has been only physically loaded and not covalently linked to the polymer.

Chitosan has been instead used in the formulation of nanoparticles for specific targeting of the CD44 receptor in cancer stem-like cells (CSLCs), 53,60,77,89,106,122 a rare tumor cell population whose resistance to therapeutic agents is a major cause of anticancer treatment failure. 170 While non-stem cancer cells resistance in 2D cultures is mainly the result of the over expression of transmembrane P-glycoprotein transporters, 171 in CSLCs a key role is played also by the tumor microenvironment. 172 Accordingly, relevant preclinical investigations of such functionalized nanocarriers required a model capable of mimicking the complex relationship between cancer cells and the surrounding environment. Thus, 3D mammary tumor spheroids (i.e., mammospheres) enriched with CSLCs have been successfully created by culturing MCF-7 cells with different growth factors and chemicals in order to promote stemness (i.e., self-renewal) and epithelial-tomesenchymal transition (EMT) as demonstrated by the down regulation of the estrogen receptors (ER) expression whose role appears to be pivotal in maintaining the epithelial differentiation. 122,173 These CD44-expressing mammospheres, which are so far one of the most advanced example of 3D system used for polymer nanocarrier evaluation, allowed to assess the capacity of chitosandecorated Pluronic127 nanoparticles to efficiently deliver the loaded doxorubicin and to bypass the CSLC drug resistance in vitro. Chitosan functionalization resulted in nanoparticles able to selectively target CD44-overexpressing cells in mammospheres (Fig. 10a) while only a minimal targeting was observed on 3D spheroids made of normal human adipose-derived stem cells. 122 The better in vitro penetration of such functionalized nanocarriers was also confirmed in vivo revealing the highest capacity of inhibition of tumor growth (Fig. 10b,c).

CD44 receptors has been targeted also by chondroitin sulfate A-deoxycholic acid-(3-Aminomethylphenyl) boronic acid (CSA-DOCA-AMPB) NPs, which in addition exploited the interaction between boronic acid and sialic acid for efficient delivery of Doxo to human lung adenocarcinoma A549 tumors spheroids. 60

Monoclonal antibody-targeted nanocarriers

Among the plethora of possible strategies for active targeting, the 2C5 monoclonal antibody (mAb 2C5) has been employed for its capacity to target several types of tumor cells thanks to the interaction with the nucleosomes originated from neighboring apoptotically died tumor cells. 174 Nucleosomes are specifically bound at the surface of tumor cells and are always present in the spent media of growing tumor-cell lines as well as in the extracellular fluid of cancer patients. 175 Thus, mAb2C5-functionalized Doxo-loaded polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) micelles have been formulated and evaluated in vitro on ovarian cancer MCTS constructed by using NCI-ADR-RES cells. 136 The key role played by this Doxo-resistant MCTS model relied with the Bcl-2 gene overexpression associated to the three dimensional organization of the tumor cells, which closely mimicked the real situation found in patients. Consistent with the beneficial effect of mAb2C5 monoclonal antibody conjugation, uniform Doxo distribution throughout the spheroids was reached only with the targeted micelles. These same PEG-PE micelles have been also functionalized with the single chain fragment variable (scFv) of the monoclonal antibody against glucose transporter-1 (GLUT-1) whose overexpression in cancer cells relies with their continuous requirement for glucose supply. Accordingly, selective targeting of this transmembrane protein might promote the ligand mediated delivery of anticancer drugs. 66,73 The in vitro evaluation on 2D monolayer of U87MG glioblastoma cells revealed that Doxo and curcumin co-encapsulation in GLUT-1-targeted micelles resulted in a significant enhancement of caspases 3 and 7 activity as compared to un-targeted micelles (mono drug-loaded and two drugs-loaded). 66 Moreover, GLUT-1 targeting improved the penetration of PEG-PE micelles into 3D glioblastoma spheroids of U87MG cells in which the Doxo and curcumin synergistic effect was confirmed by the highest cytotoxicity (approx. 70% cell death) after 5 days of treatment. 66 Estrogen receptor alpha (ER-α) mAb has been instead used to functionalize the surface of polyacrylic acid (PAA)-coated ion doped NaYF4:Yb,Er upconversion nanoparticles (UCNPs), which have been investigated as potential early-stage cancer detecting agent. 83 Such functionalization enabled to target MCF7 breast cancer cells spheroid (~500 µm) transplanted in a chick embryo chorioallantoic membrane (CAM) aiming at modelling an early stage (i.e., diameter smaller than 2 mm) breast cancer. This model not only mimicked the cell-to-cell and cell-to-microenvironment interactions but also displayed a novel vascularization around the transplanted spheroid. More convenient and easy to handle compared to in vivo animal models, this simplified system allowed a direct microscopic study of the UCNPs-mAb ability to target cancer cells and thus detect tumors in vivo at an early-stage. Indeed, following systemic administration of UCNPs-mAb via venule injection under a stereomicroscope, a strong upconversion luminescence was observed in the spheroid mass. To be noted that only cancer cells were targeted and that no accumulation in the other surrounding tissues of the embryo was observed, thus demonstrating the optimal selective capacity of such modified nanoparticles.

Aptamer-targeted nanocarriers

Nucleic acid aptamers (DNA and RNA) capable to recognize with high specificity the epithelial cell adhesion molecule (EpCAM), a type I membrane protein expressed on the surface of a variety of cancer cells 176 have been used for surface functionalization of poly(lactide-co-glycolide) (PLGA) 75 and alginate-coated chitosan nanoparticles (CHNPs) 74 whose efficiency has been evaluated in vitro on 3D MCTS models of breast 75 and colon cancer 74 . Decoration with locked nucleic acid (LNA) aptamers allowed CHNPs to reach colon cancer EpCAM-expressing cells deeply in the core of both 3D MCTS and tumor experimental models in mice. 74 Proof of their potential efficient drug carriers was provided using CHNPs loaded with the apoptotic agent SR9, a survivin antagonist. Compared to non-functionalized nanoparticles, their efficient targeting and penetration capacity resulted in a 5fold reduction of spheroid volume in vitro (after 72 h exposure) and up to 4-times lower tumor volume in colon cancer xenografts in mice (at d= 70 post tumor induction). 74

Peptide-targeted nanocarriers

Specific recognition of α ν β 3 integrins has been demonstrated to endow RDG-functionalized nanocarriers of targeting capacity toward cancer cells facilitating their internalization. 177 Accordingly, the linear or cyclic version of the RGD has been covalently linked to:

(i) micelles made by the assembly of the enzyme-sensitive peptide-linked poly(ethylene glycol) and partially hydrolyzed poly(β-benzyl l-aspartate) (PEG-GPLGVRGDG-P(BLA-co-Asp) copolymer; 138 (ii) mesoporous silica nanoparticles (MSNs) coated with poly(ethylene glycol) (PEG), polyethyleneimine (PEI) or chitosan; 84 (iii) PEG-poly(trimethylene carbonate) (PEG-PTMC) nanoparticles; 63,64 (iv) poly(amidoamine) (PAMAM) dendrimers. 146,149,150 The influence of the extent of RGD functionalization was also investigated. For instance the transfection efficiency of PAMAM-RGD dendrimers displaying various levels of RDG ligands were evaluated in a 3D spheroid model of glioma in comparison to the conventional 2D culture of U87MG cells. 149,150 In 2D cultures not any advantage was observed comparatively to the naked PAMAM, probably as a consequence of the predominant non-specific interaction mediated by the positively charged dendrimers with the cell membrane. However, the evaluation in 3D spheroids highlighted the capacity of PAMAM-RGD to strongly interfere with the α ν β 3 integrin-mediated interaction of cells with the ECM, which was directly correlated to the number of ligands conjugated to PAMAM. As a consequence of the reduced adhesion, RGD functionalization facilitated the penetration and the uptake of PAMAM dendrimers into the spheroid model although it did not result in a significant gene silencing. 149 In addition to the capacity of interaction with integrins, conferred by the RGD sequence, the so called tumor penetrating peptides (TPP) 177, 178 display a C-terminal sequence R/KXXR/K know as C-end rule (CendR). 62,63,80 Together, these two sequences mediate an active transport through tumor vessels and within the extravascular tumor tissue by interaction first with the α v β 3 -integrin and successively with the neuropilin-1 receptor (NRP). 59,80 Clearly such modification represents a valuable approach for increasing the penetration of nanocarriers in the tumor mass. 56,62,117 Among the TPP, the iRGD has been physically adsorbed onto boronic acid-rich chitosan-poly(N-3acrylamidophenyl boronic acid) (CS-PAPBA) nanoparticles, 117 or covalently linked to Pluronic F127 coated paclitaxel nanocrystals. 56 CLSM images of spheroids exposed to fluorescently TPPfunctionalized nanocarriers revealed an intense signal, which spread from the periphery toward the center of the spheroids demonstrating their superior capacity of penetration and accumulation leading to an efficient drug delivery confirmed in vitro by the reduction of spheroid volume and in vivo by inhibition of tumor progression (Fig. 11). 56,62,89,117 The use of the simple CendR motif with the RGERPPR sequence has been also proposed to increase the penetrating capacity of poly(ethylene glycol)-polyethylenimine/ plasmid DNA complexes resulting in a higher accumulation into a glioma spheroid model (U87MG cells) compared to the-non functionalized counterpart. Nevertheless whether the peptide could also be efficient in improving the transfection efficacy in this 3D model, still needs to be verified. 80 Cell penetrating properties are also displayed by the interleukin-13 peptide (IL-13p) capable of specific recognition of the IL13Rα2, a tumor-restricted receptor overexpressed in gliomas.

Functionalization of poly(ethylene glycol)-poly(ε-caprolactone) (PEG-PCL) nanoparticles with this peptide (ILNPs) resulted in an enhanced cell-uptake and penetration in U87MG spheroids. 78 A 3fold higher tumor accumulation was obtained in vivo compared to un-functionalized nanoparticles, which resulted in a better docetaxel delivery and a significant reduction of tumor weight compared to the other treatments (saline, free drug and drug-loaded naked NPs). 78 Endowing these NPs of dual targeting properties by functionalization with both IL-13p and RGD peptides further improved their penetration ability in C6 glioma spheroids as well as in vivo in orthotopic glioma-bearing mice. 79

Use of 3D MCTS to evaluate macromolecules-loaded nanomedicines

Polymer-based nanomedicines have been investigated for the delivery of proteins or DNA/RNA molecules, whose application in clinical settings is strongly limited by (i) inappropriate size and surface charge; (ii) low stability against enzymatic degradation and (iii) low cell membrane permeability. 179,180 A variety of polymer/siRNA and pDNA polyplexes have been constructed and evaluated in 3D models demonstrating their efficiency as drug carriers. Complexation has been realized for instance with (i) poly(amidoamine) (PAMAM) dendrimers; 92,149 (ii) folate-functionalized-poly(ethylene glycol)-polyamidoamine-polyethylenimine (PME-(PEG-FA)) copolymers 94 (see also 2.3.6.2); (iii) poly(L-lysine); 118 (iv) PEG-b-poly(N-substituted asparagine) copolymers(PEG-b-P[Asp(DET)); 131 triblock poly(2-ethyl-2-oxazoline)-poly(L-lactide)-g-poly(ethylenimine) (PEOz-PLA-g-PEI) polymers; 108, 109 (v) mPEG-PEI (CendR-penetrating-peptide-modified methoxy poly(ethylene glycol)-polyethylenimine) copolymers 80 (see also 2.3.6.6.); (vi) folate-poly(ethylene glycol) (PEG)-Amino Acid Modified Chitosan (CM-PFA) 107 or (vii) poly[(N,N-dimethylamino) ethyl methacrylate] (PDMAEMA)-derivatized albumin. 93,116 The pioneering studies of Kataoka and coworkers have highlighted the utility of human hepatocarcinoma multicellular spheroids (HuH-7 cells) for the long-term evaluation of the transfection efficacy achieved using polyplex micelles as gene delivery systems. 118,131 In particular core-shell type micelles assembled through electrostatic interactions between poly(L-lysine) and lactosylated poly(ethylene glycol)-siRNA conjugate (lac-PEGylated polyplexes) 118 or pDNA and PEG-block-poly(N-asparagine) copolymers (PEG-b-P[Asp(DET)]) 131 have been investigated. Only the 3D MCTS spheroids, which can be maintained in culture for several weeks, allowed to carry out an extended analysis of gene expression/suppression under conditions close to those observed in vivo in solid tumors. 118 On the contrary, such time dependent studies could not be performed on 2D monolayer cultures as consequence of cell-cell contact-induced arrest and viability decrease that prevented the monitoring of a prolonged gene expression. 131 Thus, it was possible to observe that the delivery of the RecQL1 siRNA using the lac-PEGylated polyplexes allowed an efficient suppression of gene expression, which resulted in the inhibition of spheroid growth for up to 21 days. 118 Using the same 3D model it was possible to demonstrate that pDNA-loaded PEGylated polyplexes penetrated in the spheroids and stably induced the expression of the encoded yellow fluorescent protein Venus for more than 10 days. 131 Successful prediction of in vivo transfection efficiency was obtained following evaluation in MDA-MB-231 breast cancer spheroids of heptafluorobutyric acid modified generation 4 (G4) poly(amidoamine) (PAMAM) dendrimers (G4-F735) loaded with a plasmid encoding for the TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) gene. 92 Improved gene delivery and better performance, compared to both naked pTRAIL and conventional transfection reagent poly(ethylene imine) PEI, were confirmed in vitro by the complete degradation of the MDA-MB-231 spheroids after 7 days of treatment (Fig. 12a) and in vivo by suppression of tumor growth in a subcutaneous model of luciferase expressing MDA-MB-231 cells (Fig. 12b). 92 The usefulness of stimuli-responsive approaches was assessed by Gaspar and coworkers. 108,109 Using minicircles DNA (mcDNA), micelleplexes have been constructed by self-assembly with poly(2-ethyl-2-oxazoline)-poly(L-lactide)-g-poly(ethylenimine) (PEOz-PLA-g-PEI) triblock copolymer or its bioreducible analogue (PEOz-PLA-g-PEI-SS), which allowed the formulation of stimuli-responsive micelles thanks to the introduction of redox sensitive bonds. 108 Both systems displayed good penetration ability and negligible cytotoxicity in 3D MCTS models of melanoma (B16F12 cells), cervix carcinoma (HeLa cells) 108 and breast cancer (MCF-7 cells). 109 Nevertheless, confocal images of 3D HeLa and B16F10 spheroid sections revealed that bioreducible micelleplexes enhanced GFP gene expression thanks to a higher mcDNA release following rapid intracellular reduction of the disulfide linkages.

Miscellaneous nanocarriers evaluated on 3D MCTS

Surface modification of inorganic nanoparticles with various polymer might make possible their use as theranostic systems for efficient delivery of therapeutic molecules and precise monitoring of the response. 81 In this view, coating of iron oxide nanoparticles (IONPs, MRI contrast agent) with poly(4-O-acryloyl benzaldehyde)-poly(oligoethylene glycol acrylate) P(HBA)-b-P(OEGA) block copolymers allowed their stabilization as well as the covalent conjugation of doxorubicin via a pHsensitive bond, which assured drug release in acidic environment (pH 5.5). 81 3D optical sectioning by Multi-Photon microscopy of multicellular spheroids made of lung (H129) or breast (MCF7) cancer cells clearly showed the fluorescence of Doxo-loaded IONP@P(HBA)-b-P(OEGA) nanoparticles uniformly spread across the spheroid tissue highlighting a complete penetration after 17 h of incubation; on the contrary, free Doxo accumulated in the periphery reaching a maximal depth of 40 µm only (Fig. 13). 81 In real time monitoring of tumor response to treatment is the goal pursued by Oishi and coworkers with the formulation of a PEGylated nanogel containing gold nanoparticles in the cross-linked poly[2-(N,N-diethylamino)ethyl methacrylate] (PEAMA) core in which, the fluorescein isothiocyanate (FITC) was linked to the PEG chains using the Asp-Glu-Val-Asp (DEVD) peptide sequence as caspase-3-cleavable linker. 156 Based on the Fluorescence Resonance Energy Transfer (FRET) between gold nanoparticles (i.e., fluorescent quencher) and FITC, such system behaved as a caspase-3-responsive apoptosis sensor for precise in vitro monitoring of the activity of apoptosisinducing agents. The proof of concept has been provided using human hepatocyte MCTS (HuH-7 cells), which have been incubated with nanogels for 24 h prior exposure to apoptotic drug staurosporine for 4 h. Intracellular caspase activation triggered FITC release from the nanogels and the dequenching of the fluorescent signal, which allowed to assess the early stage activation of the induced apoptotic pathway.

The application of nanodiamonds (ND) as drug delivery systems is currently strongly limited by their tendency to agglomerate and precipitate in solution. Thus whether surface modification with polymers might offer beneficial effect has been explored by grafting of poly(1-Omethacryloyl-2,3:4,5-di-O-isopropylidene-β-D-fructopyranose) (poly-(1-O-MAipFru) 62 ) onto the surface of amine-functionalized ND. Such approach should improve their stability and allow a successful loading of doxorubicin. 157 When evaluated in 2D monolayer cultures of MCF-7 and MDA-MB-231 breast cancer cells a clear superiority of the free drug over the NDs was observed. However, in 3D breast cancer MCTS (MCF-7 cells) long term exposure (8 days) to the Doxo-loaded poly(1-O-MAFru) 62 -ND resulted in higher cytotoxicity compared to the free drug which can be justified by the deeper penetration of NDs in spheroids and the sustained release of the drug. On the contrary the free Doxo was rapidly internalized by the proliferating cells of the outer layers of the spheroids and the consequent cell death hindered further penetration. 157

Combining 3D MCTS and polymer scaffolds

3D tumor models made of multicellular spheroids surrounded by a polymer scaffold have been also proposed as alternative to simple spheroids suspended in cell culture medium. In these systems the scaffold building materials create a matrix around the spheroid capable to mimic in vitro the microenvironment surrounding the tumors in vivo. Accordingly, this additional barrier may offer the possibility to better discern the capacity of various nanocarriers to diffuse and to reach the cancer cells.

One of the simplest strategies consisted in embedding preformed spheroids into a collagen gel. Such system enabled for instance to highlight the better efficacy of paclitaxel loaded into pH sensitive NPs (i.e., espansile NPs) 181 compared to the free drug, while this difference did not appear in 2D cultures. Indeed, the latter did not display any difference in the cell response independently of the drug administration method (free drug solution vs drug-loaded nanocarrier). 65 On the contrary, in 3D cultures, drug-loaded nanoparticles induced a more important slowing of the spheroid growth, which mirrored the inhibition of tumor progression obtained in vivo in tumor-bearing mice. 181 In another 3D spheroid design a single cell suspension has been mixed with collagen before gelification and cells aggregated over time inside the matrix in form of spheres. Again, while no differences were observed in 2D between the free drug (5-fluorouracil) and the drug-loaded micelles on the contrary, thanks to the presence of the collagen matrix which mimicked the tumor ECM, the 3D model enabled to reveal the limited diffusive capacity of micelles (152 nm) compared to the small molecules that led to a lower cytotoxicity. 182 Same strategy has been applied by mixing prostate cancer cells with hyaluronic acid, 183 one of the components of the stroma associated to this tumor in vivo, which not only provided structural support but also strongly influenced tumor cell morphology, gene expression and tumorigenic potential. 184 For instance, cell cultured in this 3D scaffold displayed higher expression of multidrug resistance proteins, probably as result of the limited availability of oxygen and nutrients. Accordingly, while in the 2D cultures the free drug (doxorubicin) easily reached the nucleus and exerted its therapeutic activity, in 3D the sensitivity to the free doxorubicin was reduced (5 µM vs 15 µM, respectively). Contrariwise, independently of the culture conditions the response to Doxo-loaded PEG-PCL NPs was not modified and analogous IC 50 values were measured (11µM vs 12.3 µM, respectively) probably thanks to the capacity of these NPs (54 nm diameter) to overcome the MDR while the free drug undergo to a rapid efflux, which reduced its the efficacy. 183

Combining 3D MCTS and microfluidic devices

Undoubtedly, compared to simplistic 2D cultures the above described 3D systems enabled a more predictive in vitro screening of nanoscale systems for drug delivery. Nevertheless, due to the lack of fluid dynamics these set ups mimic only a static condition. To face this issue microfluidic devices, which combine 3D culture and controlled flow conditions, have been recently developed with the aim: (i) to assess how physico-chemical parameters influence the transport through tumor biological barriers under dynamic conditions and (ii) to provide information on the optimal design required to achieve a successful tumor accumulation. 185,186 For instance, a tumor-on-a-chip device allowed the passage from a static spheroid culture to a dynamic situation by placing a spheroid in the channel of a two layers polydimethylsiloxane (PDMS) chip (Fig. 14a). 185 By tuning the flow rate in the device, fluid velocities and shear stresses similar to the blood flow in capillary vessels (75-675 µm/s) or the interstitial flow inside a tumor (0.1-3 µm/s) could be reproduced in a controllable manner. Exposure of spheroid to PEGylated nanoparticles of different sizes under stationary flow revealed interstitial accumulation only of the smaller NPs (40 nm) while the larger ones (110 nm), bigger than the ECM pores, were excluded (Fig. 14b,c). However, accumulation was only transient and nanoparticles flowed out after flushing, confirming that surface modification with PEG chains hindered the establishment of specific interactions with cells and ECM components. On the contrary no efflux was observed following NP functionalization with transferrin and 40 nm nanoparticles reached up to 15-fold increase of tumor accumulation comparatively to non-functionalized NPs. It was also observed that an increase of the flow rate resulted in accumulation in the external spheroid layer, forming a tissue-fluid interface reservoir but that did not affect the depth of NP penetration. When the NPs were then tested in vivo, in a tumor bearing mice, the same size discriminating effect was recorded, with a better accumulation of small NPs (50 nm) compared to the larger ones (160 nm). However functionalization did not led to any significant advantage and both targeted and non-targeted 50 nm NPs displayed similar tumor accumulation level (Fig. 14d,e). Such contradiction with the results

Conclusion and perspectives

Through many literature examples this review has unambiguously highlighted that simple 2D monolayers cultures do not allow complete understanding of the therapeutic potential of polymer nanomedicines while the application of 3D tumor models in preclinical evaluation would provide more accurate results, predictive of the in vivo pharmacological efficacy. The key features of the MCTS (e.g., presence of ECM, diffusive gradients, complex cell signaling, drug resistance and metabolic adaptation) undoubtedly enable to evaluate the nanomedicines in a condition closer to the clinical reality. But it cannot be ignored that the majority of tumor spheroids used in literature are made of cancer cells only and therefore they just represent a rather simplified model of the real tumorigenesis. Due to the complexity of the tumor tissues and the cross-talking between cancer cells and their microenvironment, advanced models including several cell types (e.g., endothelial cells, immune cells, fibroblast etc) and components of the ECM are urgently required. Thus, 3D cocultures 47,48,188 and microfluidic devices 111-113, 185, 186, 189 have been developed but further improvements are still needed in order to allow a wider application in preclinical investigation. In this context, the accurate characterization (e.g., cell number, long term viability of each cell components, type of ECM proteins, etc.., ) of any developed system is mandatory for allowing a reliable interpretation of the obtained results. It is indeed well acknowledged that the spheroid size 43,46 as well as the presence of stroma components 47,48,188 strongly affect the response to treatment according to the possible development of penetration barriers, [46][47][48]188 chemical gradients and/or necrosis. 43 The availability of microscopy techniques suitable for high resolution imaging of 3D cell cultures represents another challenge. Indeed, although widely used, CLSM does not allow the in toto study of large 3D samples and the obtained results refer only to penetration into the spheroids at the depth of maximum 100-150 µm. A significant improvement in 3D imaging should result from Two-Photon (TPM) and Multi-Photon Microscopies (MPM) but the low spatial resolution along the optical axis and the incompatibility of certain fluorophores with the multi-photon excitation still restrict the applicability of these methods. 140,190 In this challenging panorama, LSFM approaches (e.g., Selective Plane Illumination Microscopy (SPIM)) are emerging as techniques of choice in life sciences for the imaging of complex and highly scattering samples. [142][143][144][145] Offering the possibility to visualize the spheroids in their entirety with a sub-cellular resolution 191,192 the SPIM technique clearly enables to achieve a superior degree of information in the screening of nanomedicines pharmacological efficiency. 47 The penetration ability of nanomedicines can be assessed over the whole 3D MCTS mass and a dynamic study of the anticancer response is also possible with the time lapse imaging of living spheroids. Unfortunately, such advanced techniques require highly specialized technologies whose availability remains still limited.

It is evident that labor-intensive handling, time-consuming procedures, instrumental limitations and cost still hinder the routinely use of advanced 3D models in drug discovery programs. Noteworthy is that, to the best of our knowledge, not any promising nanomedicine currently in clinical trials has been tested in 3D MCTS during in vitro pre-clinical studies. Nevertheless, progresses in the next future are expected to rapidly support their widespread use, thus making the in vitro drug screenings more predictive and able to sieve out underperforming compounds in the early pre-clinical stage.

Introduction

In vitro models capable to faithfully mimic the complex cancer physiopathology represent an unmet need to improve the significance of preclinical research data in the early phase of drug development. It is well known that the tumor biology results from a mutual interaction between cancer cells and the surrounding microenvironment [1]. For instance, extracellular matrix (ECM) proteins, such as fibronectin (FN), are actively involved in tumor stiffness regulation [2, 3] as well as promotion of tumor growth [4, 5] and drug resistance [6, 7].

Moreover, the intricate cross-talking between cancer cells and stroma components (e.g., fibroblasts, immune and endothelial cells) regulates different features related to tumor progression [8-10]. Fibroblasts influence both tumor development and the metastatic potential of cancer cells through direct heterotypic cell-to-cell contacts and paracrine factors [11-14].

Interactions with cancer cells, fibroblasts and ECM proteins are required to allow the migration and proliferation of endothelial cells during tumor vascularization [15, 16].

Multicellular tumor spheroids (MCTS) are the most widely used 3D in vitro model in oncology preclinical research and their power of prediction of the in vivo efficacy of various chemotherapeutic agents has been clearly evidenced [17, 18]. MCTS reproduce some important key factors of real tumors such as: (i) the formation of nutrients, oxygen and metabolic waste diffusive gradients, (ii) the organization of cells in layers with different proliferation rates, (iii) the presence of cell-to-cell interactions and signalling, (iv) the expression of specific gene patterns and (v) the chemoresistance [19, 20]. Still, conventional MCTS consisting exclusively of cancer cells mimic only partially the real tumors as consequence of insufficient extracellular matrix deposition and absence of other cell types, which compose the tumor microenvironment. With the aim to overcome these limitations hetero-type MCTS consisting of 3D co-cultures of cancer cells and stroma cells have been proposed [21-24]. In addition, the embedding of mono-and hetero-type MCTS in exogenous Chapter I 76 3D matrices has been extensively used, creating a cell supportive environment and reproducing the cell-to-matrix interactions [25, 26]. Undoubtedly, the use of matrices, such as natural hydrogels (Matrigel ® and collagen I, mainly) or synthetic polymers (e.g., poly(ethylene glycol), poly(lactide-co-glycolide) and poly(N-isopropylacrylamide)), enhanced the structural stability of 3D MCTS and endowed them with higher biological relevance [27].

However, several disadvantages characterize these approaches. They include: (i) the unknown composition of natural hydrogels and the batch-to-batch variability responsible for a lack of reproducibility [21], (ii) the difficulty to harvest single spheroids once embedded in synthetic biomaterials and the need to apply experimental procedures (chemical or physical) often toxic and/or detrimental for the cells [28], (iii) the restricted accessibility of embedded spheroids, which often makes time-consuming the set-ups to carry out quantitative measurements [23, 27]. In this context, it is evident that the in vitro assessment of the role of the microenvironment on the malignant behaviour of tumors and the response to treatments is still a challenge.

Aiming to face this issue, we describe here a novel hetero-type 3D spheroid model integrating cancer cells together with cellular and acellular stroma components (i.e., fibroblasts, endothelial cells, ECM), focusing our attention on the pancreatic ductal adenocarcinoma (PDAC). PDAC belongs to the first five most lethal type of cancers in the western world and the progress in its treatment remains too slow as a consequence of the complex physiopathology of this tumor characterized by a heterogeneous cellular composition and the accumulation of a very dense fibrotic tissue (i.e., desmoplasia) [29]. In PDAC, the crosstalk between cancer and stroma cells leads to a dramatic increase in FN and collagen deposition [10, 14, 30], which causes the collapse of the tumor vessels and the formation of an aberrant and disorganised vascular network [31]. The consequent oxygen and nutrients deficiency induces the secretion of pro-angiogenic molecules that, in turn, increase the aggressiveness Chapter I 77 and the invasiveness of cancer cells [31-33]. Currently available hetero-type MCTS of PDAC are better representative of the pathological condition in comparison with 2D monolayer cultures, however they often lack an accurate characterization [34, 35] or consist of no more than two cell types [36-41]. For instance, MCTS made of cancer cells and fibroblasts have been proposed to mimic in vitro the molecular cross-talk which characterize the PDAC desmoplasia [36-38, 40, 41]. Similarly, cancer and endothelial cells were assembled in MCTS to reproduce the tumor angiogenesis and its influence on cancer cell invasiveness [39].

However, to the best of our knowledge, hetero-type pancreatic tumor spheroids including fibroblasts and endothelial cells, both essential components of the PDAC microenvironment, have not been previously reported. This was likely due to the difficulty to find the experimental conditions required to obtain viable three-cell-type PDAC multicellular spheroids. In this context, we herein report on the step-by-step construction and detailed characterization of a scaffold-free multicellular tumor spheroid consisting of a triple coculture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC). Importantly, we demonstrated that the presence of a complex microenvironment reduced the sensitivity of cancer cells to chemotherapy thus closely mimicking the resistance to treatments observed in vivo. Overall, this original scaffold-free MCTS represents a pertinent and easy-to-handle tool, which could be readily introduced in routine preclinical screening of therapeutic strategies for pancreatic cancer treatment.

Materials and methods

Cell lines

Human pancreatic cancer cells (PANC-1), human lung fibroblasts (MRC-5) and human umbilical vein endothelial cells (HUVEC) were purchased from ATCC (France) and maintained as recommended. Briefly, PANC-1 cells were maintained in Dulbecco's Modified Chapter I 78 Eagle Medium-high glucose (DMEM, Sigma Aldrich, France) supplemented with 10% heatinactivated fetal bovine serum (FBS, Gibco, France). BxPC-3 cells were cultured MRC-5 cells were cultured in Eagle's Minimum Essential Medium (EMEM, Sigma Aldrich, France) supplemented with 10% heat-inactivated FBS and 1% of 200 mM L-Glutamine solution (Sigma Aldrich, France). Luciferase-expressing human pancreatic cell line (BxPC-3-luc2) was obtained from PerkinElmer (Roissy, France) and maintained in Roswell Park Memorial Institute medium (RPMI, Sigma Aldrich, France) supplemented with 10% heat-inactivated FBS. All media were further supplemented with penicillin (50 U.mL -1 ) and streptomycin (0.05 mg.mL -1 ) (Sigma Aldrich, France). HUVEC cells were maintained in endothelial growth medium (EGM-2) consisting of endothelial basal medium (EBM-2) in which supplements and growth factors have been added according to manufacturer instruction (EGM-2 BulletKit Lonza, France). Cells were maintained in a humid atmosphere at 37 °C with 5% CO 2 . Cells were used below passage 8 after thawing and harvested at a confluence of 70-80%.

Cell transfection

Luciferase-and fluorescent protein-expressing cells were created by stable lentiviral transduction according to manufacturer protocols.

For the luciferase-transduction, PANC-1 cells (2.5 x 10 4 cells.mL -1 ) were seeded in 24-well plates (1 mL/well) and incubated for 24 h in a humid atmosphere at 37 °C with 5% CO 2 .

Transduction was performed by addition of RediFect™ Red-FLuc-Puromycin lentiviral particles (Perkin-Elmer, France) to cells. For green fluorescent protein (GFP)-transduction, MRC-5 fibroblasts (9 x 10 4 cells.mL -1 ) were seeded in 24-well plates (1 mL/well) and incubated for 24 h in a humid atmosphere at 37 °C with 5% CO 2 . Transduction was performed by addition of pLenti-C-mGFP-P2A-Puro particles (Origene, Germany) to cells.

For both cell types, a polybrene (8 µg.mL -1 )-containing medium and a multiplicity of infection (MOI) of 10 were used.

For red fluorescent protein (RFP)-transduction, HUVEC cells (5 x 10 3 cells.mL -1 ) were seeded in 24-well plates (1 mL/well) and incubated for 24 h in a humid atmosphere at 37 °C with 5% CO 2 . Transduction was performed by adding pLenti-C-mRFP-P2A-Puro particles (Origene, Germany) to cells. In this case a MOI 100 in polybrene-free medium was used.

For each experiment, transduction particle-containing medium was removed after 24 h and replaced with fresh medium containing puromycin (Thermo Fisher Scientific, France) as selection antibiotic. According to the specific cell sensitivity, puromycin was used at a concentration of 2, 1 and 0.5 µg.mL -1 for PANC-1, MRC-5 and HUVEC cells, respectively. Puromycin-containing medium was replaced every 3 days until resistant colonies could be identified. Cell bioluminescence and fluorescence were measured 48 h after infection and prior to the cryopreservation of selected cells.

Construction of 3D MCTS

Liquid overlay technique

Mono-type and hetero-type MCTS were constructed according to the liquid overlay technique [42] using 96 round-bottomed well plates (CELLSTAR ® , Sigma Aldrich, France).

Before use, 50 µL of 1.2 % (w/v) poly-2-hydroxyethyl methacrylate (poly-HEMA, Sigma Aldrich, France) ethanolic solution was added to each well, and solvent was evaporated in sterile conditions.

For the construction of the mono-type PANC-1 spheroids, cell suspensions (2.5, 5, 12.5 or 25

x 10 3 cells.mL -1 ) were prepared in DMEM complete medium and 200 µL of the cell suspensions (corresponding to 500, 1000, 2500 and 5000 cells, respectively) was transferred into each well.

Chapter I 80

For the construction of the hetero-type PANC-1:MRC-5 spheroids, suspensions of each cell type were prepared in DMEM complete medium and then 200 µL of their opportune mixture was transferred into each well. The number of PANC-1 cells was fixed at 500 cells per well.

Four PANC-1:MRC-5 ratios have been studied (i.e., 1:1; 1:2; 1:4 and 1:9).

For the construction of the hetero-type PANC-1:MRC-5:HUVEC spheroids, suspensions of each cell type were prepared in DMEM complete medium and then 200 µL of their opportune mixture was transferred into each well. The number of PANC-1 was fixed at 500 cells per well. A ratio of 1:9:4 among PANC-1:MRC-5:HUVEC has been studied. Optimization of the experimental protocol included (i) construction of a nanofilm of fibronectin-gelatin (FN-G) at the MRC-5 surface before seeding (see 2.3.2) and (ii) addition of human VEGF (50 ng.mL -1 ) (Thermo Fisher Scientific, France) to the culture medium.

Cell dispersions at different densities were prepared after automatic cell counting (Countess II, Thermo Fisher Scientific, France), according to manufacturer instructions. After cell seeding, plates were centrifuged (1100 rpm, 5 minutes, 20 °C) and then incubated in a humidified atmosphere with 5% CO 2 at 37 °C for a minimum of 48 h. For long term culture, half of the medium was replaced every 3 days.

MCTS constructed with luciferase-expressing cells were prepared according to the same protocols except for the use of phenol red-free DMEM (Sigma Aldrich, France) supplemented with 10% heat-inactivated FBS, 1% of 200 mM L-Glutamine solution, 1% of 100 mM sodium pyruvate solution (Sigma Aldrich, France), penicillin (50 U.mL -1 ) and streptomycin (0.05 mg.mL -1 ). Fluorescently-labelled spheroids were prepared using GFP-expressing MRC-5 fibroblasts and RFP-expressing HUVEC cells.

Fibronectin-gelatin coating of MRC-5

A fibronectin-gelatin nanometer sized film was constructed at the MRC-5 surface according Chapter I 81 to the previously validated Layer-by-Layer (LbL) method [43]. Briefly, after trypsinization fibroblasts were suspended in 1 mL of Tris-HCl buffer (50 mM, pH 7.4) (Sigma Aldrich, France), centrifuged (2500 rpm, 1 minute, room temperature) and then alternatively incubated for 1 minute using a Microtube Rotator (VWR, France) in 1 mL of 0.04 mg.mL -1 Human fibronectin or gelatin (Sigma Aldrich, France) Tris-HCl solutions (50 mM, pH 7.4). Each incubation was followed by a centrifugation (2500 rpm, 1 minute, room temperature) and a washing step with Tris-HCl buffer (1 mL, 50 mM, pH 7.4). A total of 9 coating steps were performed. Then, fibroblasts were suspended in cell culture medium and used to construct the triple co-culture, as above described.

Spheroid characterization

Optical imaging

Routine spheroid monitoring was performed using the AxioObserver Z1 (Carl Zeiss, Germany) inverted microscope equipped with a Peltier cooled (-40 °C) CoolSnap HQ2 CCD camera (Photometrics,Tucson, USA) and a XL incubator thermostated at 37 °C providing 5% CO 2 . Transmitted light images of spheroids were collected directly from the poly-HEMA coated plates with a Plan-Apochromat 2.5x dry objective lens, a halogen lamp and a motorized stage used on an automated mode (AxioVision software / high content acquisitions).

By using an image-processing macro, specifically created with Image-J ® software, morphometric parameters such as spheroid area, perimeter, minimum diameter, major diameter, aspect ratio and circularity were obtained. Spheroid volume (V) was calculated according to the formula V = [(a²)x(b)]/2, in which a and b represent the minor and major diameter, respectively. For the growth profile characterization, images of 128 spheroids per condition have been collected at each time point. Chapter I 82

Spheroid dissociation and single cell counting

To assess the number of cells per spheroid over time, six-to-eight MCTS were harvested using a p5000 pipette to preserve their integrity, and then pooled in a microtube. Culture medium was carefully removed and spheroids were washed with 200 µL of phosphate buffered saline (PBS, Sigma Aldrich, France). After addition of 50 µL of trypsin (Sigma Aldrich, France), samples were incubated at 37 °C for 30 minutes and pipetted every 5 minutes with a p200 pipette to facilitate the dissociation of spheroids into a single-cell suspension. The enzyme action was stopped by the addition of 50 µL of FBS-containing culture medium. The total cell number per sample was measured using the Countess II Automated Cell Counter (Thermo Fisher Scientific, France) and the average cell number per spheroid was calculated dividing it by the number of used spheroids. Experiments have been performed in triplicate.

Adenosine Triphosphate (ATP) cell viability assay

Individual MCTS (n=6) were harvested from the round bottom wells and transferred in a microtube using a p5000 pipette. Culture medium was carefully removed, spheroids were washed with PBS (200 µL) to remove any extracellular ATP and then redispersed in 50 µL of phenol red-free DMEM. The spheroid suspension (50 µL) was then transferred into whiteopaque 96-well plates (Corning ® , Sigma Aldrich, France) and 50 µL of CellTiter-Glo ® 3D reagent (Promega, France) was added into each well. Plates were protected from light and gently shaken for 10 minutes (room temperature) to induce spheroid lysis. Samples were incubated for additional 20 minutes in the dark, at room-temperature, to stabilize the bioluminescent signal, which was then recorded using a benchtop plate reader (EnSpire Alpha 2390; Perkin-Elmer, USA). The ATP concentration in each sample was quantified by using a calibration curve based on rATP standards (Promega, France) according to the manufacturer Chapter I 83 instructions.

Assessment of cell viability after exposure to doxorubicin was carried out according to the same protocol with a slight modification. Briefly, spheroids were cultured in 96 round-bottom well plates in 200 µL of medium. At determined time points, 150 µL of medium was carefully removed and 50 µL of CellTiter-Glo ® 3D Reagent was added to each well (total final volume 100 µL). Plates were gently shaken in the dark for 10 minutes to lyse the samples and incubated for additional 20 minutes at room-temperature to stabilize the bioluminescent signal.

Then, samples were individually transferred into white-opaque 96-well plates and the signal was measured using a benchtop plate reader (EnSpire Alpha 2390; Perkin-Elmer, USA) (8 replicates per condition, n=2).

In vitro luciferase assay

The bioluminescence signal produced by luciferase-expressing PANC-1 or BxPC-3 cells was measured using the Neolite Reporter Gene Assay System (Perkin-Elmer, France) according to manufacturer instructions. Briefly, spheroids were cultured in 96 round-bottom well plates in 200 µL of medium. At determined time points, 150 µL of medium was carefully removed and 50 µL of Neolite reagent was added to each well (total final volume 100 µL). Plates were protected from light and gently shaken for 10 minutes (room temperature) to induce spheroid lysis. Samples were incubated for additional 20 minutes in the dark at room-temperature to stabilize the bioluminescent signal. Samples (100 µL) were individually transferred into white-opaque 96-well plates and the signal was measured using a benchtop plate reader (EnSpire Alpha 2390; Perkin-Elmer, USA) (6 or 8 replicates per condition, at least n=3).

In another set of experiments, samples (100 µL) were individually transferred to a blackopaque 96-well plate (Corning ® , Sigma Aldrich, France) and plate images were acquired using the IVIS Lumina ® LT Series III (PerkinElmer, USA) (at least 3 replicates per condition, Chapter I 84 n=3). Images were processed using the Living Image software (PerkinElmer, USA).

Histology and immunohistochemistry

MCTS were fixed for 2 hours in 500 µL of 4% paraformaldehyde (Roti ® -Histofix 4%, Roth Sochiel EURL, France) at room temperature. After inclusion in 4% low-melting agarose (Thermo Fisher Scientific, France), spheroids were embedded in paraffin and sectioned (Plate-Forme HistIM, Institut Cochin, Paris France). Sections (5 µm) were stained with haematoxylin and eosin (H&E) according to standard protocols. For immunohistochemical staining, the heat-mediated antigen retrieval of antibodies was carried out in citrate buffer at different pH (according to manufacturer protocols) and sections (5 µm) were then incubated with monoclonal antibody to cytokeratin AE1/AE3 (mouse, dil 1:50; M351501-2 Dako, France), fibronectin (rabbit, dil 1:250; ab2413 Abcam, UK) or CD-31 (mouse, dil 1:20; M082301-2 Dako, France). Primary antibodies were detected with peroxidase-conjugated secondary antibodies by using diaminobenzidine (DAB) as chromogen and haematoxylin as counterstain.

Selective Plane Illumination Microscopy (SPIM)

4-days old fluorescently-labelled spheroids were fixed in 500 µL of 4% paraformaldehyde (Roti ® -Histofix 4%) (1 h, room temperature) and then permeabilized with 500 µL of 0.1% Triton X-100 (Sigma Aldrich, France) in PBS (1 h, room temperature). Then, Triton solution was replaced with 200 µL of PBS and cell nuclei were stained overnight with Hoechst 33342 (NucBlue™ Reagent, Thermo Fisher Scientific, France) in the dark at room temperature.

MCTS were imaged with the Lightsheet Z.1 Microscope (Carl Zeiss, Germany) equipped with a Plan-Apochromat 20x 1NA water-immersion objective lens with left and right illumination. Hoechst, GFP and RFP fluorescent signals were recorded after sample excitation at 405, 488 and 561 nm, respectively. Samples were scanned using the Zen 2014 SP1 Black-Edition software (Carl Zeiss, Germany) and optical slices of 1 µm thickness were processed for the 3D reconstruction of each sample with the Image-J ® software.

MCTS embedding in fibrin matrix

PANC 1:MRC-5:HUVEC (ratio 1:9:4, LbL, VEGF) spheroids were prepared by using RFPexpressing HUVEC cells and cultured for 4 days before the embedding in a fibrin matrix.

Individual MCTS were harvested from the round bottom wells and transferred in a microtube using a p5000 pipette. Culture medium was carefully removed and spheroids were redispersed in 250 µL of bovine fibrinogen (Sigma Aldrich, France) solution (2.5 mg.mL -1 ) in PBS with aprotinin (0.15 U. mL -1 Sigma Aldrich, France) and type I collagen (0.2 mg.mL -1 , Gibco, France) [44]. After addition of 2.5 µL of thrombin (final concentration 0.5 U/ml, Sigma, France), single spheroids were transferred into the wells of a Lab-Tek®II Chamber SlideTM (Thermo Fisher Scientific, France). The chamber slide was placed at room temperature for 5 minutes to allow fibrin polymerization and then 400 µL of EGM-2 medium was added.

Embedded MCTS were maintained in a humid atmosphere at 37 °C with 5% CO 2 for three days before to perform confocal imaging. Samples were imaged with an inverted Leica TCS SP8 microscope gated-STED (Leica, Germany) using a HC PL Fluotar CS2 10x/0.30 dry objective lens. The instrument was equipped with a WLL Laser (587 nm excitation wavelength for RFP). Red fluorescence emission was collected with a 597-800 nm wide emission slits. Transmission images were acquired with the same laser line and a PMT-trans detector. The pinhole was set at 1.0 Airy unit giving 4.29 µm optical slice thickness. 12 bit numerical images were done with Leica SP8 LAS X software (Version 2.0.1; Leica, Germany). were prepared and cultured for 4 days before treatment with a series of concentrations of drugs (doxorubicin (doxo) and gemcitabine (gem)) in culture medium. At the end of the incubation period (48 h and/or 72 h), cytotoxicity assessment was performed by (i) optical imaging (see 2.4.1); (ii) ATP quantification (see 2.4.3) and/or (iii) qualitative and quantitative measurements of the bioluminescent signal (see 2.4.4). Untreated spheroids were used as control. The viability was calculated according to the ratio of the signal of the well containing treated spheroids versus the average signal of control wells (i.e., untreated spheroids).

Statistical analysis

Data are presented as mean values with standard error of mean (s.e.m). Statistical analysis was performed with the Prism GraphPad 7.0 software and the significance was calculated using a two-way Anova method, with a Sidak's multiple comparison post-test in which p < 0.05 was considered statistically significant (*p < 0.05 **p < 0.005, *** p < 0.0005, **** indicates p < 0.0001).

Results and discussion

Construction and characterization of multicellular tumor spheroids

Mono-type multicellular tumor spheroids

MCTS consisting of cancer cells only have been constructed according to the liquid overlay

technique. We have used the PANC-1 cell line, reported as aggressive and poorly differentiated [45, 46], and whose capacity to form multicellular spheroids had been Chapter I 87 previously described [47-53]. However, to our knowledge no comprehensive characterization of the growth profile of these spheroids was available. Hence, we have prepared PANC-1 spheroids by seeding cancer cells in non-adherent poly-HEMA-coated round-bottom plates at different densities (corresponding to 500, 1000, 2500 and 5000 cells per well) and carried out a broad analysis. Optical imaging at day 4 post seeding revealed the formation of uniform and reproducible 3D structures, whose volume, ATP content and cell number have been monitored over time for a period of 17 days. Representative images of spheroids made with 500 cells (Fig. 1a), showed the spontaneous assembly of cancer cells in compact spheroids with only few surrounding scattered isolated cells (day 4). Spheroid size increased over time and a dark central region was observed at days 13 and 17 post seeding. Similar behaviour was observed for spheroids prepared by seeding higher cell numbers (Fig. S1).

The follow up of the spheroid volume clearly revealed a variation rate inversely proportional to the number of seeded cells per well (Fig. 1b). The lowest cell density (500 cells/well) resulted in the formation of spheroids characterized by the fastest proliferation rate with the major diameter that rapidly increased from 400-500 µm (day 4) to 800-900 µm (day 17), corresponding to a 10-times increase of the volume (from 0.03 to 0.3 mm 3 ). On the contrary, only a 2-fold increase was observed for spheroids prepared by seeding 5000 cells/well.

The number of cells per spheroid, representative of the proliferation capacity of PANC-1 cell in 3D conditions, followed a similar profile (Fig. 1c). Seeding of 500 and 5000 cells/well led to spheroids containing at day 4, an average of 2000 (increment by a factor of 4) and 14000 cells (increment by a factor of 2.7), respectively. The delayed proliferation of spheroids prepared at the highest cells density was also confirmed by a less rapid variation of the ATP content over time (Fig. 1d).

On the basis of these results, we have chosen for further studies spheroids made by seeding 500 cells/well. These spheroids will be from now indicated as MCTS_#1. Histological Chapter I 88 analysis of these spheroids by H&E and cytokeratin staining, at day 4 from seeding, revealed a uniform cell distribution and a compact core in which cells tightly adhered to each other (Fig. 1e,f). However, MCTS_#1 did not stain for fibronectin (Fig. 1g), clearly revealing the lack of deposition of this ECM component. The histological analysis at day 17 post seeding was in agreement with these results and no fibronectin staining was detectable (Fig. S2).

According to the upregulation of FN in PDAC stroma and its crucial involvement in cancer cells survival, invasion and drug resistance [54-56], it was evident that these spheroids made of PANC-1 cells only did not allow to appropriately mimic the in vivo complexity of the PDAC.

Hetero-type multicellular spheroids: co-culture of cancer cells and fibroblasts

To mimic the tumor-stroma interactions we further gradually increased the complexity of our model and we moved to the construction of an intermediate hetero-type MCTS consisting of cancer cells and fibroblasts. We used MRC-5 fibroblasts, previously reported as a valid alternative to pancreatic stromal cells thanks to their capacity to promote the invasive phenotype of pancreatic cancer cells and to modulate the expression of cell adhesion markers Chapter I 90 as efficiently as PDAC patient-derived fibroblasts and pancreatic stellate cells [57, 58].

Noteworthy is that this cell line has already been used in different multicellular pancreatic tumor models, and it offered the possibility to study the tumor microenvironment and investigate the cross-talk between cancer cells and stromal ones [40, 59-61]. In addition, successful reconstruction of the interaction between cancer cells and the microenvironment using fibroblasts derived from healthy tissues has also been described for breast cancer 3D models [37, 62, 63].

By keeping constant the number of PANC-1 (i.e., 500 cells per spheroid at day 0), we investigated co-cultures with fibroblasts at different ratios (1:1, 1:2, 1:4 and 1:9). In all tested conditions the two cell types spontaneously self-assembled without the need of supporting material and the size of spheroids progressively increased over time (from day 4 to day 17) (Fig. 2a, b and Fig S3). Whatever the tested ratio, the number of cells per spheroid at day 4 was lower than expected according to the seeding density, thus clearly showing that a loss of cells occurred during the first steps of the co-culture assembly (Fig. 2c). Nevertheless, once formed, all spheroids continued to grow regularly as also confirmed by the evolution of the volume and the ATP content (Fig. 2b,d). Note that co-culture with fibroblasts enhanced the metabolic activity of cancer cells compared to spheroids made of PANC-1 only, resulting in a comparable effect to that induced by primary tumor-associated fibroblasts in Transwell ® cocultures [35]. The ratio PANC-1:MRC-5 1:9, which allowed, at day 4, to construct spheroids with the highest cell number was selected for further studies. These spheroids will be from now indicated as MCTS_#2. H&E staining at day 4 of MCTS_#2 showed the assembly of the two cell types and the predominant localization in the core of elongated cells surrounded by a population of round cells (Fig. 2e). The latter was positively stained for the cytokeratin AE1/AE3 marker (specific for the detection of epithelial cells of neoplastic origin) thus revealing the peripheral Chapter I 91 localization of the PANC-1 cells. On the contrary, the core was characterized by cytokeratin-negative cells and was positively stained for the fibronectin, thus suggesting the accumulation in this area of fibroblasts responsible for the secretion of the ECM component (Fig. 2f,g). Nevertheless, at day 7 post seeding (Fig. 2h-l) all cells positively stained for the cytokeratin marker revealing the infiltration of cancer cells in the core of the spheroid previously filled by fibroblasts. The disappearance of the MRC-5 cells well correlated with the negligible fibronectin staining (Fig. 2i,l). Same results were obtained at day 17 post seeding. (Fig. S4) Cell spatial distribution at day 4 was assessed also by Selective Plane Illumination Microscopy (SPIM) imaging of MCTS_#2 constructed using GFP-expressing MRC-5 fibroblasts. SPIM allowed to observe the spheroid mass in the whole and to collect high resolution images, bypassing the limitations in terms of depth of laser penetration of traditional imaging methods such as the confocal microscopy [64-66]. The blue staining of nuclei (Hoechst 33342) showed the distribution of cells in the 3D structure (Fig. S5a) and the green fluorescence in the core confirmed the presence of fibroblasts in this region at day 4 post seeding (Fig. S5c). However, in agreement with the histological characterization, at day 7 post seeding, the reduction of the green signal indicated the progressive loss of fibroblasts and their gradual replacement by rapidly proliferating cancer cells (Fig. S5f). These results showed that the tumor microenvironment was not static and a progressive transformation occurred over time leading to the integration of cancer cells and components of the microenvironment (i.e., fibroblasts and fibronectin) for up to 4 days. A progressive loss of fibroblasts had been previously observed in lung [67] and prostate [68] MCTS as well as in spheroid co-cultures of fibroblasts and endothelial cells. [69, 70] However, to the best of our knowledge, this is the first time that modifications of the composition of long-term cultured MCTS have been precisely assessed by histological and microscopic analysis in a hetero-type pancreatic tumor model.

Hetero-type multicellular spheroids: triple co-culture of cancer cells, fibroblasts and endothelial cells

The last step in the construction of a new model consisted in the introduction of HUVEC endothelial cells and establishment of a triple co-culture. Note that hetero-type pancreatic tumor spheroids including fibroblast and endothelial components have not been previously

Chapter I 93 reported. The choice of the HUVEC has been driven by their large availability and stable features compared to tumor-derived endothelial cells, whose wide application in routine in vitro studies is still hindered by several obstacles. They include, for instance, the scarce availability of fresh tumor sample, the difficulty to isolate tumor endothelial cells with high degree of purity, the lack of well-defined culture conditions with consequent phenotype mutation, their short life span and thus the possibility to use them for a limited number of passages only [71].

Firstly, PANC-1 cancer cells (500 cells per spheroid at day 0) were mixed with MRC-5 fibroblasts and HUVEC at 1:9:4 ratio and seeded on poly-HEMA-coated plates according to the liquid overlay technique already used for MCTS_#1 and MCTS_#2. Cell assembly and spatial organization of the three cellular components were regularly monitored as early as 2 days after seeding. Also in this case, immunostaining revealed a peripheral localization of the cytokeratin-positive cells (i.e., PANC-1 cells) while the cytokeratin-negative ones and fibronectin were localised in the core of the spheroid (Fig. S6a,b). However, this assembly was characterized by weak hetero-type cell-to-cell contacts as attested by the frequent loss of PANC-1 cells during sample preparation. In addition, CD31 immunostaining, used as a marker of the endothelial cells, highlighted only a feeble presence of HUVEC cells (Fig. S6c); MCTS_#1 and MCTS_#2 spheroids at day 4 post seeding were used as negative controls (Fig. S7). Two days after (day 4), spheroids were more compact (Fig. S6d,e) but the CD31 staining was negligible (Fig. S6f). As we evidenced above, a reorganisation of the cell composition with time was observed also in these spheroids, with a progressive accumulation of PANC-1 cells in the core of the spheroids together with a decrease of the fibronectin content (Fig. S6d,e). To promote integration and residence of endothelial cells within the spheroid, the preparation method was slightly modified. Briefly, before mixing with PANC-1 and HUVEC, a Layer-by-Layer (LbL) nanofilm of fibronectin and gelatin (FN-G) was constructed at the Chapter I 94 surface of the MRC-5 fibroblasts, according to a previously validated technique [72]. The FN-G coating on the cell membrane, which mimicked the natural ECM acting as an adhesive surface [73-75], had already been successfully applied to the construction of stable multilayer multicellular tissues on Transwell ® inserts [43, 76-80]. Such coating was capable of promoting cell adhesion into the spheroids, facilitating the triple co-culture with the HUVEC cells. Compared to the traditional protocol, this approach allowed to increase the number of CD31-positive cells, which after two days of culture formed a discontinuous layer at the fibroblasts/cancer cells interface. However, the latter were only loosely attached to the external spheroid boundaries (Fig. S8a-c). At day 4, spheroids were more compact, but also in this case the presence of endothelial cells was strongly reduced (Fig. S8d-f). As expected, the FN-G nanofilm supported a better assembly of the three cell types in the spheroid, but it was not enough to ensure their prolonged 3D co-culture. Of note, because of the presence of the adhesive FN-G layer, these spheroids were less fragile and could be more easily handled, thus facilitating their routinely use at day 4. Accordingly, this preparation method was maintained for all the successive experiences. Although collagen, rather than gelatin, is the main component of the pancreatic tumor ECM, the latter has been chosen for the cell coating due to its solubility in neutral medium, which ensures a higher cytocompatibility. On the contrary, the acidic solution of collagen is not well suitable for the culture condition due to the low pH value. Moreover, being the gelatin obtained by collagen denaturation, it maintains a chemical composition very similar to that of the parent molecule.

Significant improvement was then obtained by culturing the LbL-assembled spheroids in medium supplemented with VEGF (50 ng.mL -1 ), a well-known endothelial survival factor [81] and angiogenesis inducer [82]. In addition, VEGF already showed a beneficial effect on the viability and sprouting of HUVEC spheroids [34, 83, 84]. In these experimental conditions, at day 2, a thick layer of endothelial cells was observed around the fibronectin-rich Chapter I 95 cytokeratin-negative core (Fig. S9c). On the contrary, no stable adhesion of cancer cells occurred, and they could be easily lost during sample preparation (Fig. S9). A more compact spheroid was, instead, obtained after a culture period of 4 days (Fig. 3a). PANC-1 cancer cells, that are cytokeratin-positive cells, mostly organized in an external layer surrounding cytokeratin-negative cells and with which cell-to-cell hetero contacts were established (Fig. 3b). Fibronectin staining revealed homogenous distribution of this ECM protein in the core (Fig. 3c) in which a network of CD31-positive endothelial cells was also well visible (Fig. 3d).

The latter closely resembled to the collapsed vessels observed in PDAC [31, 33]. Thus, after opportune optimization of the experimental conditions (that is, LbL coating and VEGF supplement) we successfully constructed a hetero-type pancreatic MCTS based on a triple coculture of cancer cells, fibroblasts and endothelial cells. These spheroids (PANC-1:MRC-5:HUVEC, LbL, VEGF) will be from now indicated as MCTS_#3. Histological analysis showed that the optimal triple co-culture was achieved at day 4. At day 7, all cells were positively stained for the cytokeratin (Fig. 4a), as previously observed also for MCTS_#2 (Fig. 2h-l), and no more fibronectin and HUVEC were detectable (Fig. 4b,c). The spatial distribution of the three cell types at day 4 was assessed in detail by SPIM imaging of MCTS_#3 constructed using GFP-expressing MRC-5 fibroblasts and RFPexpressing HUVEC cells (Fig. 5). The uniform blue staining revealed a homogeneous distribution of cells in the 3D structure (Fig. 5b). The green fluorescence confirmed the presence of fibroblasts in the core (Fig. 5c), which correlated with histology images showing a core made of cytokeratin-negative cells and rich in fibronectin (Fig. 3b,c). Red-fluorescent HUVECs were also visible in the centre of the spheroid confirming their strong association with the fibroblasts (Fig. 5d). The slightly elongated shape of HUVEC suggested their potential capacity, in opportune conditions to develop vessel-like networks (Fig S10 ), as previously observed by Boutin and co-workers in their imaging analysis of rat cortical spheroids [85]. In agreement with the histological characterization, SPIM imaging of MCTS_#3 spheroids at day 7 showed the progressive loss of the stromal cells (Fig. 5e-h).

Chapter I 97 According to the stability of the triple co-culture, characterization of MCTS_#3 was carried out for up to 7 days from seeding. Morphology and growth kinetic were microscopically assessed and optical-images showed spheroids with a dense core and a ruffled peripheral cell layer whose compactness increased over time (Fig. 6a). Since day 2, the triple co-culture led to the formation of larger spheroids as compared to MCTS_#1 and MCTS_#2 (Fig. S11) and they reached a mean volume of 0.09 mm 3 (~ 600 µm in diameter) at day 4 (Fig. 6b). At this time point, MCTS_#1 and MCTS_#2 showed a mean volume of 0.03 and 0.04 mm 3 , respectively (Fig. 1 and2). Variation of the number of cells over time revealed the same trend (Fig. 6c). Also in this case, single cell counting in MCTS_#3 evidenced that not all cells assembled to form the spheroids but that a certain loss occurred, and the number of cells per Chapter I 98 spheroid at day 2 was lower than the seeded one. This corroborated the hypothesis of the existence of a critical initial number of cells for establishing heterotypic interactions and stable 3D assembly. The strong influence of the microenvironment on the spheroid metabolic activity was confirmed by the ATP content (Fig. 6d) which, at day 4, was up to two times higher than in spheroids made of only cancer cells (0.54 and 1.2 µM for MCTS_#1 and MCTS_#3). This influence, likely mediated by the direct 3D cell-to-cell contact as well as by the production of growth factors and cytokines [60, 86] persisted also at day 7 when spheroids where mainly composed of cancer cells (Fig. 4). Among the secreted soluble factors, the hepatocyte growth factor (HGF) might play a key role. It has been demonstrated that by binding to its transmembrane receptor c-Met, the HGF may activate several signaling pathways involved in pancreatic cancer cells growth and invasion, tumor progression, desmoplastic reaction and resistance to treatment [87-89]. In addition, previous studies have reported a high secretion of HGF by MRC-5 fibroblasts, which was responsible of an enhanced proliferation and migration of several types of cancer cells [35, 90-92]. Thus, although further investigation would be required to confirm this hypothesis, it is possible that the proliferation of PANC-1 might be mediated by the HGF secreted by the MRC-5 cells.

Overall, these results demonstrated that the structural and functional interactions between cancer cells and several stromal components have been successfully recreated in MCTS_#3.

Cytotoxic studies: effect of the tumor microenvironment

It is well known that the tumor microenvironment strongly influences the drug sensitivity of cancer cells and may affect the efficacy of treatments [29]. Therefore, we aimed to verify whether the MCTS_#3 model was capable or not to reproduce such influence. Firstly, as proof of concept we assessed the cell viability in MCTS_#3 and MCTS_#1 after a 48 h and 72 h exposure to a single dose of doxorubicin (0.5 µM, IC 50 for PANC-1 cells grown in 2D monolayer). Because of their stability and 3D compact structure, spheroids at day 4 were used for the onset of the treatment. Spheroids were constructed using luciferase-expressing PANC-1 cells, enabling the luciferase activity to be quantified to specifically monitor the cytotoxicity of doxorubicin on cancer cells. After 48 h of incubation, the survival of cancer cells was similar in both types of spheroids and only a general decrease of ~10% could be measured in comparison to their respective untreated controls (Fig. 7a). However, after longer exposure Chapter I 100 (72 h) higher survival was observed in MCTS_#3. Sensitivity of cancer cells was influenced by the presence of the tumor microenvironment (Fig. 7a). Bioluminescence images of these spheroids were in agreement with the quantitative results. Only a weak difference of the pseudocolor was observed between control (untreated) and doxo-treated MCTS_#3. On the contrary, for MCTS_#1 the colour changed from green to light blue (lowest values) after treatment which underlined the higher sensitivity to doxorubicin (Fig. 7b). Accordingly, the overall spheroid viability, as measured by the ATP content, was also significantly different at 72 h and confirmed the key role of the tumor microenvironment on the sensitivity of the spheroids to the treatment (Fig. 7c). In addition, the anti-proliferative effect of the drug has been also microscopically assessed by collecting bright-field images of spheroids (n=64 per condition). Quantitative information of the growth inhibition was provided by the variation of the spheroid volume after treatment [93]. Once again data revealed the lowest anticancer efficacy of the drug on MCTS_#3 and further corroborated the influence of the microenvironment on the proliferation of the tumor mass (Fig. 6d). The higher sensitivity of MCTS_#1 compared to MCTS_#3 was observed also after exposure of spheroids, constructed using luciferase-expressing PANC-1 cells, to a series of doxorubicin concentrations (Fig. S12a). Similar results were obtained after incubation with gemcitabine, in spite of the different mechanism of actions of the two anticancer drugs (Fig. S12b). Indeed, the doxorubicin exerts its cytotoxic activity by intercalation between DNA base pairs blocking DNA replication and transcription, blockade of topoisomerase II activity as well as generation of free radicals, [94] while, on the other hand, gemcitabine acts as nucleoside analogue [95].

Noteworthy is that after exposure to doxorubicin and gemcitabine, MCTS_#3 displayed higher resistance to treatments also when compared to MCTS_#2, thus confirming the crucial role of a complex tumor microenvironment consisting of multiple cell types (Fig. S13).

We have then modified the culture condition to assess whether similar results could be Chapter I 101 obtained by using another pancreatic cell line. Thus, both mono-type and hetero-type (i.e., triple co-culture) multicellular spheroids, have been constructed replacing the PANC-1 cell line with the BxPC-3 one (indicated as MCTS_#1 BxPC-3 and MCTS_#3 BxPC-3, respectively). However, exposure of these spheroids to growing concentrations of doxorubicin and gemcitabine did not induce a dose/effect response. In addition, for each concentration tested, we observed a higher viability of mono-type spheroids compared to the hetero-type triple coculture (Fig. S14). This was attributed to the more compact structure of MCTS_#1 BxPC-3 (Fig. S15), which artefactually opposed to the diffusion/penetration of the drug through the tumor mass. These results clearly highlight that accurate selection of the cell types used for the construction of the spheroids and characterization of the resulting cell assembly are a key requisite to develop valuable tools for accurate preclinical screening of therapeutic strategies for pancreatic cancer treatment.

Conclusions

The failure of treatments and the poor prognosis of pancreatic ductal adenocarcinoma are related to the extremely complex physio-pathology of this tumor, combining abundant fibrosis Chapter I 103 and collapsed vasculature, which strongly limits drugs accessibility. In this study, we reported on the construction and detailed characterization of a scaffold-free 3D model capable to integrate in a hetero-type multicellular spheroid the cancerous component of the pancreatic tumor as well as its microenvironment made of fibroblasts, endothelial cells and extracellular matrix. To our knowledge, no analogous model combining the fibrotic tissue and a collapsed vessel-like structure, both hallmark of the PDAC, has been previously constructed. Evaluation of spheroid growth and response to treatment confirmed the influence of the microenvironment on drug sensitivity of pancreatic cancer cells, and showed the capacity of this model to replicate the resistance to treatments often observed in vivo. Overall, we believe this easy to handle spheroid model, reproducing crucial features of the PDAC, could find application in standardized drug screening protocols as well as in the assessment of cancer cells-stroma interactions and tumor angiogenesis. E-mail address: simona.mura@u-psud.fr

Introduction

An efficient chemotherapeutic treatment relies on the capacity of drugs, either in free from or loaded in nanoscale systems (i.e., nanomedicines), to move through the tumor tissue and reach the target cells [1]. However, the complex physiopathology of solid tumors makes the penetration not straightforward. It is well known that the uncontrolled growth of cancer cells generates a solid pressure able to compress and obstruct blood and lymphatic vessels [2] and that this aberrant vasculature strongly limits cancer cell accessibility to nutrients, oxygens and also reduces their exposure to drugs [3]. If intravenous injected drugs/nanomedicines succeeded to extravasate, the high interstitial fluid pressure and the thick extracellular matrix (ECM) would further hamper their path to the target cells. Consequently, they often accumulate in the perivascular space only [4, 5] and the drug concentration in the tumor does not reach the optimal therapeutic level [6, 7].

Although nanomedicines can improve the drug pharmacokinetic and reduce side effects related to a nonspecific systemic distribution, they frequently suffer from the difficulty to efficiently cross the tumor biological barriers [8-10]. It has been demonstrated that different physicochemical properties strongly influence the penetration of drug delivery systems into the tumor mass and various strategies have been proposed to maximize their performances [11-13]. However, the predictive in vitro assessment of the capacity of tissue penetration still represents one of the main difficulties in the preclinical screening of any new nanomedicine [6]. It is evident that such information cannot arise from 2D cell cultures. Indeed, in conventional uptake studies, drugs and nanomedicines are added on the top of cancer cells Chapter II 128 grown on flat monolayers and the only barrier they have to cross to reach the intracellular compartment is the cell membrane. Useful data can instead be collected using 3D cell culture models which, reproducing in vitro the tumor and its microenvironment, are more representative of the biological reality. Among them, multicellular tumor spheroids (MCTS) have attracted a great deal of attention thanks to the capacity to mimic key features of nonvascularized tumors, such as the close contact among cells and their self-organization in layers with different proliferation rates [14, 15]. In addition, cells in spheroids secrete ECM proteins thus allowing to recreate a microenvironment that acts as biological barrier able to interfere with the transport phenomena and block the diffusion of drugs and NPs [11, 16].

However, the thickness of such 3D models represents a technical challenge making quite difficult the qualitative and quantitative in vitro assessment of the penetration of fluorescent drugs and NPs [17]. In order to overcome these issues, the penetration is often evaluated on spheroid slices sectioned at different depths from paraffin-embedded or frozen samples which are then imaged by conventional fluorescence microscopy [18-20]. However, spheroid sectioning is laborious and time consuming [21]. Although this strategy allows to obtain high quality images, it does not give a representative reconstruction of complex and heterogeneous 3D structures [22]. Alternatively, the penetration of fluorescent molecules into MCTS is largely assessed with the Confocal Laser Scanning Microscopy (CLSM) through a virtual stack of optical sections [23-27]. In the CLSM set-up, the same lens is used for the excitation of dyes and the detection of the emitted signals and the pinhole system allows to remove the out-of-focus light making possible the optical sectioning of the 3D samples [28]. However, the image resolution is generally restricted to depth distances of 60 -80 µm [23, 25, 27] because both the excitation and emission lights, passing through the spheroid perpendicularly to the image plane, encounter a pronounced scattering [29]. The different refractive indices (Tyndall effect) and the non-uniform distribution of cells and extracellular components in the Chapter II 129 tissue cause wavelength-dependent phenomena of light scattering and absorption that degrade the quality of the fluorescent signals [30]. As a consequence, the number of emitted photons able to arise from the scanned sample decays exponentially with the increasing depth [31]. This problem could be partially solved by the use of higher wavelengths with two-or multiphotons microscopies [32-34]. However, due to the exposition of the whole sample to the excitation light during the imaging of each optical section, these techniques suffer in general from the production of strong photobleaching and phototoxic effects [35].

In this context, the Light Sheet Fluorescence Microscopy (LSFM) has recently emerged as a technique suitable for the imaging of large biological specimens with high scattering properties [22, 36-38]. This approach overcomes the limitations of traditional microscopies and allows deep sample scan at high imaging speed with minimal photodamage [28, 36]. An illumination system (made of one or two illumination lenses) creates a light sheet of few microns thickness that scans the sample and excites only the fluorophores in the focused plane [39]. Since the detection lens is placed perpendicularly to the light sheet, the out-of-focus light is strongly reduced allowing a better definition of the scanned section (Fig. 1a) [29, 40].

Thanks to the separation of illumination and detection pathways, only the detection objective is responsible for the lateral resolution [29, 41]. The optical sectioning of large samples is then easily obtained by moving them through the focal plane. This translation along the optical axis of the detector creates a z-stack of images that can be used for the 3D reconstruction [42, 43]. In recent years, the LSFM in combination with 3D models has been widely used in preclinical drug discovery to study morphological changes and toxic effects at a cellular level [17, 30, 43-46] but only few studies have proposed this technique to evaluate the penetration of drugs in simple spheroids made of one cell type [47, 48]. Whether such approach would be suitable also in systems with higher complexity was matter of question.

In this study we have investigated the performance of these imaging methodologies to assess Chapter II 130 the penetration of doxorubicin (doxo) and doxo-loaded NPs into a hetero-type MCTS model of pancreatic cancer recently developed in our team [49]. This model integrates both the cancerous component of the pancreatic tumor (cancer cells) and cellular/acellular components of the microenvironment (that is, fibroblasts, endothelial cells and extracellular matrix) and therefore represents a valuable model capable to mimic the obstacles to diffusion. The advantages of the LSFM compared to the traditional CLSM in terms of deep imaging and isotropic resolution were largely confirmed. Overall, the results collected from the multi-view imaging (Fig. 1b) provide a powerful example of the feasibility of the LSFM application in vitro on complex hetero-type MCTS and strongly encourage its use since the earliest phases of preclinical screening of nanomedicines for tumor treatment.

Materials and methods

Cell lines

Human pancreatic cancer cells (PANC-1, ATCC, France) were maintained in Dulbecco's Modified Eagle Medium-high glucose (DMEM, Sigma Aldrich, France) supplemented with Chapter II 131 10% heat-inactivated fetal bovine serum (FBS, Gibco, France). Human lung fibroblasts (MRC-5, ATCC, France) were cultured in Eagle's Minimum Essential Medium (EMEM, Sigma Aldrich, France) supplemented with 10% heat-inactivated FBS and 1% of 200 mM L-Glutamine solution (Sigma Aldrich, France). Human umbilical vein endothelial cells (HUVEC, ATCC, France) were maintained in endothelial growth medium (EGM-2) consisting of endothelial basal medium (EBM-2) in which supplements and growth factors were added according to manufacturer instruction (EGM-2 BulletKit Lonza, France). Cells were held in incubator with a humid atmosphere at 37 °C with 5% CO 2 , used below passage 8 after thawing and harvested at a confluence of 70-80%.

MCTS formation

Hetero-type MCTS containing pancreatic cancer cells, fibroblasts and endothelial cells were constructed according to the liquid overlay technique [50] in poly-2-hydroxyethyl methacrylate (poly-HEMA)-coated (1.2 % (w/v)) 96 round-bottomed well plates (CELLSTAR ® , Sigma Aldrich, France).

A seeding ratio of 1:9:4 among PANC-1:MRC-5:HUVEC was used for the construction of both small (500:4500:2000 cells per well) and large (5000:45000:20000 cells per well) spheroids. As previously described [49], a fibronectin-gelatin nanometer-sized film was created at the MRC-5 surface before seeding according to the layer-by-layer method [51].

Briefly, after trypsinization, fibroblasts were suspended in 1 mL of Tris-HCl buffer (50 mM, pH 7.4) (Sigma Aldrich, France), centrifuged (2500 rpm, 1 minute, room temperature) and then alternatively incubated for 1 minute using a microtube rotator (VWR, France) in 1 mL of 0.04 mg.mL -1 Human fibronectin or gelatin (Sigma Aldrich, France) Tris-HCl solutions (50 mM, pH 7.4). Each incubation was followed by a centrifugation (2500 rpm, 1 minute, room temperature) and a washing step with Tris-HCl buffer (1 mL, 50 mM, pH 7.4). A total of 9 coating steps were performed. Then, fibroblasts were suspended in cell culture medium and used to construct the triple co-culture. Suspensions of each cell type (that is, PANC-1 cells, coated-MRC-5 and HUVEC) were prepared in DMEM complete medium supplemented with the human vascular endothelial growth factor (VEGF, 50 ng.mL -1 ) (Thermo Fisher Scientific, France) and then 200 µL of their opportune mixture was transferred into each well. Cell counting was performed with the automatic cell counting (Countess II, Thermo Fisher Scientific, France), according to manufacturer instructions. After cell seeding, plates were centrifuged (1100 rpm, 5 minutes, 20 °C) and then incubated in a humidified atmosphere with 5% CO 2 at 37 °C for 4 days.

Optical imaging and volume measurement

Optical images were acquired using the AxioObserver Z1 (Carl Zeiss, Germany) inverted microscope equipped with a Peltier cooled (-40 °C) CoolSnap HQ2 CCD camera (Photometrics,Tucson, USA) and a XL incubator thermostated at 37 °C providing 5% CO 2 .

By using a halogen lamp and a motorized stage in automated mode (AxioVision software / high content acquisitions), transmitted light images of spheroids were collected directly from the poly-HEMA coated plates with a Plan-Apochromat 2.5x dry objective lens.

Through an image-processing macro, specifically created with the Image-J ® software, the minimum and major diameters of 40 spheroids were measured. Spheroid volume (V) was calculated according to the formula V = [(a²)*(b)]/2, in which a and b represent the minor and major diameter, respectively.

Doxorubicin-loaded nanoparticles preparation and characterization

The drug-initiated method was used to grow a polyisoprene (PI) chain from an initiator bearing gemcitabine (gem) [52, 53]. Then, a nitroxide exchange reaction was applied to post-Chapter II 133 functionalize the polymer with doxorubicin (doxo) [54, 55]. In this way, a gem-PI-doxo heterotelechelic (dual functionalized) polymer prodrug was synthesized (Fig. 7) [55].

Nanoparticles were formulated according to the nanoprecipitation metod. In brief, 2.5 mg of the polymer were dissolved in 0.5 mL of tetrahydrofuran and quickly added to 1 mL MilliQ water. The organic solvent was evaporated at room temperature under vacuum (Buchi Rotavapor, France). The nanoparticle diameter was measured by dynamic light scattering (DLS) with a Nano ZS from Malvern (173° scattering angle) at a temperature of 25 °C after 1:20 dilution in MilliQ water. The surface charge of the nanoparticles was investigated by zeta (ζ)-potential (mV) measurement at 25 °C after a 1:10 dilution with 1 mM NaCl by applying the Smoluchowski equation and using the same instrument. The morphology of the nanoparticles was observed by Cryogenic Transmission Electron Microscopy (cryo-TEM). 5 µL drop of nanoparticle suspension (0.5 mg.mL -1 ) was deposited on a Lacey Formvar/carbon 300 mesh copper microscopy grid (Ted Pella). The excess of the drop was removed with a blotting filter paper and the remaining thin film within the holes of the grid was vitrified by using liquid ethane. Samples were then observed using a JEOL 2100HC microscope.

Spheroid penetration assessment with CLSM

Small hetero-type MCTS (500:4500:2000 cells per well) were prepared and cultured for 4 days before incubation with doxorubicin at a concentration of 20 µM in culture medium.

After 3 h of incubation at 37 °C, MCTS were harvested, transferred in a microtube and then the culture medium was carefully removed. Spheroids were washed once with 200 µL of phosphate buffered saline (PBS, Sigma Aldrich, France) and fixed in 500 µL of 4% paraformaldehyde (Roti ® -Histofix 4%) (1 h, room temperature). Samples were finally imaged with an inverted Confocal Laser Scanning Microscope LSM 510-Meta (Carl Zeiss, Germany) using a Plan-Apochromat 20x/0.75 objective lens, equipped with an argon laser (488 nm Chapter II 134 excitation wavelength). The red fluorescence emission was collected with a 560 nm long pass (LP) emission filter. The pinhole was set at 1.0 Airy unit. 12-bit numerical images were acquired with the LSM 510 software version 3.2.

Spheroid penetration assessment with LSFM

Small (500:4500:2000 cells per well) and large (5000:45000:20000 cells per well) heterotype MCTS were prepared and cultured for 4 days before incubation with doxo or doxoloaded NPs, (20 µM in culture medium). Small and large MCTS were harvested after 3 h and 4 h of incubation at 37 °C, respectively. The culture medium was carefully removed and, after a single washing step in 200 µL of PBS, spheroids were fixed in 500 µL of 4% paraformaldehyde (Roti ® -Histofix 4%) (1 h, room temperature). To facilitate the post-imaging z-stack fusion, nuclei of large MCTS were stained. Thus, after fixation, MCTS were permeabilized with 500 µL of 0.1% Triton X-100 (Sigma Aldrich, France) in PBS (1 h, room temperature). Then, the Triton solution was replaced with 200 µL of PBS and spheroids were stained at least for 24 h with Hoechst 33342 (NucBlue™ Reagent, Thermo Fisher Scientific, France) in the dark at room temperature. Spheroids were then kept in the dark with the nuclei staining solution until the moment of imaging. To perform the LSFM imaging, samples (small and large MCTS) were embedded in 0.8 % low-melting agarose (Thermo Fisher Scientific, France) by using a glass cylindrical capillary and then images were acquired with a Lightsheet Z.1 Microscope (Carl Zeiss, Germany) equipped with a Plan-Apochromat 20x 1NA waterimmersion objective lens with left and right illumination. Red (doxorubicin) and blue (Hoechst) fluorescent signals were recorded after sample excitation at 488 and 405 nm, respectively. Samples were scanned using the Zen 2014 SP1 Black-Edition software (Carl Zeiss, Germany) with a 575-615 nm LP emission filter for doxo and a 420-470 nm LP emission filter for Hoechst. For small spheroids, collected optical slices were processed for the 3D reconstruction by maximum intensity projection (MIP) with the Image-J ® software.

With the large MCTS, a 3D tomography was obtained with the Imaris Stitcher ® software (Bitplane AG, Zurich -Switzerland) by fusion of two z-stacks collected from two independent views of the same sample (0° and 180°) (Fig. 2). The Imaris ® software (Bitplane AG, Zurich -Switzerland) was used to render the 3D representation of the entire spheroid and select single optical sections.

Results and discussion

Penetration studies with CLSM

Pancreatic tumor spheroids made of cancer cells and stromal components were constructed by seeding 500 PANC-1 cancer cells, 4500 MRC-5 fibroblasts and 2000 HUVEC cells per well (1:9:4 ratio) in non-adherent poly-HEMA-coated round-bottom plates. As previously described, a 3D model integrating the three cellular components and capable to mimic the heterogeneity of the pancreatic tumor was obtained after four days of culture. At this time point, spheroids showed an average diameter of 600 µm [49].

We have used this model to investigate the drug penetration by incubating spheroids with doxorubicin, an anticancer drug showing high DNA affinity [58] and fluorescent properties useful for imaging [6]. After 3 h of incubation, the penetration of doxo was firstly assessed by Confocal Laser Scanning Microscopy using a z-stack of 149 focal plans and a step pass of 0.98 µm. Virtual slices were recorded until complete loss of the fluorescent signal and subsequently rendered as a maximum intensity projection (MIP) for the 3D reconstruction (Fig. 3a). The total scanned portion was approximately of 145 µm. The MIP clearly showed the intercalation of doxorubicin in the DNA, which resulted in a strong red fluorescence in all the cell nuclei at the spheroid surface. However, the MIP did not provide information about the depth of penetration, whose appreciation required the analysis of single optical sections from the z-stack. The optical section taken at ~ 5 µm depth, showed the doxo internalization and accumulation in the nuclei of cells of the external layer of the spheroid, which appeared quite rough and somehow irregular (Fig. 3b). Doxo penetration could be observed up to 50 µm depth, even if a slight decrease of the fluorescent signal was already evident (Fig. 3c).

Then, the continuous loss of the doxo fluorescence in the deepest layers strongly reduced the quality of the imaging in the spheroid core (Fig. 3d) and at a distance of around 100 µm from the spheroid surface, the real degree of drug penetration could not be estimated. The absence of signal in the inner part of the spheroid was to ascribe i) to the absence of doxorubicin (that is, the drug was unable to reach this area) or ii) to the scattering and absorption of the 3D tissue. This could not be verified by CLSM, clearly demonstrating and confirming the limitation of this imaging technique in the investigation of drug diffusion into spheroids.

Penetration studies with LSFM

Can doxorubicin penetrate through these spheroids entirely until their core? An unequivocal answer to this question could not be obtained by CLSM. Thus, hetero-type MCTS incubated with doxorubicin following the same experimental set up (i.e., concentration of 20 µM, 3 h) were imaged with the Carl Zeiss Lightsheet Z.1 microscope. This Selective Plane Illumination Microscope (SPIM) was equipped with two lenses for a double-sided illumination of the sample and a light-sheet pivoting system able to create a regular exposition and to reduce stripe artefacts [40, 59]. The spheroid was embedded in an agarose column by filling a glass capillary connected to a motor that allowed the physical rotation of the sample for the acquisition of z-stacks from different rotation angles [30, 60]. The MIP of the whole spheroid (observation angle 0°) was reconstructed from 521 optical sections recorded every 0.73 µm. This projection (Fig. 4a), confirming the results obtained by CLSM (Fig 3a), clearly demonstrated the capacity of doxorubicin to penetrate in the cells and then move to the nuclei.

While CLSM failed to assess the drug penetration in the deeper layers, herein the analysis of single planes highlighted the higher performance of the LSFM. Indeed, the light sheet allowed to scan the spheroid from the surface to the core without loss of the fluorescent signal (Fig. 4b-d) and the collected images showed that the drug was able to diffuse uniformly in the Chapter II 138 peripheral layers (Fig. 4b), as well as, in the inner ones (Fig. 4d). Once arrived in the centre, the spheroid was turned of 180° to image with high resolution also the other half of the sample. Such multi-view imaging is a specific feature of the LSFM and, interestingly, it requires only a short frame of time thanks to the employment of a Charge-Coupled Device (CCD) camera [40, 57]. 535 other optical sections with a step pass of 0.73 µm were collected and allowed to definitively demonstrate the presence of a fluorescent signal in the whole spheroid and confirmed that doxorubicin was capable to diffuse through the entire sample (Fig. 4e-h). Overcoming the limited isotropic resolution of the conventional confocal technique, the LSFM permitted a highly informative imaging thus clarifying the misleading results obtained with the CLSM. In order to further prove the potentiality of the LSFM technique, we applied it to the imaging of larger spheroids, which in addition offered the opportunity to test the 3D tomography as post-imaging reconstruction [57, 61] through the fusion of two z-stacks independently recorded at 0° and 180°.

Hetero-type MCTS with a diameter of ~ 1 mm (mean volume of 0.5 mm 3 ) were constructed by keeping constant the cell ratio (1:9:4, PANC-1:MRC-5:HUVEC) but increasing 10 times the cell number (Fig. 5). Following spheroid incubation with doxorubicin (20 µM, 4 h), the cell nuclei were stained with Hoechst 33342 which allowed to display the whole 3D mass during the acquisition and facilitated the merging of the z-stacks during the processing. Problems of light scattering and absorption are frequent with such large specimens [38, 43]. Thus, images from the different optical planes were collected until the first shadowing of the blue fluorescent signal occurred.

Then, the sample rotation of 180° and the acquisition of the second z-stack ensured the imaging of the whole spheroid. The final fusion of the two z-stacks generated a single data set of 324 focal planes spaced 2.90 µm along the z-axis. Their 3D complete reconstruction Chapter II 140 enabled to appreciate the spherical volume of the sample in which the red fluorescence of the drug matched with all visible cell nuclei (Fig. 6a-c). The doxorubicin penetration in the whole spheroid was demonstrated by three different optical sections, recorded at depths of 113, 403 and 861 µm (Fig. 6d-i). To be noted that section 2 was obtained from the middle of the sample and evidenced the capacity of doxorubicin to diffuse deeply and reach the core of the spheroid (Fig. 6e,h). The red fluorescence was visible in the central part of the sample though, as consequence of strong scattering phenomena, the signal was shadowed and hindered the clear distinction of the doxorubicin in the nuclei.

Overall these results confirmed the penetration capacity of this small molecule into large spheroids and data were in accordance with the penetration depth demonstrated with other advanced microscopic systems [16, 48]. In the last decades, nanoscale drug delivery systems have been proposed to overcome the limitations associated to the administration of drugs in free form (e.g., rapid drug metabolization, excretion, nonspecific cell distribution and insurgence of resistance phenomena) and also doxorubicin has been loaded in various systems such as liposomes, polymer nanoparticles or micelles [62-66]. In this context, our group is working since several years on the formulation of polymer prodrug nanoparticles and recently, innovative heterotelechelic polyisoprene nanoparticles were formulated using as building block a linear polymer whose end chains were conjugated to two anticancer drugs (i.e., doxorubicin and Chapter II 142 gemcitabine) [55]. By simple nanoprecipitation of the organic solution of this polymer in water, spherical nanoparticles, with a mean diameter of 100 nm, narrow size distribution, and negative surface charge (-25 mV) were obtained (Fig. 7). Taking advantage of the fluorescent properties of doxorubicin, we have investigated the capacity of these NPs to diffuse through the tumor mass. The NP penetration was investigated following the incubation (20 µM equiv. doxorubicin, 4h) with the large spheroids. Afterwards, spheroids were imaged by SPIM and 295 optical sections from the entire spheroid were collected and merged. Images were recorded every 2.90 µm by two independent z-stacks at 0° and 180°. Opposite to what was observed with the free drug, after 4h of incubation, no red fluorescence signal was detectable (Figure 8). To be noted that, when these polyisoprene NPs dual functionalized with doxorubicin and aminoglutethimide were incubated with 2D monolayers of cancers cells, their cellular internalization was observed even at earlier time points using a conventional confocal microscope [55].

The fast penetration of the free doxorubicin is likely to be attributed to the rapid diffusion in the ECM and in the interstitial spaces between cells, before being internalized and avidly bind to the DNA [6, 58]. A similar behavior was likely hindered by the hydrophobic surface of the Chapter II 143 nanoparticles and their larger size, demonstrating the influence of the physico-chemical features on the capacity of diffusion in this 3D tumor model harboring a dense ECM. This was in agreement with the previous observation of Kataoka and coworkers which showed that large micelles (100 nm diameter) hardly diffused in fibrosis-rich pancreatic tumors in vivo [67].

Overall these data clearly highlight that a reliable prediction of the capacity of nanoparticles to reach the target cells and efficiently delivery their cargo requires the use of models capable to reproduce the complexity of the tumor tissue. In addition, when moving to such models, opportune powerful imaging techniques are imperatively needed to nullify the risk of false interpretation of the experimental data as can happen by using confocal microcopy only.

Conclusions

In this study we have demonstrated that the application of the Light Sheet Fluorescence Microscopy allowed to overcome the limitations of the traditional confocal imaging and thus precisely discriminate the capacity of free doxorubicin and drug-loaded nanocarriers to penetrate (or not) in a complex 3D tumor model of pancreatic tumor. Thus, by sample imaging with a Selective Plane Illumination Microscope we succeeded in the monitoring of the fluorescent signal in the whole spheroid without loss of resolution and thanks to the complete 3D tomography of the MCTS, we confirmed that the doxorubicin was capable to reach the spheroid core. Moreover, we did not observed penetration of nanoparticles in this stroma rich model, while they have been easily visualized intracellularly when incubated with cancer cells cultured in 2D. This work demonstrated well that the increase in complexity of the in vitro models must be accompanied by an increase of the power of the instruments used for their analysis. Herein, presented results must strongly encourage the use of opportune tools to carry out a reliable preclinical screening in vitro of nanomedicines.

Introduction

In the last decades, different drug delivery strategies have been exploited in the treatment of solid tumors to avoid the nonspecific distribution of drugs in the whole body and promote their accumulation at the target site. However, therapeutic compounds are generally injected intravenously and from the site of administration to that of action, they have to overcome several biological barriers in a multistep process dependent on various parameters [1]. Firstly, the movement and the trajectory of drugs and nanomedicines in the vascular network and, consequently, their interaction with the vessel wall are strongly affected by the geometry of vessels and the shear stress induced by the blood flow [2]. Then, the extravasation of these anticancer agents is generally attributed to the enhanced permeability of the tumor vasculature which, coupled to a reduced drainage, allows their accumulation in the tumor mass. However, the extent of this so called enhanced permeability and retention (EPR) effect is extremely variable among patients [2, 3]. Finally, once extravasated, drugs and nanomedicines have to diffuse through the tumor mass to reach the target cells. In this last step, further obstacles are represented by the presence of a dense extracellular matrix (ECM), thigh cell-to-cell interactions and a high interstitial fluid pressure [3].

Frequently, the poor understanding of these events causes the failure in vivo of drug delivery systems although they seemed extremely promising in vitro [4, 5]. The cause of these misleading results has probably to be identified in the still too limited use, during the in vitro preclinical studies, of relevant models capable to reproduce the biological barriers [6]. The routine investigation relies on cytotoxicity and internalization studies that are generally carried out on 2D monocultures of cancer cells. Recently, several groups have introduced 3D models such as multicellular tumor spheroids (MCTS). Often, these MCTS are prepared using only cancer cells and despite the 3D architecture, they remain a simplistic model far from the complexity of human tissues in which different cell types interact together in a 3D microenvironment perfused by an intricate vascular network [7, 8]. Undoubtedly, the use of hetero-type MCTS capable to mimic the interactions between epithelial cells, stromal components and proteins of the extracellular matrix [9-12] has improved the reliability and the predictive capacity of the in vitro studies, enabling a better evaluation of the nanomedicine penetration capacity and their antitumor activity [13]. However, they lack of fluid dynamics and only mimic a static condition [14].

Microfluidic and 3D bioprinting techniques have been applied to generate organ-on-a-chip (mainly liver, brain, gut, breast, heart and kidney) able to recapitulate at different levels the complexity of the human physiopathology [15-19]. Endothelial walls, tissue barriers and cellto-cell interactions have been recreated in many microfluidic platforms [8, 20, 21] and they allowed i) the modelling of vascularized organs [22-25], ii) the mimicry of the vascular pathophysiology [26, 27], iii) the investigation of angiocrine signaling [28, 29], iv) the assessment of tumor-induced angiogenesis, cell invasion and metastasis [14, 23, 30-35] and v) the evaluation of the anticancer and/or antiangiogenic efficacy of drugs and nanomedicines delivered by controlling the flow through the device microchannels [36-38]. Moreover, it has been demonstrated that fibroblasts and endothelial cells embedded in ECM hydrogels (used as scaffolds) could generate perfusable microvascular networks when co-cultured in specific microfluidic devices [14, 39, 40]. The recreation in these devices of shear stress, growth factor gradients and cellular interactions promoted the spontaneous organization of the endothelial cells in open vessels with similar morphology and permeability of those observed in vivo [39, 41]. To the best of our knowledge, only three examples of spheroids on chip endowed with a vascular network made of ramified structures, bifurcations and narrow capillaries have been described. However, in these systems the spheroids are made only of cancer cells [42] or a co-culture of fibroblasts and endothelial cells [14, 43, 44].

In this context, we sought to exploit our recently developed hetero-type MCTS model of pancreatic tumor, based on a triple co-culture of cancer cells, fibroblasts and endothelial cells [9]. Combination of this model with a network of functional microvessels in a microfluidic device (i.e., the MCTS-on-a-chip), would allow to dispose of a more relevant system well suitable for the preclinical screening of penetration and efficacy of different anticancer agents.

To reach this goal we have planned to construct a chip bearing i) a central chamber containing gel-embedded fibroblasts, which will host the spheroid, ii) several flanking channels alternatively filled with fibroblasts and endothelial cells and iii) a system of medium channels and reservoirs to ensure both the nutrients/oxygen support and the perfusion of the tested compounds. The gradient of factors secreted by fibroblasts is expected to trigger and support the self-organization of the endothelial cells to form functional vessels into the hydrogel scaffold [39].

In this chapter, we report the first part of this work that relies with the set up and optimization of an easy photo-lithography protocol for the creation of molds with a highly defined geometry. Their use in soft-lithography allowed the rapid production of PDMS devices whose intact structure prevented the leakage of the filling hydrogels from the channels. The system in now ready to test the seeding of the different cell types and their spatially controlled organization to reproduce the different components of the pancreatic tumor and its microenvironment (e.g., compact tumor mass, fibrosis and tumor-associated vessels).

The perfusion of drugs and nanomedicines in the device will be achieved i) by a gravity flow generated and tuned through the differences of volume between the reservoirs or ii) by controlling the injection with a system of pumps. The tested compounds will move into the Chapter III 157 self-assembled vessels before reaching the hetero-type MCTS and thus, it will be possible to explore the impact of the fluid flow on their delivery, penetration and uptake into the tumor tissue. We expect this model would allow to achieve a more physiological understanding of the barriers to the therapeutic agents enabling a rapid identification of the strategies that could ensure the most efficient drug delivery and consequently the highest treatment efficacy.

Materials and methods

MCTS-on-a-chip fabrication

The entire process is schematised in figure 1. Briefly, molds with negative relief patterns of SU-8 photoresist on a silicon (Si) wafer were prepared by photo-lithography (see section 2.1.1) and then, they were used to fabricate final devices of PDMS by soft-lithography (replica molding) (see section 2.1.2). Fig. 1. Representation of the entire fabrication process for a microfluidic PDMS chip. Firstly, a mold with a relief pattern of the photoresist is created on silicon wafer by photo-lithography: a uniform film of photoresist is spin-coated on a silicon wafer, a mask with a microscale pattern is overlaid and this system is exposed to the ultraviolet (UV) light in order to transfer the pattern. The design is finally developed by dissolving the uncross-linked photoresist. Then, the mold is used to transfer a complementary pattern to the PDMS stamp with softlithography (replica molding). Adapted with permission from ref. [15]. Copyright 2014 Springer Nature.

SU-8 photo-lithography process

Two SU-8 compositions (Microchem Corporation, USA) in cyclopentanone solvent were chosen for these studies: SU-8 3050 and SU-8 2050. Their viscosity (12000 cSt and 12900 cSt, respectively) allows the deposition of thick films for the fabrication of high aspect/ratio structures. The photoresists were spin-coated on Si wafers of 4 inches diameter and 525 µm thickness (Neyco, France) by tuning opportunely the speed to obtain a layer of the desired thickness. Depending on this parameter, the prebaking times, the UV exposition, the postbaking times and the development period were further adapted by following manufacturer instructions. The UV exposition was performed by using the MJB4 Mask Aligner (SÜSS MicroTec SE, Germany). The printed patterns were developed through the dissolution of the unpolymerized SU-8 with the SU-8 developer solvent (Microchem Comporation, USA) Chapter III 159 followed by a final washing step in isopropanol. The thickness of the relief topography was measured with a Dektak Stylus Profiler (Bruker Corporation, USA). The quality of the developed patterns was assessed by optical microscopy using an inverted microscope (Zeiss, Germany) equipped with a Plan-Apochromat 4x dry objective lens and a halogen lamp.

Soft-lithography (replica molding) process

A 10:1 (w/w) mixture of RTV615 PDMS base and curing agent (Momentive Performance Materials Inc., USA) was poured on the molds and degassed under vacuum for 2 h.

After the PDMS polymerization in a dry oven (60 °C, at least 2 h), the resulting PDMS stamp was separated from the mold. Reservoirs, hydrogel injection ports and the tumor-stroma central chamber opening were punched out with biopsy punches of 5 mm, 1 mm and 3 mm in diameter, respectively. The PDMS was cleaned with nitrogen gas and then was covalently bonded to an acetone and isopropanol-washed glass coverslip (75 x 26 mm, 1 mm thickness) through an oxygen plasma treatment (20 s) (PDC-32G Plasma Cleaner, Harrick Plasma, USA) followed by heating in dry oven (60 °C , 1 h). Before the injection of the hydrogel, the hydrophobicity of the PDMS devices was restored by keeping them in dry oven at 80 °C for at least 24 h.

Injection of acellular hydrogel: feasibility study

The 2.5 mg.mL -1 bovine fibrinogen (Sigma Aldrich, France) solution was prepared in phosphate buffered saline (PBS, Sigma Aldrich, France) with aprotinin (0.15 U.mL -1 Sigma Aldrich, France) and type I collagen (0.2 mg.mL -1 , Gibco, France) [39]. After the addition of thrombin (Sigma, France) to a final concentration 0.5 U.ml -1 , 10 µL of the fibrinogen solution was immediately injected into a single channel. The filled device was placed at room temperature for 5 minutes to allow the fibrin polymerization.

Chapter III 160

Results and discussion

Design of the MCTS-on-a-chip

It has been previously reported [39] that a system of parallel channels, alternatively filled with hydrogel-embedded fibroblasts, medium and hydrogel-embedded endothelial cells, allowed the spontaneous organization of the last ones in functional microvessels. The presence of pillars between the channels ensured both the physical separation and the paracrine communication among the different cell types, while the intermediate medium channels generated the gradient of fibroblast-secreted factors, required to trigger the formation of the tubular lumenized structures [33, 39, 43]. Accordingly, we have designed a microfluidic chip in which a tumor-stroma chamber (TS) containing both a spheroid and fibroblasts embedded in a fibrin matrix was flanked by a system of vascular channels (V1 and V2). As previously reported, two different configurations of vascular channels could induce the microvessels formation according to the angiogenesis or the vasculogenesis process [23, 39]. The former represents the development in vivo of new vessels through the sprout of endothelial cells from the existing vasculature, while the latter refers to the formation of completely new vessels from the differentiation of progenitor cells [45]. Herein, in the angiogenesis configuration (pattern A, Fig. 2) the endothelial cells will be seeded as monolayer on the side wall of channels V1 and V2 (red arrows in Fig. 2). Their sprouting toward the central TS chamber will be triggered by the angiogenesis factors secreted by the fibroblasts and the hetero-type spheroid, here located. In the vasculogenesis configuration (pattern V, Fig. 3), instead, the endothelial cells will be embedded in a fibrin matrix and then seeded in channels V1 and V2 (red arrows in Fig. 3). Thanks to the factors arising from the central TS chamber as well as from the side stroma channels filled with fibrin-embedded fibroblasts (S1 and S2) the cells will spread from the centre to the walls of channel V1 and V2 delimited by pillars.

Fabrication of the microfluidic device

Although the process of SU-8 deposition is described in manufacturer datasheets, different facilities and experimental conditions lead often to dissimilar results [46, 47]. Therefore, an accurate optimization of the photo-lithography protocol was required for the reproducible creation of thick SU-8 films on silicon wafers used, then, as casting mold for the fabrication of microfluidic devices by soft-lithography. The SU-8 is an acid-catalyzed negative photoresist containing highly branched epoxy groups suitable for the fabrication of structures Chapter III 163 with the required high aspect/ratio [46]. As previously reported, indeed, channels with heights between 50 µm and 250 µm (high aspect/ratio structures) allow to reduce the internal pressure during the manual hydrogel injection [48] and facilitate the rapid formation of microvessels by ensuring the filling with a sufficient number of cells [33, 39, 40, 49].

Two different plastic contact-masks were designed and used to transfer on the photoresistcoated substrate the specific patterns corresponding to the angiogenesis (Fig. 4) and vasculogenesis (Fig. 5) configuration. In both configurations, the tumor-stroma chamber (TS) was 3 mm width and 6 mm long and trapezoidal pillars, spaced every 100 µm, divided the chamber from the adjacent channels. Vessels channels (V) had 0.7 and 1.2 mm width in the angiogenesis (Fig. 4) and vasculogenesis (Fig. 5) pattern respectively and they were delimited by hexagonal pillars placed at a 100 µm distance one from each other, as previously reported [39]. To be noted that the 100 µm gaps between the pillars prevented the leaking of the hydrogel from the filled channel thanks to the surface tension established with the PDMS hydrophobic surfaces [48].

In both configurations, medium reservoirs and hydrogel injection ports had a diameter of 5 mm and 1 mm, respectively. Chapter III 166 Therefore, to create a master with structures of 100 µm height, the negative photoresist SU-8 3050 was dispensed onto the Si substrate by using a spin-coater with a speed of 1000 rpm (acceleration of 500 rpm/s) for 30 s as suggested by the manufacturer. This single layer of photoresist was prebaked first at 65 °C for 10 min to evaporate the solvent and promote the adhesion and, then, at 95 °C for 50 min to densify the SU-8. The obtained sample was cooled and dried at room temperature for 1 h. After placing the mask in hard contact, the UV exposure at 365 nm for 60 s activated the photolytic reaction by producing the photoacid initiator able to protonate the SU-8 epoxides (Fig. 6). The application of heat in the postbaking step (3 min at 65 °C followed by 10 min at 95 °C) amplified the cross-linking process and completed the transfer of the mask pattern to the photoresist. The SU-8 microstructures were finally obtained with the development process during which the dissolution of the unpolymerized SU-8 (portions not exposed to UV light) converted the UV-printed latent design into a patterned surface that will serve as a mold during the softlithography. Note that the SU-8 mold will present the inverse pattern of the desired device structure. The overall height reached with this protocol #1 (Table 1) was ~ 130 µm, but unfortunately the optical imaging of the surface highlighted an incomplete pattern transfer after 6 min of development. Well defined profiles were obtained only for the largest details of the design, such as medium channels and reservoirs (Fig. 7a,b). A lower resolution was evident for the interspaces between adjacent channels (Fig. 7c) and only a partial development was observed for the trapezoidal and hexagonal pillars (narrow openings in the molds) (Fig. 7d).

To increase the possibilities to obtain a well-developed pattern a modification of the protocol, relying on the deposition of two thinner layers of SU-8 3050 on the silicon wafer followed by a single UV exposition, was investigated. With the aim to reach a final film of 100 µm height, the manufacturer instructions to construct 50 µm thick SU-8 layers were followed.

Accordingly, the spin-coating deposition was performed at 2000 rpm (acceleration of 500 rpm/s) for 30 s and the prebake carried out at 65 °C for 5 min and at 95 °C for 20 min. After leaving the 1 layer-coated wafer at room temperature for 1 h, the second SU-8 layer was deposed according to the same experimental conditions. This thick double film was exposed at 365 nm for 60 s in hard contact with the mask and, then, postbaked for 3 min at 65 °C and for 10 min at 95 °C. Nevertheless, also in this case, the development (10 min) led to an incomplete pattern with, in addition, very irregular heights (from 90 µm to 150 µm). These set-ups (protocol #1 and #2, Table 1) did not allow to obtain well defined molds, thus the fabrication method was further modified.

The single layer method was maintained but the deposition of SU-8 3050 was carried out in two subsequent cycles. Firstly, the photoresist was homogeneously distributed on the Si wafer at 1000 rpm for 30 s with an acceleration of 500 rpm/s and then, in the second cycle, the rotational speed was increased to 1500 rpm with an acceleration of 300 rpm/s and maintained for 30s in order to reach the desired film thickness (100 µm). According to manufacturer instructions, the coated wafer was prebaked at 95 °C for 40 min and then irradiated. The other modification was related to the UV light exposition which was carried out for 30 s without filter in order to use the maximum laser intensity and by placing the mask in strong vacuum contact to limit the light pass and improve the detail resolution. Indeed, the air gap between the mask and the photoresist is responsible for the light diffraction and, often, could cause variations in the pattern design [51]. Afterwards, the sample was postbaked (2 min at 65 °C and 5 min at 95 °C) and finally developed for 15 min. Compared to the first protocol (protocol #1, Table 1), this approach (protocol #3, Table 1) increased the pattern resolution of large (reservoirs and channels) and small (pillars) details, which were developed in both angiogenesis (pattern A, Fig. 8a) and vasculogenesis (pattern V, Fig. 8b) configurations. The obtained molds had regular heights of ~ 70 µm and, intending to obtain also a film with higher thickness, we tested again the two layers approach but we decided to irradiate subsequently each single layer (protocol #4, Table 1). Briefly, for the first layer, the SU-8 3050 was spin-coated onto the silicon wafer with an initial speed of 1000 rpm (acceleration of 500 rpm/s, for 30 s) ramped to a final speed of 3000 rpm (acceleration of 300 rpm/s, for 30 s) to reach the targeted thickness of 50 µm. Once prebaked (3 min at 65 °C and 12 min at 95 °C) and dried at room temperature (1 h), the coated-wafer was placed in vacuum contact with the mask and exposed to the UV light for 22 s in order to print the pattern. After the postbaking of the first SU-8 layer (3 min at 65 °C and 9 min at 95 °C), the second one was dispensed by following the same protocol. For the second UV exposure, the optical mask was aligned with the first printed pattern and the vacuum contact was established. The design transfer in the second SU-8 layer was performed with the above detailed protocol of UV exposition and postbaking of the first layer. After a development of 10 min, we obtained a pattern with good resolution. This result confirmed that the improvement in the feature resolution can be achieved by i) the reduction of the air gap between the photoresist and the mask during the UV exposition in vacuum contact and ii) the increase in the UV light intensity by the filter removal. However, also in this case, the double layer approach with the SU-8 3050 (protocol #4, Table 1) generated patterns characterized by irregular heights (~ 70 µm for some portions and ~ 90 µm for other ones). Although none of the tested condition was satisfying enough, we decided to attempt the fabrication of a PDMS device by soft-lithography (replica molding) in both the angiogenesis and vasculogenesis configurations using the molds created with the protocol #3 (Table 1). The injection of the hydrogel in these two devices revealed a leakage in almost all channels delimited by the trapezoidal and hexagonal pillars. It was evident that the gaps between pillars were larger than 100 µm and the 3D structures in PDMS were probably incomplete and not able to fully delimit the channels. To be noted that pillars and other small details of the PDMS stamp correspond to openings in the SU-8 molds. The incomplete pattern transfer with our experimental conditions was probably due to the excessively narrow size of these openings.

In the attempt to create devices with well delimited channels that can be easily produced and replicated, we simplified the mask design (Fig. 9 and 10) by avoiding too small details and creating channels with more regular and thicker boundaries (orange arrow in Fig. 9 and10).

Accordingly, the trapezoidal and hexagonal pillars of the original pattern were replaced with simpler ones having rectangular shape (width: 200 µm and height: 300 µm) (Fig. 9 and10).

These new pillars, placed at 100 µm distance one from each other, would i) prevent the leakage of the injected hydrogel, ii) act as physical supports for the formation of vessels and iii) permit the free diffusion of growth factors. We also increased the width of the central chamber TS and the vessel channels V (4 mm and 1.4 mm, respectively) to facilitate the hydrogel injection by reducing the friction forces with the PDMS walls. In addition, we abandoned the photoresist SU-8 3050 and moved to the SU-8 2050 that was reported to have a better coating quality and offered the possibility to produce high aspect/ratio structures. The first step consisted in the creation of a single layer of 50 µm height.

According to the manufacturer instructions (protocol #5, Table 1) we dispensed the photoresist onto the silicon wafer with two consecutives spin cycles: i) 500 rpm for 10 s, acceleration of 100 rpm/s ii) 1500 rpm for 30 s, acceleration of 300 rpm/s

The mask of the vasculogenesis configuration was chosen to perform these tests because it represented the most complex design with the largest number of channels and pillars. After the prebaking (3 min at 65 °C and 9 min at 95 °C) and the drying step (room temperature, 1 h), the new mask was placed in vacuum contact with the sample and the system exposed to UV for 15 s without filter (maximum light intensity). The postbaking (2 min at 65 °C and 7 min at 95 °C) and the 5 min development completed the protocol. Figure 11 shows the good resolution of the relief pattern. Through this process we obtained defined channels and reservoirs as well as rectangular openings with a very good level of precision. The measure of the film thickness confirmed the creation of regular heights of ~ 55 µm.

Taking advantage of these positive results with one SU-8 2050 layer, we tested also the deposition of two consecutive layers (protocol #6, Table 1) to fabricate a mold with higher structures. Briefly, each layer was spin-coated and prebaked according to the above described settings (protocol #5, Table 1). A single UV exposure of 40 s was performed when the second layer was dried (room temperature, 1 h) and, in order to complete the cross-linking, the thick double film was postbaked at 65 °C for 5 min and at 95 °C for 10 min. After a development of 10 min a clear pattern was obtained, as already seen with the single-layer approach (protocol #5, Table 1), and we fabricated features with homogeneous thickness of ~ 180 µm although a bit higher than expected according to the overlap of two layers. In our experimental settings, the SU-8 2050 showed better coating proprieties and was well suitable for the creation of uniform thick films with the double layer approach. The combination of the new mask designs with the optimized SU-8 photo-lithography protocol (e.g., photoresist SU8-2050, UV exposition in vacuum contact and without filter) allowed the construction of a mold with well-defined pattern.

A new device was finally fabricated through soft-lithography (replica molding) by using the one-layer mold (protocol #5, Table 1). Injection of the fibrin hydrogel revealed the integrity of the structures and no hydrogel leakage was observed. The gel remained confined in the channel of injection and the rectangular pillars perfectly hindered its diffusion into the flanking channels. These preliminary data suggest that the system could ensure good spatial control of the different cellular components that will compose the final MCTS-on-a-chip

General discussion

During this PhD work, the potentiality of the 3D cell culture to mimic in vitro the complexity of the real tumor tissues has been explored with the aim to recreate the biological barriers encountered in vivo by the drug delivery systems. In particular we focused on the development of a 3D model of pancreatic ductal adenocarcinoma. Hallmark of this disease is an extensive desmoplastic reaction, which acts as a tortuous barrier, sequestrating free drugs and nanomedicines, blocking their diffusion, dramatically hampering the drug bioavailability and thus limiting the effectiveness of the treatments [1, 2]. Accordingly, there is the need of relevant models which would enable i) to carry out fundamental studies to gain knowledge on the mechanisms of transport of nanomedicines into the tumor and ii) to identify the ones able to overcome the stromal barrier leading to a better exposure of tumors to the therapeutic agents and, finally, to a clinical benefit. In addition, they displayed similar capacity than immortalized pancreatic stellate cells to i) induce morphological changes in pancreatic cancer spheroids and ii) modulate the cancer cell expression of E-cadherin, beta catenin and Ezrin [4]. To be noted that the MRC-5 cell line has already been combined with pancreatic cancer cells in 2D and 3D co-cultures and these combinations have offered the possibility to study the tumor microenvironment and to successfully investigate the cross talk between cancer and stromal cells [5-8]. It is possible that these hetero-type MCTS would show higher biomimetic features if the MRC-5 will be replaced with cancer associated fibroblasts (CAFs), which represent the major component of the pancreatic tumor microenvironment and are involved in proliferation, invasion, metastasis, angiogenesis and chemoresistance of the cancer in vivo [9, 10]. However, although CAFs would be more representative of the tumor microenvironment, they are less accessible and more difficult to obtain than fibroblasts arising from healthy tissues. This justifies the larger employ of the latter for the construction of 3D models (e.g., pancreas and breast tumors) [11-13]. The endothelial component of the 3D triple co-culture consisted of Human Umbilical Vein Endothelial Cells (HUVEC), whose choice has been driven by the large accessibility and ease of handling. We are aware that recent studies have demonstrated the existence of some differences between the endothelial cells of the pancreatic tumor vessels and their healthy counterpart [14-16]. However, several obstacles hamper the wide application of tumorderived endothelial cells for routine in vitro studies. They include, for instance: i) the scarce availability of fresh tumor samples, ii) the difficulty to isolate tumor endothelial cells with high degree of purity, iii) the lack of well-defined culture conditions to maintain the cell phenotype and iv) the short life span of these primary cells and thus, the possibility to use them for only a limited number of passages [17]. Accordingly, no examples of 3D pancreatic tumor models made with these cells have been developed yet.

We applied the liquid overlay technique to construct the spheroids and an opportune optimization of the experimental conditions has been required to integrate the three cell types in a compact 3D model. Firstly, a layer-by-layer nanometric fibronectin-gelatin film was created on the fibroblast surface according to a previously validated technique, developed by colleagues of the University of Osaka (Japan) with whom our team collaborates since several years [18]. This film formed an adhesive surface that mimicked the natural extracellular matrix and supported the assembly of the spheroid by promoting the interaction between the three cell types. However, the cell integration was stable for no more than 48 h and, afterwards, the endothelial cells were rapidly lost as indicated by the negligible CD31

immunostaining. The medium supplementation with the vascular endothelial growth factor (VEGF), well-known for its positive effects on endothelial cell survival and angiogenesis [19, 20], promoted their residence and allowed to successfully obtain a triple 3D co-culture. At day 4 post seeding these spheroids were compact and the cancer cells surrounded a core rich in fibronectin in which the endothelial cells formed a ramified vessel-like network. This resembled the collapsed vessels observed in the pancreatic tumor in vivo.

The stability over time and the evolution of these hetero-type MCTS have been monitored in detail through histological analysis, microscopic imaging and viability assay. Results highlighted that this in vitro tumor model was not static but evolved. We observed, indeed, a progressive infiltration of cancer cells in the core of the spheroid initially filled by fibroblasts.

The best integration of cancer cells and the microenvironment components (e.g., fibroblasts, endothelial cells and extracellular matrix) was achieved at day 4 post seeding and this was then chosen as starting point for all the further studies. The knowledge of the exact cellular composition at this time point ensured the relevance of the results obtained using this model, which clearly arose from its complex composition.

The exposure of spheroids, made only of cancer cells or based on the triple co-culture, to two different cytotoxic drugs such as gemcitabine and doxorubicin has revealed the influence of the microenvironment. Its presence in the hetero-type MCTS led, indeed, to a lower sensitivity of cancer cells to the treatments mimicking the resistance often observed in vivo.

The relevance of the developed model was confirmed also by proving the angiogenesis capacity of the endothelial component. It has been observed that, in opportune conditions (i.e., embedding in a fibrin-collagen matrix), the endothelial cells were capable to organise in simple vessel-like structures through the process of sprouting. These results suggest the possibility to use these hetero-type MCTS also for the evaluation of the antiangiogenic effects of different therapeutic strategies.

In Chapter II we have demonstrated the advantages arising from the use of the Light Sheet Fluorescence Microscopy (LSFM) to investigate the penetration of free doxorubicin and doxo-loaded nanoparticles within the hetero-type pancreatic MCTS. The LSFM is an emerging microscopic technique that allows the 3D reconstruction of large biological samples having high scattering properties [21].

We have highlighted the capacity of doxorubicin to completely penetrate spheroids with a diameter of up to 1 mm bypassing the technical limitations encountered with the traditional Confocal Laser Scanning Microscopy (CLSM). Indeed, CLSM allowed to image only the first 100 µm of the sample and led to uncertain results as consequence of a rapid loss of the drug fluorescent signal due to the strong light scattering and absorption. On the contrary, the LSFM minimized these phenomena thanks to the creation of a light sheet with a thickness of few microns that scans the sample perpendicularly to the detection lens [22]. The image of the whole spheroid has been reconstructed in 3D with an accurate post-imaging fusion process of two independent z-stacks obtained from the multi-view scan of the sample. These highinformative results have confirmed the ability of doxorubicin to penetrate the whole spheroid in spite of their large size. Only in the inner part, the fluorescent signal was slightly shadowed.

At this sample depth, spheroids were highly compact and the phenomena of scattering and absorption were more intense and lowered the number of photons collected by the detection lens, thus causing a reduction of the image resolution [23, 24]. The optical tissue clearing has been proposed as a technique to overcome the limitation associated to the imaging of complex biological samples that include multiple structures with different refractive indices. Several protocols are available in literature and, basically, they are pre-imaging chemical treatments able to remove high refractive components from the sample (e.g., lipids) improving light penetration, imaging resolution and contrast [25]. Although extremely laborious, these methods could be eventually applied also to the spheroids, before the LSFM imaging, in order to improve the fluorescence detection and the resolution in the core.

Moving forward in the demonstration of the usefulness of this imaging method, we have provided a proof of concept that the combination of the LSFM and hetero-type MCTS can reveal differences in the penetration ability of doxorubicin and a nanoscale drug delivery system by using, as a model, the doxorubicin-loaded polyisoprene-based nanoparticles (NPs) developed in our team. Results have shown that these NPs had inferior penetration ability compared to the free drug in the same experimental conditions and have evidenced that the 3D complex structure reproduced by the hetero-type MCTS represented an obstacle to the diffusion of these NPs. It has to be noted that the internalization of similar polyisoprene-based NPs into single cells was easily observed by confocal microscopy when the uptake studies have been carried out on traditional 2D monolayer cultures [26]. Such discrepancy clearly highlights the need to dispose of pertinent models and tools to reliably assess the penetration of nanoparticles through the entire tumor mass and further studies would allow to identify the ones capable of the most efficient drug delivery.

according to literature data and were characterized by the presence of hexagonal and trapezoidal openings [27-29]. However, such structures were extremely complicated to reproduce and we failed in transferring a complete pattern on the SU-8 molds with our experimental protocol. Indeed, the final PDMS devices, created by soft-lithography using these first molds, have shown the hydrogel leakage after injection due to the not perfect finishing of the channels. Such evidences have pushed us to change and simplify the whole mask design by avoiding small features (e.g., trapezoidal and hexagonal openings) and creating channels with more regular boundaries. Rectangular openings characterized the motifs of the new masks and, finally, their simpler shape has been transferred on the SU-8 layer with a good resolution by using the previously optimized experimental conditions (e.g., photoresist SU8-2050, UV exposition without filter and in vacuum contact). Once replicated the motif in the PDMS stamp, we have observed that i) the rectangular pillars, placed at a 100 µm distance one from each other, prevented the leakage of the injected hydrogel and ii) the new channels with thick boundaries ensured the integrity of the whole device structure. At last, we succeeded in combining a standardized photo-lithography protocol and a simple chip design suitable for an easy and reproducible construction of the microfluidic PDMS device that will be used to create the complete MCTS-on-a-chip.

General conclusions and outlook

This Aiming to study the penetration of drugs and nanomedicines in a high informative manner, we have applied the Light Sheet Fluorescence Microscopy (LSFM) to the imaging of the hetero-type MCTS. The complex structure and the heterogeneous composition of our model made it a reliable example of biological barrier suitable for such penetration studies but, at the same time, it has represented a real challenge for the conventional Confocal Laser Scanning Microscopy (CLSM). We have demonstrated the advantages of using the LSFM in terms of deep laser penetration and high imaging resolution. The observed differences between the penetration ability of doxorubicin and doxorubicin-loaded nanoparticles emphasized the biological relevance of the hetero-type MCTS and should prompt the routine use of this microscopic approach in drug/nanomedicine preclinical screening.

In the attempt to move a step closer to the in vivo situation, we have also decided to work on the creation of a 3D model suitable for studing the penetration and efficacy of drugs and nanomedicines under dynamic conditions. The microfluidic techniques have offered the possibility to design a device potentially able to integrate the hetero-type MCTS and a network of self-assembled microvessels (i.e., MCTS-on-a-chip). After optimization of the photo-lithography protocol, we have succeeded in the creation of a poly(dimethylsiloxane) (PDMS) device characterized by intact microchannels and a well-defined pattern, which should ensure the cell seeding in a spatially controlled manner.

The assembly of this innovative device, which unfortunately has not been realised during this PhD for time reasons, will be the object of future studies. The formation of a functional microvessel network will be assessed as well as its functional integration with the hetero-type General conclusions and outlook 195 MCTS. Afterwards, the MCTS-on-a-chip would be used to investigate the capacity of nanomedicines to penetrate in the tumor mass under controlled in vitro flow conditions.

In conclusion, the proposed 3D in vitro models and related analytical/imaging techniques would represent very useful tools to assess the ability of nanomedicines to move across multiple biological barriers and efficiently deliver their cargo. Different experiments could now be set up to evaluate the impact of size, surface properties and shape of nanocarriers on tumor accumulation under static and dynamic conditions.

We expect these systems would enable a rapid identification of the nanomedicine features that allow to achieve the most efficient drug delivery and, thus, the highest treatment efficacy.

Then, only the nanomedicines displaying the best performance would be evaluated in vivo, leading to a reduction of the need for animals during preclinical studies and enabling to abide by the 3Rs guiding principles for the use of animals in research.

Introduction

The efficacy of conventional therapies is often limited by a non-specific cell/tissue distribution of drugs, and their rapid metabolization and/or excretion from the body. In this context, nanoscale systems for drug delivery (i.e., nanomedicines) have (2010-2015). Patrick Couvreur's contributions in the field of nanomedicine are highly recognized and respected around the world (H-index 84 and over 28 000 citations). His research is interdisciplinary, being at the interface between physico-chemistry of colloids, polymer chemistry, materials science and pharmacology with applications in oncology and neurosciences. He is a member of several Academies both in France and abroad. received in the last few decades tremendous attention because they hold the potential to overcome these limits, providing a solution to medical challenges that urgently require novel therapeutic strategies. [1][2][3] Nanomedicines can indeed improve the therapeutic index of the transported drug by (i) offering protection from degradation, (ii) enabling its controlled release and distribution and (iii) increasing its bioavailability. In particular, with cancer being one of the leading causes of death worldwide, much work has been done in this field with the aim of proposing more efficient treatments.

Gianpiero Lazzari

Accordingly, a plethora of well-engineered nanomedicines surface-modified with cell targeting ligands 4 or endowed with stimuli-responsiveness 5 have been designed. However, despite the encouraging results observed in preclinical experimental models as well as in clinical trials, 6,7 the introduction of nanoscale drug delivery systems in clinical practice is not straightforward, 8,9 and only a limited number of nanomedicines have reached the marketplace. These include, for instance, lipid and natural protein-based nanomedicines such as Doxorubicin (Doxo)-loaded liposomes (i.e., Doxil®, Myocet®) and paclitaxel albumin-bound nanoparticles (NPs) (i.e., Abraxane®). It is noteworthy that so far no nanomedicine based on synthetic polymers has been introduced in the market despite the high versatility offered by the macromolecular synthesis and the possibility of opportunely tuning the polymers' properties (e.g., composition, structure, functionalization, degradability etc.). [10][11][12][13] It has to be noted that reaching the biological targets and ensuring sufficient drug delivery and accumulation is extremely challenging as a consequence of the multiple biological barriers that characterize tumors and their microenvironment, which are crossed by nanomedicines either at low efficiency or not at all. 14,15 However, such biological transport barriers are generally not taken into account during the in vitro preclinical evaluation of nanomedicines, the majority of which is still routinely carried out in two dimensional (2D) monocultures of isolated cancer cells. Despite their relative ease of handling, these cultures do not show any structural architecture and lack the complex physiology and the microenvironment of real tumor tissues, which consist of different cell types (e.g., fibroblasts, macrophages, endothelial cells, immune cells) embedded in an extracellular matrix (ECM) mainly composed of fibrous proteins and proteoglycans. [16][17][18] Cells cultured in 2D monolayers display altered gene expression and activation of signaling pathways when compared to cells grown in the native tumor tissue. 19 Moreover, growing in a single layer, they do not replicate either (i) the cell-to-cell and cell-to-ECM interactions [20][21][22] or (ii) the oxygen, nutrients and pH gradients, 23 which play a crucial role in tumor progression, chemoresistance and metastatic spread. 24 Accordingly, there is an urgent need for more relevant models capable of closely mimicking the heterogeneity and the microenvironment of the in vivo conditions, thus allowing a more predictive in vitro evaluation of nanomedicines. In this context, three dimensional (3D) culture models such as multicellular spheroids (MCTS), polymer scaffolds and microfluidic systems have been proposed as an alternative approach to overcome the aforementioned limitations. [25][26][27][28][29][30][31][32] 3D tumor models are indeed capable of recapitulating some key features of real tumors, thus representing a valuable tool for (i) a more accurate preclinical screening of nanomedicines and (ii) the identification of candidates with the highest chances of success, which would be further evaluated in vivo. According to their predictive capacity, 3D models would enable to limit the number of animals required in preclinical studies allowing to abide by the 3Rs guidelines. 33,34 Although the application of 3D models for a systematic assessment of the therapeutic efficacy of nanomedicines is still at the beginning, their superiority to 2D models is nowadays clearly acknowledged. This review will provide the reader with an overview of the application of multicellular tumor spheroids, the widest employed 3D tumor model so far, for the evaluation of nanomedicines, highlighting their usefulness as a discriminating tool. In agreement with the scope of the journal, attention has been focused on drug delivery systems either made of polymers or in which polymers have been used for surface modifications. ate complexity between 2D in vitro cell cultures (i.e., cell monolayers) and in vivo solid tumors with which they share important similarities. Since their first introduction in the early 1970s by Sutherland and coworkers, 35,36 the application of multicellular tumor spheroids in drug discovery has grown exponentially, offering the possibility of screening a large variety of different molecules, [37][38][39] and they are currently considered a suitable 3D model for drug evaluation in the oncology field. 37,40 The physiological communication and the signaling established between cells growing in close contact make possible to reproduce in spheroids key aspects of the tumor and its microenvironment such as: (i) different cell proliferative rates, (ii) specific gene expression, (iii) deposition of ECM components, (iv) cell-to-cell and cell-to-microenvironment interactions and (v) drug resistance. [40][41][42] Still, one limitation of MCTS is the fact that they mimic only the avascular region of in vivo tumor tissues, thus leaving out relevant aspects of real tumorigenesis such as the surrounding vasculature, the immune system components and the fluid dynamics. 38,41 Large spheroids (∼400-500 μm diameter) display an internal layered cell distribution analogous to that observed in solid tumors. This is the result of mass transport limitations, which interfere with the diffusion of oxygen, nutrients and metabolic wastes through the spheroid creating specific gradients. 43,44 Accordingly, thanks to the easier access to oxygen and nutrients, highly proliferating cells are located in the external layer of spheroids and correspond to tumor cells close to capillaries in vivo. Conversely, quiescent cells characterize the middle layer, because cell metabolism decreases progressively with the increasing distance from the spheroid periphery. Finally, a critical situation characterizes the spheroid core in which the oxygen depletion (i.e., hypoxia), the nutrient shortage and the metabolic waste accumulation result in cell necrosis (Fig. 1). 40,[43][44][45] The organization in cell layers and the presence of diffusive gradients, acting as microenvironmental stresses, force the inner cells to a specific metabolic adaptation, 43 responsible for the observed impaired therapeutic efficacy of various anticancer drugs or drug-loaded nano-carriers. 40,46 For instance, cells in the hypoxic region are resistant to drugs which promote cellular death through reactive oxygen species, while the existence of necrotic and quiescent cells reduces the therapeutic efficacy of drugs active against the proliferating ones. 40 Another important feature of MCTS is the presence of a network of structural (i.e., collagen and elastin) and adhesive (i.e., fibronectin and laminin) ECM proteins embedded in a gel of glycosaminoglycans and proteoglycans. 40,42 This microenvironment acts as a regulating factor influencing cell proliferation, differentiation and tumor growth. 41,43 In addition, it closely mimics the physical barriers found in real solid tumors, which obstruct the free penetration of drugs through the whole mass. 40 Undoubtedly, the presence of this tumor microenvironment represents an advantage compared to the conventional monolayer cultures although it has to be noted that ECM components found in MCTS present a different cellular origin compared to those present in the in vivo tumor tissues. In the former, they are secreted by the same cancer cells forming the spheroids while in the latter they are secreted by tumor-associated fibroblasts (TAF). 42 To face this issue, spheroids composed of cancer cells and components of the supportive stroma have been recently proposed. 47,48 These hetero-type multicellular spheroids better reproduce the cellular heterogeneity of the tumor tissues and are expected to allow a more reliable evaluation of cancer cell-microenvironment interactions and their impact on the therapeutic outcomes. 43,[47][48][49]

3D multicellular tumor spheroids

3D MCTS culture methods

Different techniques for spheroid construction are currently available and many of them have been optimized for largescale production under highly reproducible conditions. 40 Principally, they involve the use of cell attachment-resistant surfaces or physical forces to promote cell-to-cell interactions and support the 3D spheroid formation (Fig. 2). These techniques will be briefly discussed in the following paragraphs while for a more detailed and extensive description the interested reader could refer to recently published reviews. 38,41,45,50,51 2.1.1. Liquid overlay methods. This technique mainly relies on the use of non-adherent 96-well plates, in which a coating with poly-2-hydroxyethyl methacrylate ( poly-HEMA) [52][53][54][55][56][57][58] or agarose prevents cell attachment. This method allows the preparation of both mono and hetero-type spheroids, whose size and morphology can be finely tuned by varying the number of cells seeded in each well. 48,50,51,86 The large number of produced spheroids and the ease of handling make this approach applicable to different kinds of highthroughput investigations. 51,86 For instance, cells could be seeded using a bulk liquid dispenser or a liquid handling robot, which also offers the possibility of scaling-up the spheroid fabrication. 37 Compared to the agitation-based approaches, the liquid overlay methods require a lower volume of media and testing materials (e.g., drugs). 50,87 However, a major drawback is the time-consuming plate-coating step. Although pre-coated low cell adhesion plates are commercially available (e.g., Ultra-low Attachment Surface from Corning®; PrimeSurface low adhesion culture plate from Sumitomo Bakelite Co.; Lipidure®-coated plates developed by NOF Corporation), their use increases the overall costs. 38 2.1.2. Hanging drop methods. This technique exploits the surface tension to hang small cell suspension droplets (20-50 µL) on the underside of a tissue culture dish lid. 44,51 Then, gravity drives cell accumulation at the tip of the drop (liquid-air interface) and induces their rapid aggregation into a single spheroid. 44,50 By changing the cell density, control of spheroid size has been achieved with a high reproducibility for numerous cell lines 38 both in mono and co-cultures. 47 However, the volume of the seeding suspension is generally limited and does not provide enough nutrients for a long term culture. 44,50 Therefore, once formed, spheroids need to be transferred into 96-well plates or embedded in polymer matrices for further studies. [88][89][90][91][92][93][94][95] Not only such transfer can affect the integrity of spheroids but it clearly makes this technique extremely labor intensive and time-consuming, thus preventing large scale application. 44,50,51 Commercially available platforms (InSphero GravityPlus™ technology and PERFECTA3D™ hanging drop plates of 3D Biomatrix) can reduce the workload and enable experiments to be carried out in a high-throughput manner, although with a significant increase of the expenses.

2.1.3. Agitation-based approaches. Spinner flasks and rotational culture systems (i.e., NASA Rotary Cell Culture System) are bioreactors in which cell aggregation and spheroid formation are mediated by a continuous spinning that keeps in motion a cell suspension supporting cell-to-cell interactions and avoiding their attachment to the container wall. 38 The movement of the medium provides a controlled environment and maintains the transport of nutrients and waste allowing (i) long-term culture and (ii) large-scale production. However, although by tuning the cell-seeding density, spinning rate and culture time it is possible to influence the average spheroid diameter, 44 the overall control of the number of cells per spheroid and their size is extremely poor. 38,50 Therefore, manual selection of similarly sized spheroids and their transfer into different supports are mandatory before any further assay (e.g., cytotoxicity, penetration, etc.). [96][97][98] It has to be noted that only cell lines that can withstand high shear stress can be cultured in spinner flasks. 38,50 For less resistant cells types, the NASA Rotary Cell Culture System characterized by lower shear forces, thanks to the simultaneous rotation of the culture chamber together with its content, should be instead preferred. 38,44 However, both approaches require specialized equipment and a large amount of culture media, which therefore limits their wide application. 38 2.1.4. Patterned surfaces and microfluidic devices. Patterned surfaces in combination or not with microfluidics devices have been recently developed and offer a new range of advanced strategies for spheroid construction. 37,38,[99][100][101][102] For instance, spheroids with a defined size and composition have been generated using arrays of microwells fabricated with the micromolding or the photolithography technique. 37,44,45,103,104 Low attachment surfaces have been obtained using non-adherent materials such as the poly(diméthylsiloxane) (PDMS) 46,105,106 or applying a coating of agarose [107][108][109][110] or Pluronic® F-127. 48 Compatible with traditional well plate formats and microscopes, such microwell plates are suitable for high-throughput screening. 111 Similarly, the formation of cellular aggregates in a controlled environment and with high simplification of the liquid handling procedures has been obtained with microfluidic systems containing various micro-sized chambers and channels. 45,50,102 Requiring only limited amounts of cells, media and reagents they are extremely attractive and convenient for drug screening applications; however, the impossibility to retrieve and extensively characterize the formed spheroids is a general drawback of these approaches. 38,111 A further level of complexity is achieved with microfluidic devices displaying (i) distinct compartments loaded with different cell types (e.g., epithelial cells, endothelial cells and fibroblast), (ii) collagen gel inserts and (iii) variously shaped channels, which ensure the cell-to-cell chemical communication, thus mimicking the complex in vivo-like organizations. [111][112][113] Nevertheless, to date, the complexity and the costs of the equipment required for their use have hindered their wide application in the preclinical investigation of nanomedicines.

End-point assessment in 3D MCTS

Thanks to the capacity to recreate key features of real tumors, 3D MCTS have been largely used for the assessment of the efficacy of various therapeutic strategies. In this context, assays and detection methods specific for 3D cultures are highly required to carry out an accurate and predictive evaluation (Table 1). Their detailed description being outside the scope of this review, the most common techniques are only summarized in the next paragraphs, while a comprehensive presentation can be found in recently published articles. 37,[39][40][41]45 Optical microscopy is the simplest and most widely used technique for the visual assessment of morphological changes in spheroids. 52,[55][56][57][58][60][61][62][63][64][65]70,73,74,77,84,[89][90][91][92][93]95,107,110,[114][115][116][117][118][119][120][121][122][123][124][125][126] Following exposure to various treatments, the measurement of the variation of morphometric parameters (e.g., mean diameter, minimum diameter, maximum diameter, volume, area and circularity) of spheroids from bright field images is used for a quantitative analysis of the extent of growth inhibition. 37,87 After interruption of the treatment, the spheroid growth delay might be calculated in comparison with untreated samples as the time lag required to reach the quintuple spheroid volume of day 4 (5 × V d4 ; assuming V d4 as the starting spheroid volume at the onset of treatment). 87 The assays currently used in 2D monolayer cultures as extensively validated methods to assess the drug cytotoxicity mainly rely on the measurement of (i) cell membrane integrity (i.e., LDH assay) 127 or (ii) intracellular metabolic activity (e.g., MTT, WTS-1, AlamarBlue® assays). 128 Some of them have been adapted to spheroids and are currently commercially available. Among them, the quantifications of the acid phosphatase (APH) activity and the intracellular adenosine triphosphate (ATP) level are well-suited assays that do not require preliminary dissociation of spheroids into single-cell suspensions. 37,45 The APH assay is a simple and inexpensive technique based on the quantification of cytosolic acid phosphatase activity in viable cells through the measurement of the absorption of the p-nitrophenol, obtained by APH hydrolyzation of p-nitrophenyl phosphate, at 405 nm using any standard plate detection reader. 37,129 The APH assay has been validated to measure spheroid viability and cytotoxic effects of both free drugs 87,129 and drug-loaded nanocarriers. 82,95,115,124 The ATP assay exploits the oxidation of the luciferin, the substrate of the firefly luciferase enzymes, in the presence of O 2 , Mg 2+ and ATP. This reaction brings the luciferin to an electronically excited state, which then decays with the emission of a photon of light (i.e., bioluminescence). Thus, when substrate and enzyme are added to cell cultures the light output, which is directly proportional to the intracellular ATP content (marker of metabolically active cells), allows to quantify the number of viable cells. Ready-to-use assay kits, which exploit the high sensitivity and the low background of the bioluminescence signal 45 (e.g., CellTiter-Glo® 3D Cell Viability Assay (Promega); Molecular Probes® ATP Determination Kit (Invitrogen)), are currently available. The CellTiter-Glo® 3D Cell Viability Assay represents a time-effective and well standardized assay since the addition of a single reagent directly to the 3D cell culture results in cell lysis and simultaneous generation of the bioluminescence. 130 This assay is especially formulated with a robust lytic capacity to overcome 3D MCTS obstacles such as tight cell-cell junctions and the presence of the extracellular matrix. 130 The relatively simple workflow and data analysis make this ATP assay scalable to high-throughput screenings of drug-loaded nanocarrier efficacy. 125 Qualitative assessment of proliferating or dead/necrotic cells can be performed by Confocal Laser Scanning Microscopy (CLSM) imaging following spheroid incubation with various live/dead reagents. 46,118,131 Preliminary spheroid dissociation into single cells is instead required for a quantitative measurement by Fluorescence-Activated Cell Sorting (FACS). 70 Staining cell dispersions of dissociated spheroids with fluorescently labeled Annexin V (AnnexinV-FITC) and propidium iodide (PI) has also been largely used to detect early apoptotic and dead/ necrotic cells, respectively. 96,[132][133][134] Apoptosis can be detected also on spheroid cross-sections by using the Terminal deoxynucleotidyl Transferase (TdT) dUTP Nick-End Labeling (TUNEL) method. The TdT recognition of the blunt ends of double stranded DNA breaks, which characterizes the late stages of apoptosis, 135 catalyzes the addition of biotinylated dUTPs that are then visualized using streptavidin-conjugated detection agents (i.e., peroxidase or fluorescent markers). 56,136 Monitoring of fluorescent drugs and dye-labeled nanocarriers by CLSM has been largely applied for evaluating their uptake and penetration in individual spheroids. 63,66,69,76,95,105,133 Unfortunately, compared to conventional 2D cultures, the 3D structure represents a technical challenge for conventional instrumentation. Indeed, the increases of spheroid size and thickness result in a loss of image quality due to the scattering, reflection and absorbance of light. 37,41 The layered structure of spheroids limits the inner scanning depth and, as a consequence, the penetration ability of different systems can be compared only in a portion of the spheroid generally corresponding to a depth of 80-100 µm from the spheroid surface. 63,76,95,131,137 Although more time consuming, better information on the real depth of penetration can be provided by the acquisition of fluorescent images from serial sections obtained from fixed spheroids. 46,71,88,[96][97][98]138,139 Compared to traditional CLSM, significant improvements in the evaluation of the penetration into living tumor spheroids can be obtained with Two-Photon (TPM) or Multi-Photon microscopy (MPM) in which the energies of two (or more) photons are combined to promote the transition of a fluorescent marker to an excited state. 140 The use of an excitation wavelength in the NIR region (700-1000 nm) offers the possibility of increasing the laser penetration into 3D MCTS (up to 500-800 μm according to the instrumentation) with low phototoxic effects. 50,140 These techniques have already been applied to visualize with high resolution the diffusion through spheroids of various polymer-based nanocarriers. 70,81,85,120,137,141 Imaging of the cellular processes and drug delivery in spheroids with an even higher 3D isotropic resolution and with limited photodamage is the promise of Light-Sheet Fluorescent Microscopy (LSFM) methods such as the Selective Plane Illumination Microscopy (SPIM) 47,142,143 method already applied for the imaging of large biological samples. 144,145 However, so far, LSFM methods have not found any application in the preclinical investigation of polymer nanomedicines.

Use of 3D MCTS to screen polymer nanocarriers with different physico-chemical properties

The distinguishing characteristics of polymer nanocarriers, such as chemical composition, size, shape and surface properties, might strongly affect their capacity to diffuse into tumors and therefore have a profound impact on their anticancer efficacy. 158 Accordingly, these parameters must be taken into account during the design and evaluation of any novel nanomedicine. In this context the 3D MCTS, thanks to their similarity in morphology and biological microenvironment to solid tumors, have already been used as a robust tool for easy polymer nanoparticle screening, 70,97 and to accurately predict the in vivo behavior of the nanocarriers as a function of their specific physico-chemical properties. 139 The most relevant results will be discussed in the following sections. 2.3.1. The size effect. The influence of size on the penetration profile of nanoscale systems has been clearly highlighted and, according to the general trend observed in vitro with 3D MCTS and further confirmed in vivo, it is evident that the diffusion capacity is inversely correlated to the particle size. 56,72,97,98,139,148 For instance, nanogels obtained by chelating ligand-modified hyaluronic acid were capable of deeper penetration into MKN74 gastric cancer spheroids compared to the polymer in the linear form and their small size (21 nm) ensured a uniform distribution over the entire 3D mass. 106 Similarly, following release from poly(ethylene glycol) (PEG)poly(D,L-lactide) (PLA) nanoparticles, into which they have been loaded, small polyamidoamine (PAMAM) dendrimers (G2, 2 nm) penetrated efficiently into the core of KB papilloma cell spheroids (diameter ∼50 µm) as compared to their larger counterparts (G4 (5 nm) and G7 (7 nm)) whose florescent signal was detected only in the peripheral rim. 72 It is noteworthy that the size plays a major role in the capacity of tumor penetration even in the case of surface functionalization with specific ligands. By using a murine breast cancer MCTS model (4T1 cells) it was observed that RGD functionalization endowed Pluronic F127-coated paclitaxel (Ptx) nanocrystals with a better penetration ability compared to the free drug (Taxol) and to the non-functionalized carriers. However, for nanocrystals displaying similar functionalization, a smaller size (10 nm vs. 70 nm) conferred a greater advantage in terms of depth of penetration (Fig. 3a). 56 It has to be noted that no difference was observed when the particles were evaluated on a 2D culture model, thus highlighting the usefulness of the 3D MCTS to discern the crucial role of the size. TUNEL staining of the spheroid's cross-sections revealed that deeper penetration was associated with a more extensive cell apoptosis, which caused inhibition of spheroid growth and their further collapse following prolonged drug exposure (Fig. 3b andc). 56 Despite these results, the identification in vitro of the optimal size of a nanocarrier for achieving the highest drug delivery in vivo is extremely complex because although MCTS closely mimic the tumor tissues, they lack predictive value in terms of pharmacokinetics and biodistribution. Accordingly, the evaluation of nanomedicines in animal tumor models still remains a mandatory step to assess these parameters. It has indeed been shown that the requirements for tumor penetration and tumor retention are often in contradiction with each other. Thus, despite being unfavorable for pene- tration in the tumor mass, due to their considerable diffusional hindrance, 159 nanocarriers with a size of around 100 nm are the most advantageous in improving pharmacokinetics and extravasation. 160 On the other hand, smaller nanocarriers show much better penetration in the tumor interstitial space. 139,148 However, for extremely small particles (≤5.5 nm), rapid clearance from tumors and short half-life have been observed. 161 To face this issue, the ideal drug delivery system should be able to shrink and adapt its size in response to the encountered microenvironment. Interestingly, size-switchable stimuli-responsive nanoparticles able to overcome multiple tumor barriers have been developed by various groups. 58,76,119,133,134,138,141 For instance, pH sensitive nanoparticles were formulated by the molecular assembly of platinum (PtIV)-prodrug conjugated polyamidoamine (PAMAM) dendrimers with two amphiphilic polymers containing either ionizable pH-responsive amide bonds (i.e., polycaprolactone-2propionic-3-methylmaleic anhydride (PCL-CDM), PCL-CDM-PAMAM/Pt nanoparticles) 134 or tertiary amine groups (i.e., poly(ethylene glycol)-b-poly(2-azepane ethyl methacrylate) (PEG-b-PAEMA) PEG-b-PAEMA-PAMAM/Pt) (Fig. 4a). 133 Size variation as a function of pH has been evaluated on pancreatic cancer multicellular spheroids (BxPC3 cells). At physiological pH the clustered nanoparticles (i.e., pHsensitive cluster nanobombs (SCNs/Pt)) displayed a size of around 100 nm, while the pH drop (from 7.4 to 6.5-7) in the tumor extracellular space triggered an instantaneous disassembly of these pH sensitive nanoparticles in small Pt-PAMAM prodrugs (≈5 nm) able to penetrate deeply and uniformly into the spheroid mass (Fig. 4b). Then, once internalized, the intracellular redox environment led to the release of the active molecule (i.e., Pt(II) species) resulting in significant cell apoptosis. As expected, a higher cell viability was observed when spheroids were incubated with pH-insensitive nanoparticles (i.e., ICNs), which demonstrated a limited capacity of penetration and drug delivery as a consequence of their stable size (Fig. 4b). Whether such advantage was maintained in vivo was then assessed after an intravenous injection of both nanoparticles in an experimental model of pancreatic cancer (BxPC3 cancer cells) characterized by an important desmoplastic reaction and a limited permeability. 162 Tumor accumulation studies confirmed a higher capacity of penetration of pH-sensitive nanoparticles, which diffused in the tumor inter- stitium, after vessel extravasation, for several hundreds of nanometers. In contrast, the pH-insensitive NPs accumulated in the tumor vessels with little penetration in the tumor mass (Fig. 4c) leading to an inefficient drug delivery. In agreement with the in vitro results, an extensive apoptosis was detected in tumor sections of mice treated with the pH-sensitive nanoparticles, thus confirming the predictive potential of the 3D multicellular spheroids.

2.3.2. The shape effect. 3D tumor spheroids have found application also in the early screening of nanocarriers with different shapes (i.e., spherical vs. elongated such as filaments, rod-like or worm-like vectors). However, whether this parameter may positively or negatively affect the nanocarrier penetration, the cellular uptake and the therapeutic efficacy still remains a matter of debate. 85,120,147 For instance, when wormlike poly(ethylene oxide-b-ε-caprolactone) (PEO-PCL) photosensitizer-loaded micelles were compared to spherical ones in 3D models of colon (HCT-116 cells) and head and neck cancer (FaDu cells), surprisingly no advantage in terms of photodynamic therapy efficiency was observed, in contradiction with the previously reported superior uptake of so-called "filomicelles". 163 Nevertheless, the different size of the studied systems, as well as other parameters such as the nanocarrier length, rigidity and surface properties, might explain such differences and would have to be further explored. 120 The influence of length was studied by Stenzel and coworkers, who investigated rod-shaped poly(1-O-methacryloylβ-D-fructopyranose)-b-poly-(methyl methacrylate) ( poly(1-O-MAFru)-b-PMMA)-based micelles, clearly showing that, among the various fructose-coated rod-like micelles, the shortest ones displayed the highest capacity of penetration in a 3D MCTS model of breast cancer cells (MCF-7). 147 Similarly, in order to better highlight the role of these parameters, poly (ethylene glycol) diacrylate (PEGDA)-based anionic nanohydrogels were synthesized by jet and flash imprint lithography in the form of disc-shaped nanocylinders and cuboidal nanorods of two different sizes (low and high aspect ratio; aspect ratio (H/D) = height/diameter). 85 The resulting nanohydrogels displayed negative charge of ∼-55 mV, which should limit the interactions with cell membranes and serum proteins and promote their penetration into HEK293 spheroids (human embryonic kidney cells). Fluorescence intensity analysis of two-photon microscopy images revealed two fold higher accumulation near the spheroid outer half of the disc-shaped nanocylinders with the lowest aspect ratio (H/D ∼0.3, 325 nm diameter and 100 nm height) (Fig. 5f ) compared to both nano- cylinders with higher aspect ratio (H/D ∼0.45) (Fig. 5e) and nanorods (Fig. 5g andh). Such a preferential penetration might be attributed to their larger surface contact area, which promoted the interaction with the cells and the diffusion (either passive or active) across the 3D tumor mass.

2.3.3. The charge effect. As observed for shape and size, the surface charge of the nanocarriers might also affect tissue penetration and efficient drug delivery. 72,82,164,165 For instance, the penetrating capacities of PAMAM dendrimers were discriminated according to their size (see section 2.3.1), but also a charge effect was clearly observed showing that only the amino-modified ( positively-charged) ones were capable of significant accumulation in the outer layers of breast cancer (MCF7 cells) tumor spheroids while negatively and neutral ones were completely excluded (Fig. 6a). 72 It is noteworthy that small G2-NH2 dendrimers penetrated deeper than the larger G7-NH2 ones (214 ± 36 μm vs. 81 ± 8.31 μm), but surface accumulation was directly proportional to the dendrimer generation (G7 > G2) and therefore to the surface charge density (i.e., number of NH2 groups) (Fig. 6b). This behavior was correlated to the different strength of dendrimer-cell interactions: while it was negligible for the G2 dendrimers leading to low accumulation and fast penetration by paracellular diffusion, the firmer interaction of G7 ones led to a strong uptake, which however translated to a low diffusion via a transcellular mechanism.

The key role of a positive surface charge in the accumulation into multicellular spheroids has been shown also by comparing gold nanorods (AuNRs) (55 nm length × 14 nm diameter) coated with either (i) cetyltrimethylammonium bromide (CTAB) and poly(diallyldimethylammonium chloride) (PDDAC), or (ii) polystyrene sulfonate (PSS) displaying a surface charge of +40-50 mV or -25 mV, respectively. 82 When incubated with MCF-7 breast cancer MCTS, the coating with a cationic polymer ensured the highest gold accumulation, which was expected to induce strong photothermal cytotoxicity after NIR irradiation as was observed in 2D monolayer cultures. However, despite their tendency to be largely retained, irradiation of these positively charged AuNRs led to a 40% lower hyperthermia efficacy compared to the negatively charged PSS-coated AuNRs. Such unexpected behavior was related to the penetration capacities of the different AuNRs. Indeed, cationic polymer-coated AuNRs highly accumulated in the spheroids but only in the outer region, and simple surface adsorption was also observed. Moreover, the interaction with negatively-charged serum proteins led to an increase of their size, preventing diffusion. In contrast, the negative charge of PSS-coated AuNRs allowed a more homogeneous distribution in the spheroid core, which resulted in higher viability loss and destruction of the inner compact spheroid structure.

2.3.4. The role of the chemical composition. 3D multicellular spheroids have been used to discern whether the chemical composition of the polymer building blocks could affect the drug delivery efficacy of the resulting nanoparticles. 60,91,96,152,153,155 Thus, curcumin-loaded biodegradable and non-biodegradable nanoparticles prepared by selfassembly of bovine serum albumin (BSA) conjugated with poly-(ε-caprolactone) (PCL) or poly(methyl methacrylate) (PMMA) were investigated in LNCaP prostate cancer MCTS in comparison with conventional 2D cultures. 91 The latter did not reveal appreciable differences between the two types of nanoparticles, which were both rapidly taken up by cells and induced similar cytotoxicity. In contrast, the MTCS allowed the detection of the faster and deeper accumulation of BSA-PMMA NPs, which occurred via a rapid sequence of endo and exocytosis events. However, such a high penetration rate hindered sufficient intracellular drug release, thus leading to a lower cytotoxicity compared to the BSA-PCL NP, whose biodegradability ensured, in contrast, high intracellular drug concentrations (Fig. 7a). Non-biodegradable BSA-PMMA NPs induced a growth inhibition comparable to the biodegradable NPs, only when tested at 5 times higher drug concentration (Fig. 7b).

Other examples of anticancer drug-loaded biodegradable nanocarriers evaluated on 3D spheroids included milk protein nanoparticles 153 and micelles composed of either (i) pseudo block copolymers formed by the assembly of β-cyclodextrin terminated multiarmed poly(N-vinylpyrrolidone) (PVP) and adamantane functionalized polycaprolactone (PCL) 155 or (ii) poly (ethylene oxide)-poly [(R)-3-hydroxybutyrate]-poly (ethylene oxide) triblock copolymers. 96 The capacity of penetration of these nanosystems in 3D models was consistent with further in vivo experiments, thus supporting the predictive capacity of the tumor spheroids. 96,153,155 2.3.5. The role of crosslinking. The capacity of amphiphilic block copolymers to self-assemble in water as micelles has been largely applied in efficient drug delivery. 90,120,167,124,166 Micelles display a typical core-shell structure and a variety of crosslinking strategies have been proposed to reach a higher stability. For instance, the irreversible crosslinking of poly (ethylene glycol methyl ether acrylate)-b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA) micelles with 1,8-diaminooctane 90,124 protected against disassembly, leading to better cellular uptake when compared to uncrosslinked counterparts. However, such a crosslink induced a reduction of the drug release, leading to micelles with lower cytotoxicity compared to the free drugs when evaluated on 2D monolayer cell cultures of human prostate carcinoma (LNCaP cells). 124 Analogous results have been obtained when glycol dimethacrylate (EGDMA) crosslinked poly(ethyleneoxide-b-3-caprolactone) (PEO-PCL) micelles have been investigated on HCT-116 (human colorectal carcinoma) and FaDu cells (human squamous cell carcinoma). 120 In contrast, the aforementioned micelles were characterized by superior antitumor activities to the free drug in different MCTS made with HCT-116, FaDu or LNCaP cells, 120,124 clearly demonstrating the limited predictive potential of 2D cultures in the assessment of the real value of nanocarriers as drug delivery systems.

Whether the crosslink could affect the mechanism and depth of penetration as well as the cytotoxicity of drug-loaded micelles has been recently investigated by the group of Stenzel using pancreatic (AsPc-1 cells) multicellular tumor spheroids. 90 Hence, 1,8-diaminooctane-crosslinked poly(N-(2-hydroxypropyl) methacrylamide-co-methacrylic acid)-block-poly (methyl methacrylate) (P(HPMA-co-MAA)-b-PMMA) micelles (CKM) were compared to their uncrosslinked version (UCM) (Fig. 8a). Results revealed that CKM were capable of moving through the cell layers via a transcellular process and delivered higher doxorubicin amounts to the spheroid core which resulted in greater cytotoxicity compared to the UCM (Fig. 8bd). The latter quickly disassembled after penetration into the cells of the outer layers, releasing the loaded drug and causing cell death. As a result, no further micelle transcytosis could occur. The lower efficacy of the free drug (evaluated in terms of DNA content and inhibition of spheroid growth) correlated with its limited diffusion. 90 Nevertheless, crosslinking is not always the best strategy to improve drug cytotoxicity. Thus, the evaluation of the effect of the reversible disulfide core-crosslinker cystamine in micelles formed by the poly(ethylene glycol methyl ether acrylate)-bpoly(carboxyethyl acrylate) (POEGMEA-b-PCEA) block copolymer 95 led to results that were in clear contrast to those previously published by the same group of Stenzel. 90,124 Indeed, micelles with the highest level of crosslinking were capable of the deepest penetration in LNCaP prostate multicellular tumor spheroids, but displayed the lowest cytotoxicity. This behavior could be explained by their too compact structure that hindered intracellular reductive agents (e.g., glutathione) from diffusing in the core of the micelles, thus slowing down cross-linked micelle disassembly and release of the loaded drug. 95 Compared to the 2D monolayer cultures, the 3D spheroid model would provide a reliable correlation between micelle penetration and cytotoxicity, thus allowing figuring out the real contribution of the crosslinking in terms of drug delivery efficiency.

2.3.6. The role of targeting ligands. Functionalization of the polymer nanocarrier surface with various ligands has been largely exploited as a strategy for selective cell targeting. 4 As discussed in the following sections, MCTS are currently widely used in order to assess the effectiveness of such modifications and evaluate the tumoral behavior of targeted nanoparticles.

2.3.6.1. Transferrin-targeted nanocarriers. Overexpression of transferrin receptor (TfR) has been detected in different types of rapidly proliferating tumors. 168 Accordingly, opportune surface functionalization with transferrin (Tf ) has been applied to efficiently deliver drugs to cancer cells. Such modification clearly enhanced the cellular uptake and penetration depth of polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) micelles 67,68 and poly(amidoamine) (PAMAM) dendrimers 61 in MCTS models of ovarian carcinoma 67,68 and glioma 61 compared to the non-functionalized counterparts. More efficient delivery of the loaded drugs ( paclitaxel (Ptx) 67,68 or doxorubicin (Doxo)) 61 resulted in a significant inhibition of cell proliferation confirmed by reduction of spheroid volume and metabolic activity (ATP content). A 6-fold reduction of the IC 50 value (8.92 µM vs. 1.35 µM) was measured on the 3D spheroids incubated with Tf-functionalized Ptx-loaded PEG-PE micelles (as compared to non-targeted ones) but these values were higher than those observed in 2D monolayers. These results reflect a reduction of efficacy due to the existence in the MCTS of physical barriers to diffusion and a different cell sensitivity due to the 3D spatial organization. 67 Reflectance confocal microscopy and synchrotron X-ray fluorescence microscopy (XFM) have been used to monitor, in a quantitative and qualitative manner, the capacity of penetration into 3D breast cancer spheroids (MCF-7 cells) of transferrin-decorated polymer-modified gold nanoparticles (100 nm). 105 Images revealed that although functionalization with human Tf increased the amount of internalized NPs compared to controls (i.e., bovine transferrin functionalized particles and naked ones), after 48 h the penetration of functionalized NPs was limited to a depth of 50 µm, thus representing a real issue for further therapeutic applications due to their incapacity to diffuse in the tumor core. 105 2.3.6.2. Folic acid-targeted nanocarriers. As previously observed for the transferrin receptor, also the folate receptor is largely expressed in cancer cells, thus making the functionalization with folic acid (FA) a widely applied approach to enhance the ligand-mediated uptake by cancer cells. 55,57,94,107 3D tumor spheroids have been employed to assess the influence of the folic acid density at the NP surface on the cell targeting capacity, internalization and tumor penetration of polymer/DNA complexes ( polyplexes) made by the assembly of DNA with poly(amidoamine)-poly(ethylenimine) (PME) copolymers conjugated to FA functionalized PEG (PME-(PEG-FA)). FA functionalization should enable overcoming the reduction of cellular uptake caused by the PEG chains (the so-called PEGdilemma) 169 and make these systems valuable tools for efficient gene delivery. Divalent modification (PME-(PEG 3.4k -FA 2 ) 1.72 ) resulted in a higher receptor mediated uptake and a better penetration in HEK293 T human embryonic kidney spheroids compared to mono-functionalized (PME-(PEG 3.4k -FA 1 ) 1.66 ) and non-functionalized nanocarriers (PME-(PEG 3.5k ) 1.69 ) (Fig. 9a-c). 94 Indeed, although all polyplexes were detected up to 380 µm depth, the divalent FA modification allowed achieving a better cell internalization (Fig. 9d).

The in vitro-in vivo predictive capacity of the 3D models in the evaluation of the FA functionalized nanocarriers was recently reported. Indeed, the higher capacity of penetration in vitro into 3D neuroblastoma spheroids (SH-SY5Y cells) of FA-decorated Doxo-loaded soy protein NPs (SP-NPs) 55 or carboxymethyl chitosan-N-3-acrylamidophenylboronic acid (CMCS-PAPBA) 57 NPs resulted in the highest inhibition of tumor growth in H22 tumor-bearing mice.

2.3.6.3. Carbohydrate-targeted nanocarriers. The natural affinity of dextran for highly glycosylated surfaces has been exploited for achieving targeted delivery of ald-dex-Doxo nanoparticles (i.e., aldehyde-dextran polymer conjugated to doxorubicin via a pH sensitive bond) to SK-N-BE(2) cells by the interaction with the glycocalyx at their surface. 126 Investigation of these NPs in 3D neuroblastoma spheroids (∼400-500 μm) enabled to discern their potential as drug delivery systems, while it was not evident under 2D conditions where the free drug displayed superior cytotoxicity. Such a difference disappeared in the spheroid model in which a drastic reduction of the free Doxo efficacy was observed while that of the alddex-Doxo nanoparticles remained unvaried and resulted in a more efficient reduction of tumor outgrowth. Such a difference was related to the different capacity to overcome the encountered biological barriers: after 24 h the free Doxo penetrated up to 50 µm in the spheroids while nanoparticles were detected at that depth after 1 h only and diffused in the whole mass at 4 h. Interestingly, such a capacity was exclusive of alddex-Doxo nanoparticles and was not observed when cells were incubated with dextran based NPs in which the drug has been only physically loaded and not covalently linked to the polymer.

Chitosan has been instead used in the formulation of nanoparticles for specific targeting of the CD44 receptor in cancer stem-like cells (CSLCs), 53,60,77,89,106,122 a rare tumor cell population whose resistance to therapeutic agents is a major cause of anticancer treatment failure. 170 While non-stem cancer cell resistance in 2D cultures is mainly the result of the overexpression of transmembrane P-glycoprotein transporters, 171 in CSLCs a key role is played also by the tumor microenvironment. 172 Accordingly, relevant preclinical investigations of such functionalized nanocarriers required a model capable of mimicking the complex relationship between cancer cells and the surrounding environment. Thus, 3D mammary tumor spheroids (i.e., mammospheres) enriched with CSLCs have been successfully created by culturing MCF-7 cells with different growth factors and chemicals in order to promote stemness (i.e., self-renewal) and epithelial-to-mesenchymal transition (EMT) as demonstrated by the downregulation of the estrogen receptors (ER) expression whose role appears to be pivotal in maintaining the epithelial differentiation. 122,173 These CD44-expressing mammospheres, which are currently one of the most advanced examples of the 3D systems used for polymer nanocarrier evaluation, allowed assessing the capacity of chitosan-decorated Pluronic127 nanoparticles to efficiently deliver the loaded doxorubicin and to bypass the CSLC drug resistance in vitro. Chitosan functionalization resulted in nanoparticles able to selectively target CD44-overexpressing cells in mammospheres (Fig. 10a) while only a minimal targeting was observed in 3D spheroids made of normal human adipose-derived stem cells. 122 The better in vitro penetration of such functionalized nanocarriers was also confirmed in vivo revealing their highest capacity of inhibition of tumor growth (Fig. 10b andc).

CD44 receptors have been targeted also by chondroitin sulfate A-deoxycholic acid-(3-aminomethylphenyl) boronic acid (CSA-DOCA-AMPB) NPs, which in addition exploited the interaction between boronic acid and sialic acid for efficient delivery of Doxo to human lung adenocarcinoma A549 tumors spheroids. 60 Penetration observed in vitro by Confocal Laser Scanning Microscopy (CLSM) was confirmed in vivo in A549tumor bearing mice by three-dimensional near-infrared fluorescence (NIRF) imaging and resulted in significant suppression of tumor development.

2.3.6.4. Monoclonal antibody-targeted nanocarriers. Among the plethora of possible strategies for active targeting, the 2C5 monoclonal antibody (mAb 2C5) has been employed for its capacity to target several types of tumor cells thanks to the interaction with the nucleosomes originating from neighboring apoptotically died tumor cells. 174 Nucleosomes are specifically bound to the surface of tumor cells and are always present in the spent media of growing tumor-cell lines as well as in the extracellular fluid of cancer patients. 175 Thus, mAb2C5-functionalized Doxo-loaded polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) micelles have been formulated and evaluated in vitro on ovarian cancer MCTS constructed by using NCI-ADR-RES cells. 136 The key role played by this Doxo-resistant MCTS model relied on the Bcl-2 gene overexpression associated with the three dimensional organization of the tumor cells, which closely mimicked the real situation found in patients. Consistent with the beneficial effect of mAb2C5 monoclonal antibody conjugation, uniform Doxo distribution throughout the spheroids was achieved only with the targeted micelles. These same PEG-PE micelles have been also functionalized with the single chain fragment variable (scFv) of the monoclonal antibody against glucose transporter-1 (GLUT-1) whose overexpression in cancer cells relies on their continuous requirement for glucose supply. Accordingly, selective targeting of this transmembrane protein might promote the ligand mediated delivery of anticancer drugs. 66,73 The in vitro evaluation on a 2D monolayer of U87MG glioblastoma cells revealed that Doxo and curcumin co-encapsulation in GLUT-1-targeted micelles resulted in a significant enhancement of caspase 3 and 7 activity as compared to un-targeted micelles (mono-drug-loaded and two-drug-loaded). 66 Moreover, GLUT-1 targeting improved the penetration of PEG-PE micelles into 3D glioblastoma spheroids of U87MG cells in which the Doxo and curcumin synergistic effect was confirmed by the highest cytotoxicity (approx. 70% cell death) after 5 days of treatment. 66 An estrogen receptor alpha (ER-α) mAb has been instead used to functionalize the surface of polyacrylic acid (PAA)coated ion doped NaYF4:Yb,Er upconversion nanoparticles (UCNPs), which have been investigated as a potential earlystage cancer detecting agent. 83 Such functionalization enabled targeting of MCF7 breast cancer cell spheroids (∼500 μm) transplanted in a chick embryo chorioallantoic membrane (CAM) aiming at modelling an early stage (i.e., diameter smaller than 2 mm) breast cancer. This model not only mimicked the cell-to-cell and cell-to-microenvironment interactions but also displayed a novel vascularization around the transplanted spheroid. Being more convenient and easy to handle compared to in vivo animal models, this simplified system allowed a direct microscopy study of the UCNPs-mAb's ability to target cancer cells and thus detect tumors in vivo at an early-stage. Indeed, following a systemic administration of UCNPs-mAb via venule injection under a stereomicroscope, a strong upconversion luminescence was observed in the spheroid mass. It should be noted that only cancer cells were targeted and that no accumulation in the other surrounding tissues of the embryo was observed, thus demonstrating the optimal selective capacity of such modified nanoparticles.

2.3.6.5. Aptamer-targeted nanocarriers. Nucleic acid aptamers (DNA and RNA) capable of recognizing with high specificity the epithelial cell adhesion molecule (EpCAM), a type I membrane protein expressed on the surface of a variety of cancer cells, 176 have been used for surface functionalization of poly(lactide-co-glycolide) (PLGA) 75 and alginate-coated chitosan nanoparticles (CHNPs) 74 whose efficiency has been evaluated in vitro on 3D MCTS models of breast 75 and colon cancers. 74 Decoration with locked nucleic acid (LNA) aptamers allowed CHNPs to reach colon cancer EpCAM-expressing cells deeply in the core of both 3D MCTS and tumor experimental models in mice. 74 Proof of their being potential efficient drug carriers was provided using CHNPs loaded with the apoptotic agent SR9, a survivin antagonist. Compared to non-functionalized nanoparticles, their efficient targeting and penetration capacity resulted in a 5-fold reduction of spheroid volume in vitro (after 72 h exposure) and up to 4 times lower tumor volume in colon cancer xenografts in mice (at d = 70 post tumor induction). 74 2.3.6.6. Peptide-targeted nanocarriers. Specific recognition of α ν β 3 integrins has been demonstrated to endow RDG-functionalized nanocarriers with a targeting capacity toward cancer cells facilitating their internalization. 177 Accordingly, the linear or cyclic version of the RGD has been covalently linked to:

(i) micelles made by the assembly of the enzyme-sensitive peptide-linked poly(ethylene glycol) and partially hydrolyzed poly(β-benzyl L-aspartate) (PEG-GPLGVRGDG-P(BLA-co-Asp)) co-polymer; 138 (ii) mesoporous silica nanoparticles (MSNs) coated with poly(ethylene glycol) (PEG), polyethyleneimine (PEI) or chitosan; 84 (iii) PEG-poly(trimethylene carbonate) (PEG-PTMC) nanoparticles; 63,64 (iv) poly(amidoamine) (PAMAM) dendrimers. 146,149,150 The influence of the extent of RGD functionalization was also investigated. For instance, the transfection efficiency of PAMAM-RGD dendrimers displaying various levels of RDG ligands was evaluated in a 3D spheroid model of glioma in comparison with the conventional 2D culture of U87MG cells. 149,150 In 2D cultures no advantage was observed compared to the naked PAMAM, probably as a consequence of the predominant non-specific interaction mediated by the positively charged dendrimers with the cell membrane. However, the evaluation in 3D spheroids highlighted the capacity of PAMAM-RGD to strongly interfere with the α ν β 3 integrinmediated interaction of cells with the ECM, which was directly correlated to the number of ligands conjugated to PAMAM. As a consequence of the reduced adhesion, RGD functionalization facilitated the penetration and the uptake of PAMAM dendrimers into the spheroid model although it did not result in a significant gene silencing. 149 In addition to the capacity of interaction with integrins, conferred by the RGD sequence, the so-called tumor penetrating peptides (TPP) 177,178 display a C-terminal sequence R/ KXXR/K known as the C-end rule (CendR). 62,63,80 Together, these two sequences mediate an active transport through tumor vessels and within the extravascular tumor tissue by interaction first with the α v β 3 -integrin and then with the neuropilin-1 receptor (NRP). 59,80 Clearly such modification represents a valuable approach for increasing the penetration of nanocarriers in the tumor mass. 56,62,117 Among the TPP, the iRGD has been physically adsorbed onto boronic acid-rich chitosan-poly(N-3-acrylamidophenyl boronic acid) (CS-PAPBA) nanoparticles, 117 or covalently linked to Pluronic F127 coated paclitaxel nanocrystals. 56 CLSM images of spheroids exposed to fluorescently TPP-functionalized nanocarriers revealed an intense signal, which spread from the periphery toward the center of the spheroids, demonstrating their superior capacity of penetration and accumulation leading to an efficient drug delivery confirmed in vitro by the reduction of spheroid volume and in vivo by the inhibition of tumor progression (Fig. 11). 56,62,89,117 The use of the simple CendR motif with the RGERPPR sequence has also been proposed to increase the penetrating capacity of poly(ethylene glycol)-polyethylenimine/plasmid DNA complexes resulting in a higher accumulation into a glioma spheroid model (U87MG cells) compared to the nonfunctionalized counterpart. Nevertheless whether or not the peptide could also be efficient at improving the transfection efficacy in this 3D model still needs to be verified. 80 Cell penetrating properties are also displayed by the interleukin-13 peptide (IL-13p) capable of specific recognition of the IL13Rα2, a tumor-restricted receptor overexpressed in gliomas. Functionalization of poly(ethylene glycol)-poly (ε-caprolactone) (PEG-PCL) nanoparticles with this peptide (ILNPs) resulted in an enhanced cell-uptake and penetration in U87MG spheroids. 78 A 3-fold higher tumor accumulation was obtained in vivo compared to un-functionalized nanoparticles, which resulted in a better docetaxel delivery and a significant reduction of tumor weight compared to the other treatments (saline, free drug and drug-loaded naked NPs). 78 Endowing these NPs with dual targeting properties by functionalization with both IL-13p and RGD peptides further improved their penetration ability in C6 glioma spheroids as well as in vivo in orthotopic glioma-bearing mice. 79

Use of 3D MCTS to evaluate macromolecules-loaded nanomedicines

Polymer-based nanomedicines have been investigated for the delivery of proteins or DNA/RNA molecules, whose application in clinical settings is strongly limited by (i) inappropriate size and surface charge; (ii) low stability against enzymatic degradation and (iii) low cell membrane permeability. 179,180 A variety of polymer/siRNA and pDNA polyplexes have been constructed and evaluated in 3D models demonstrating their efficiency as drug carriers. Complexation has been realized for instance with (i) poly(amidoamine) (PAMAM) dendrimers; 92,149 (ii) folate-functionalized-poly(ethylene glycol)-polyamidoamine-polyethylenimine (PME-(PEG-FA)) copolymers 94 (see also section 2.3.6.2); (iii) poly(L-lysine); 118 (iv) PEG-b-poly(Nsubstituted asparagine) copolymers(PEG-b-P[Asp(DET)); 131 (v) triblock poly(2-ethyl-2-oxazoline)-poly(L-lactide)-g-poly(ethylenimine) (PEOz-PLA-g-PEI) polymers; 108,109 (vi) mPEG-PEI (CendRpenetrating-peptide-modified methoxy poly(ethylene glycol)polyethylenimine) copolymers 80 (see also section 2.3.6.6); (vii) folate-poly(ethylene glycol) (PEG)-Amino Acid Modified Chitosan (CM-PFA) 107 or (viii) poly[(N,N-dimethylamino) ethyl methacrylate] (PDMAEMA)-derivatized albumin. 93,116 The pioneering studies of Kataoka and coworkers have highlighted the utility of human hepatocarcinoma multicellular spheroids (HuH-7 cells) for the long-term evaluation of the transfection efficacy achieved using polyplex micelles as gene delivery systems. 118,131 In particular, core-shell type micelles assembled through electrostatic interactions between poly (L-lysine) and lactosylated poly(ethylene glycol)-siRNA conjugate (lac-PEGylated polyplexes) 118 or pDNA and PEG-block-poly (N-asparagine) copolymers (PEG-b-P[Asp(DET)]) 131 have been investigated. Only the 3D MCTS spheroids, which can be maintained in culture for several weeks, allowed carrying out an extended analysis of gene expression/suppression under conditions close to those observed in vivo in solid tumors. 118 In Fig. 11 contrast, such time dependent studies could not be performed on 2D monolayer cultures as a consequence of cell-cell contact-induced arrest and viability decrease that prevented the monitoring of a prolonged gene expression. 131 Thus, it was possible to observe that the delivery of the RecQL1 siRNA using the lac-PEGylated polyplexes allowed an efficient suppression of gene expression, which resulted in the inhibition of spheroid growth for up to 21 days. 118 Using the same 3D model it was possible to demonstrate that pDNA-loaded PEGylated polyplexes penetrated in the spheroids and stably induced the expression of the encoded yellow fluorescent protein Venus for more than 10 days. 131 A successful prediction of in vivo transfection efficiency was obtained following an evaluation in MDA-MB-231 breast cancer spheroids of heptafluorobutyric acid modified generation 4 (G4) poly(amidoamine) (PAMAM) dendrimers (G4-F735) loaded with a plasmid encoding for the TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) gene. 92 Improved gene delivery and better performance, compared to both naked pTRAIL and conventional transfection reagent poly (ethylene imine) (PEI), were confirmed in vitro by the complete degradation of the MDA-MB-231 spheroids after 7 days of treatment (Fig. 12a) and in vivo by suppression of tumor growth in a subcutaneous model of luciferase expressing MDA-MB-231 cells (Fig. 12b). 92 The usefulness of stimuli-responsive approaches was assessed by Gaspar and coworkers. 108,109 Using minicircle DNA (mcDNA), micelleplexes have been constructed by selfassembly of poly(2-ethyl-2-oxazoline)-poly(L-lactide)-g-poly(ethylenimine) (PEOz-PLA-g-PEI) triblock co-polymer or its bioreducible analogue (PEOz-PLA-g-PEI-SS), which allowed the formulation of stimuli-responsive micelles thanks to the introduction of redox sensitive bonds. 108 Both systems displayed a good penetration ability and negligible cytotoxicity in 3D MCTS models of melanoma (B16F12 cells), cervix carcinoma (HeLa cells) 108 and breast cancer (MCF-7 cells). 109 Nevertheless, confocal images of 3D HeLa and B16F10 spheroid sections revealed that bioreducible micelleplexes enhanced GFP gene expression thanks to a higher mcDNA release following rapid intracellular reduction of the disulfide linkages. 108 2.5. Miscellaneous nanocarriers evaluated on 3D MCTS Surface modification of inorganic nanoparticles with various polymers might make possible their use as theranostic systems for efficient delivery of therapeutic molecules and precise monitoring of the response. 81 In this view, coating of iron oxide nanoparticles (IONPs, MRI contrast agent) with poly(4-Oacryloyl benzaldehyde)-poly(oligoethylene glycol acrylate) P(HBA)-b-P(OEGA) block copolymers allowed their stabilization as well as the covalent conjugation of doxorubicin via a pHsensitive bond, which assured drug release in an acidic environment ( pH 5.5). 81 3D optical sectioning by multiphoton microscopy of multicellular spheroids made of lung (H129) or breast (MCF7) cancer cells clearly showed the fluorescence of Doxo-loaded IONP@P(HBA)-b-P(OEGA) nanoparticles uniformly spread across the spheroid tissue highlighting a complete penetration after 17 h of incubation; in contrast, free Doxo accumulated in the periphery reaching a maximal depth of 40 μm only (Fig. 13). 81 Real time monitoring of tumor response to treatment is the goal pursued by Oishi and coworkers with the formulation of a PEGylated nanogel containing gold nanoparticles in the crosslinked poly[2-(N,N-diethylamino)ethyl methacrylate] (PEAMA) core in which fluorescein isothiocyanate (FITC) was linked to the PEG chains using the Asp-Glu-Val-Asp (DEVD) peptide sequence as a caspase-3-cleavable linker. 156 Based on the Fluorescence Resonance Energy Transfer (FRET) between gold nanoparticles (i.e., fluorescent quencher) and FITC, such a system behaved as a caspase-3-responsive apoptosis sensor for precise in vitro monitoring of the activity of apoptosis-inducing agents. The proof of concept has been provided using human hepatocyte MCTS (HuH-7 cells), which have been incubated with nanogels for 24 h prior to exposure to the apoptotic drug staurosporine for 4 h. Intracellular caspase activation triggered FITC release from the nanogels and the dequenching of the fluorescent signal, which allowed assessing the early stage activation of the induced apoptotic pathway.

The application of nanodiamonds (ND) as drug delivery systems is currently strongly limited by their tendency to agglomerate and precipitate in solution. Thus whether surface modification with polymers might offer a beneficial effect has been explored by the grafting of poly(1-O-methacryloyl-2,3:4,5di-O-isopropylidene-β-D-fructopyranose) ( poly-(1-O-MAipFru) 62 ) onto the surface of amine-functionalized ND. Such an approach should improve their stability and allow a successful loading of doxorubicin. 157 When evaluated in 2D monolayer cultures of MCF-7 and MDA-MB-231 breast cancer cells, a clear superiority of the free drug over the NDs was observed. However, in 3D breast cancer MCTS (MCF-7 cells) long term exposure (8 days) to the Doxo-loaded poly(1-O-MAFru) 62 -ND resulted in higher cytotoxicity compared to the free drug, which can be justified by the deeper penetration of NDs in spheroids and the sustained release of the drug. In contrast, the free Doxo was rapidly internalized by the proliferating cells of the outer layers of the spheroids and the consequent cell death hindered further penetration. 157

Combining 3D MCTS and polymer scaffolds

3D tumor models made of multicellular spheroids surrounded by a polymer scaffold have also been proposed as an alternative to simple spheroids suspended in cell culture medium. In these systems the scaffold building materials create a matrix around the spheroid capable of mimicking in vitro the microenvironment surrounding the tumors in vivo. Accordingly, this additional barrier may offer the possibility of discerning better the capacity of various nanocarriers to diffuse and to reach the cancer cells.

One of the simplest strategies consisted in embedding preformed spheroids into a collagen gel. Such a system enabled for instance highlighting the better efficacy of paclitaxel loaded into pH sensitive NPs (i.e., espansile NPs) 181 compared to the free drug, while this difference did not appear in 2D cultures. Indeed, the latter did not display any difference in the cell response independently of the drug administration method (free drug solution vs. drug-loaded nanocarrier). 65 In contrast, in 3D cultures, drug-loaded nanoparticles induced a more important slowing of the spheroid growth, which mirrored the inhibition of tumor progression obtained in vivo in tumor-bearing mice. 181 In another 3D spheroid design a single cell suspension has been mixed with collagen before gelification and cells aggregated over time inside the matrix in the form of spheres. Again, while no differences were observed in 2D between the free drug (5-fluorouracil) and the drug-loaded micelles, in contrast, thanks to the presence of the collagen matrix which mimicked the tumor ECM, the 3D model enabled revealing the limited diffusive capacity of micelles (152 nm) compared to the small molecules that led to a lower cytotoxicity. 182 The same strategy has been applied by mixing prostate cancer cells with hyaluronic acid, 183 one of the components of the stroma associated with this tumor in vivo, which not only provided structural support but also strongly influenced tumor cell morphology, gene expression and tumorigenic potential. 184 For instance, cells cultured in this 3D scaffold displayed higher expression of multidrug resistance proteins, probably as a result of the limited availability of oxygen and nutrients. Accordingly, while in the 2D cultures the free drug (doxorubicin) easily reached the nucleus and exerted its therapeutic activity, in 3D the sensitivity to the free doxorubicin was reduced (5 µM vs. 15 µM, respectively). Contrariwise, independently of the culture conditions the response to Doxo-loaded PEG-PCL NPs was not modified and analogous IC 50 values were measured (11 µM vs. 12.3 µM, respectively) probably thanks to the capacity of these NPs (54 nm diameter) to overcome the MDR while the free drug undergoes a rapid efflux, which reduced its efficacy. 183

Combining 3D MCTS and microfluidic devices

Undoubtedly, compared to simplistic 2D cultures the abovedescribed 3D systems enabled a more predictive in vitro screening of nanoscale systems for drug delivery. Nevertheless, due to the lack of fluid dynamics these setups mimic only a static condition. To face this issue, microfluidic devices, which combine 3D culture and controlled flow conditions, have been recently developed with the aim: (i) to assess how physicochemical parameters influence the transport through tumor biological barriers under dynamic conditions and (ii) to provide information on the optimal design required to achieve a successful tumor accumulation. 185,186 For instance, a tumor-on-a-chip device allowed the passage from a static spheroid culture to a dynamic situation by placing a spheroid in the channel of a two layer poly(diméthylsiloxane) (PDMS) chip (Fig. 14a). 185 By tuning the flow rate in the device, fluid velocities and shear stresses similar to the blood flow in capillary vessels (75-675 µm s -1 ) or the interstitial flow inside a tumor (0.1-3 µm s -1 ) could be reproduced in a controllable manner. Exposure of spheroids to PEGylated nanoparticles of different sizes under stationary flow revealed interstitial accumulation only of the smaller NPs (40 nm) while the larger ones (110 nm), bigger than the ECM pores, were excluded (Fig. 14b andc). However, accumulation was only transient and nanoparticles flowed out after flushing, confirming that surface modification with PEG chains hindered the establishment of specific interactions with cells and ECM components. In contrast, no efflux was observed following NP functionalization with transferrin and 40 nm nanoparticles showed up to 15-fold increase of tumor accumulation compared to non-functionalized NPs. It was also observed that an increase of the flow rate resulted in accumulation in the external spheroid layer, forming a tissue-fluid interface reservoir but that did not affect the depth of NP penetration. When the NPs were then tested in vivo in a tumor bearing mice, the same size discriminating effect was recorded, with a better accumulation of small NPs (50 nm) compared to the larger ones (160 nm). However, functionalization did not lead to any significant advantage and both targeted and non-targeted 50 nm NPs displayed similar tumor accumulation levels (Fig. 14d ande). Such contradiction with the results obtained in vitro clearly highlighted a limit of the 3D models, which cannot fully reproduce the complexity of living organisms and the behavior of NPs after intravenous administration. Thus, although it is evident that 3D models would allow a more relevant preclinical screening of nanomedicines compared to 2D cultures, at present they cannot completely replace the in vivo experimentation.

Another proposed approach consisted in the loading of Matrigel-embedded preformed spheroids in the central channel of a microfluidic device, while continuous medium addition in the lateral channels recreated some blood flow conditions. This allowed to assess under dynamic conditions treatment responses to doxorubicin in free form and loaded into micelles (Fig. 15). 187

Conclusion and perspectives

Through many literature examples this review has unambiguously highlighted that simple 2D monolayers cultures do not allow a complete understanding of the therapeutic potential of polymer nanomedicines while the application of 3D tumor models in preclinical evaluation would provide more accurate results, predictive of the in vivo pharmacological efficacy. The key features of the MCTS (e.g., presence of ECM, diffusive gradients, complex cell signaling, drug resistance and metabolic adaptation) undoubtedly enable evaluating the nanomedicines under conditions closer to the clinical reality. But it cannot be ignored that the majority of tumor spheroids used in the literature are made of cancer cells alone and therefore they just represent a rather simplified model of real tumorigenesis. Due to the complexity of the tumor tissues and the cross-talk between cancer cells and their microenvironment, advanced models including several cell types (e.g., endothelial cells, immune cells, fibroblasts etc.) and components of the ECM are urgently required. Thus, 3D co-cultures 47,48,188 and microfluidic devices [111][112][113]185,186,189 have been developed but further improvements are still needed in order to allow wider application in preclinical investigation. In this context, the accurate characterization (e.g., cell number, long term viability of each cell component, type of ECM protein, etc.) of any developed system is mandatory for allowing a reliable interpretation of the obtained results. It is indeed well acknowledged that the spheroid size 43,46 as well as the presence of stroma components 47,48,188 strongly affect the response to treatment according to the possible development of penetration barriers, [46][47][48]188 chemical gradients and/or necrosis. 43 The availability of microscopy techniques suitable for high resolution imaging of 3D cell cultures is another challenge. Indeed, although widely used, CLSM does not allow the in toto study of large 3D samples and the obtained results refer only to penetration into the spheroids at the depth of a maximum of 100-150 µm. A significant improvement in 3D imaging should result from Two-Photon (TPM) and Multi-Photon Microscopy (MPM) studies but the low spatial resolution along the optical axis and the incompatibility of certain fluorophores with the multi-photon excitation still restrict the applicability of these methods. 140,190 In this challenging panorama, LSFM approaches (e.g., Selective Plane Illumination Microscopy (SPIM)) are emerging as techniques of choice in life sciences for the imaging of complex and highly scattering samples. [142][143][144][145] Offering the possibility of visualizing the spheroids in their entirety with a sub-cellular resolution 191,192 the SPIM technique clearly enables achieving a superior degree of information in the screening of nanomedicines' pharmacological efficiency. 47 The penetration ability of nanomedicines can be assessed over the whole 3D MCTS mass and a dynamic study of the anticancer response is also possible with the time lapse imaging of living spheroids. Unfortunately, such advanced techniques require highly specialized technologies whose availability still remains limited.

It is evident that labor-intensive handling, time-consuming procedures, instrumental limitations and costs still hinder the routine use of advanced 3D models in drug discovery programmes. It is noteworthy that, to the best of our knowledge, no promising nanomedicine currently in clinical trials has been tested in 3D MCTS during in vitro preclinical studies. Nevertheless, advances in the near future are expected to rapidly support their widespread use, thus making the in vitro drug screenings more predictive and able to sieve out underperforming compounds in the early preclinical stage.

a b s t r a c t

The preclinical drug screening of pancreatic cancer treatments suffers from the absence of appropriate models capable to reproduce in vitro the heterogeneous tumor microenvironment and its stiff desmoplasia. Driven by this pressing need, we describe in this paper the conception and the characterization of a novel 3D tumor model consisting of a triple co-culture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC), which assembled to form a hetero-type multicellular tumor spheroid (MCTS). By histological analyses and Selective Plain Illumination Microscopy (SPIM) we have monitored the spatial distribution of each cell type and the evolution of the spheroid composition. Results revealed the presence of a core rich in fibroblasts and fibronectin in which endothelial cells were homogeneously distributed. The integration of the three cell types enabled to reproduce in vitro with fidelity the influence of the surrounding environment on the sensitivity of cancer cells to chemotherapy. To our knowledge, this is the first time that a scaffold-free pancreatic cancer spheroid model combining both tumor and multiple stromal components has been designed. It holds the possibility to become an advantageous tool for a pertinent assessment of the efficacy of various therapeutic strategies.

Statement of Significance

Pancreatic tumor microenvironment is characterized by abundant fibrosis and aberrant vasculature. Aiming to reproduce in vitro these features, cancer cells have been already co-cultured with fibroblasts or endothelial cells separately but the integration of both these essential components of the pancreatic tumor microenvironment in a unique system, although urgently needed, was still missing. In this study, we successfully integrated cellular and acellular microenvironment components (i.e., fibroblasts, endothelial cells, fibronectin) in a hetero-type scaffold-free multicellular tumor spheroid. This new 3D triple co-culture model closely mimicked the resistance to treatments observed in vivo, resulting in a reduction of cancer cell sensitivity to the anticancer treatment. Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

In vitro models capable to faithfully mimic the complex cancer physiopathology represent an unmet need to improve the significance of preclinical research data in the early phase of drug development. It is well known that the tumor biology results from a mutual interaction between cancer cells and the surrounding microenvironment [1]. For instance, extracellular matrix (ECM) proteins, such as fibronectin (FN), are actively involved in tumor stiffness regulation [2,3] as well as in promotion of tumor growth [4,5] and drug resistance [6,7]. Moreover, the intricate crosstalking between cancer cells and stroma components (e.g., fibroblasts, immune and endothelial cells) regulates different features related to tumor progression [8-10]. Fibroblasts influence both the tumor development and the metastatic potential of cancer cells through direct heterotypic cell-to-cell contacts and paracrine factors [11-14]. Interactions with cancer cells, fibroblasts and ECM proteins are required to allow the migration and the proliferation of endothelial cells during tumor vascularization [15,16].

Multicellular tumor spheroids (MCTS) are the most widely used 3D in vitro model in oncology preclinical research and their power of prediction of the in vivo efficacy of various chemotherapeutic agents has been clearly evidenced [17,18]. MCTS reproduce some important key factors of real tumors such as: (i) the formation of nutrients, oxygen and metabolic waste diffusive gradients, (ii) the organization of cells in layers with different proliferation rates, (iii) the presence of cell-to-cell interactions and signalling, (iv) the expression of specific gene patterns and (v) the chemoresistance [19,20]. Still, conventional MCTS consisting exclusively of cancer cells mimic only partially the real tumors as consequence of an insufficient extracellular matrix deposition and the absence of other cell types, which compose the tumor microenvironment. With the aim to overcome these limitations hetero-type MCTS consisting of 3D co-cultures of cancer cells and stroma cells have been proposed [21-24]. In addition, the embedding of mono-and hetero-type MCTS in exogenous 3D matrices has been extensively used, creating a cell-supportive environment and reproducing the cell-to-matrix interactions [25,26]. Undoubtedly, the use of matrices, such as natural hydrogels (Matrigel Ò and collagen I, mainly) or synthetic polymers (e.g., poly(ethylene glycol), poly(lactide-coglycolide) and poly(N-isopropylacrylamide)), enhanced the structural stability of 3D MCTS and endowed them with higher biological relevance [27].

However, several disadvantages characterize these approaches. They include: (i) the unknown composition of natural hydrogels and the batch-to-batch variability responsible for a lack of reproducibility [21], (ii) the difficulty to harvest single spheroids once embedded in synthetic biomaterials and the need to apply experimental procedures (chemical or physical) often toxic and/or detrimental for the cells [28], (iii) the restricted accessibility of embedded spheroids, which often makes time-consuming the set-ups to carry out quantitative measurements [23,27]. In this context, it is evident that the in vitro assessment of the role of the microenvironment on the malignant behaviour of tumors and the response to treatments is still a challenge.

Aiming to face this issue, we describe here a novel hetero-type 3D spheroid model integrating cancer cells together with cellular and acellular stroma components (i.e., fibroblasts, endothelial cells, ECM), focusing our attention on the pancreatic ductal adenocarcinoma (PDAC). PDAC belongs to the first five most lethal type of cancers in the western world and the progress in its treatment remains too slow as a consequence of the complex physiopathology of this tumor characterized by a heterogeneous cellular composition and the accumulation of a very dense fibrotic tissue (i.e., desmoplasia) [29]. In PDAC, the crosstalk between cancer and stroma cells leads to a dramatic increase in FN and collagen deposition [10,14,30], which causes the collapse of the tumor vessels and the formation of an aberrant and disorganised vascular network [31]. The consequent oxygen and nutrients deficiency induces the secretion of pro-angiogenic molecules that, in turn, increase the aggressiveness and the invasiveness of cancer cells [31-33]. Currently available hetero-type MCTS of PDAC are better representative of the pathological condition in comparison with 2D monolayer cultures, however they often lack an accurate characterization [34,35] or consist of no more than two cell types [36-41]. For instance, MCTS made of cancer cells and fibroblasts have been proposed to mimic in vitro the molecular cross-talk which characterizes the PDAC desmoplasia [36-38,40,41]. Similarly, cancer and endothelial cells were assembled in MCTS to reproduce the tumor angiogenesis and its influence on cancer cell invasiveness [39]. However, to the best of our knowledge, hetero-type pancreatic tumor spheroids including fibroblasts and endothelial cells, both essential components of the PDAC microenvironment, have not been previously reported. This was likely due to the difficulty to find the experimental conditions required to obtain viable three-cell-type PDAC multicellular spheroids. In this context, we herein report on the step-by-step construction and detailed characterization of a scaffold-free multicellular tumor spheroid consisting of a triple co-culture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC). Importantly, we demonstrated that the presence of a complex microenvironment reduced the sensitivity of cancer cells to chemotherapy thus closely mimicking the resistance to treatments observed in vivo. Overall, this original scaffold-free MCTS represents a pertinent and easy-to-handle tool, which could be readily introduced in routine preclinical screening of therapeutic strategies for pancreatic cancer treatment.

Materials and methods

Cell lines

Human pancreatic cancer cells (PANC-1), human lung fibroblasts (MRC-5) and human umbilical vein endothelial cells (HUVEC) were purchased from ATCC (France) and maintained as recommended. Briefly, PANC-1 cells were maintained in Dulbecco's Modified Eagle Medium-high glucose (DMEM, Sigma Aldrich, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, France). MRC-5 cells were cultured in Eagle's Minimum Essential Medium (EMEM, Sigma Aldrich, France) supplemented with 10% heat-inactivated FBS and 1% of 200 mM L-Glutamine solution (Sigma Aldrich, France). The luciferase-expressing human pancreatic cell line (BxPC-3-luc2) was obtained from PerkinElmer (Roissy, France) and maintained in Roswell Park Memorial Institute medium (RPMI, Sigma Aldrich, France) supplemented with 10% heat-inactivated FBS. All media were further supplemented with penicillin (50 U.mL À1 ) and streptomycin (0.05 mg.mL À1 ) (Sigma Aldrich, France). HUVEC cells were maintained in endothelial growth medium (EGM-2) consisting of endothelial basal medium (EBM-2) in which supplements and growth factors have been added according to manufacturer instruction (EGM-2 BulletKit Lonza, France). Cells were maintained in a humid atmosphere at 37 °C with 5% CO 2 . Cells were used below passage 8 after thawing and harvested at a confluence of 70-80%.

Cell transfection

Luciferase-and fluorescent protein-expressing cells were created by stable lentiviral transduction according to manufacturer protocols.

For the luciferase-transduction, PANC-1 cells (2.5 Â 10 4 cells. mL À1 ) were seeded in 24-well plates (1 mL/well) and incubated for 24 h in a humid atmosphere at 37 °C with 5% CO 2 . Transduction was performed by addition of RediFect TM Red-FLuc-Puromycin lentiviral particles (Perkin-Elmer, France) to cells. For green fluorescent protein (GFP)-transduction, MRC-5 fibroblasts (9 Â 10 4 cells. mL À1 ) were seeded in 24-well plates (1 mL/well) and incubated for 24 h in a humid atmosphere at 37 °C with 5% CO 2 . Transduction was performed by addition of pLenti-C-mGFP-P2A-Puro particles (Origene, Germany) to cells. For both cell types, a polybrene (8 mg.mL À1 )-containing medium and a multiplicity of infection (MOI) of 10 were used.

For red fluorescent protein (RFP)-transduction, HUVEC cells (5 Â 10 3 cells.mL À1 ) were seeded in 24-well plates (1 mL/well) and incubated for 24 h in a humid atmosphere at 37 °C with 5% CO 2 . Transduction was performed by adding pLenti-C-mRFP-P2A-Puro particles (Origene, Germany) to cells. In this case a MOI of 100 in polybrene-free medium was used.

For each experiment, transduction particle-containing medium was removed after 24 h and replaced with fresh medium containing puromycin (Thermo Fisher Scientific, France) as selection antibiotic. According to the specific cell sensitivity, puromycin was used at a concentration of 2, 1 and 0.5 mg.mL À1 for PANC-1, MRC-5 and HUVEC cells, respectively. Puromycin-containing medium was replaced every 3 days until resistant colonies could be identified. Cell bioluminescence and fluorescence were measured 48 h after infection and prior to the cryopreservation of selected cells.

Construction of 3D MCTS

Liquid overlay technique

Mono-type and hetero-type MCTS were constructed according to the liquid overlay technique [42] using 96 round-bottomed well plates (CELLSTAR Ò , Sigma Aldrich, France). Before use, 50 mL of 1.2% (w/v) poly-2-hydroxyethyl methacrylate (poly-HEMA, Sigma Aldrich, France) ethanolic solution was added to each well, and solvent was evaporated in sterile conditions.

For the construction of the mono-type PANC-1 spheroids, cell suspensions (2.5, 5, 12.5 or 25 Â 10 3 cells.mL À1 ) were prepared in DMEM complete medium and 200 mL of the cell suspensions (corresponding to 500, 1000, 2500 and 5000 cells, respectively) was transferred into each well.

For the construction of the hetero-type PANC-1:MRC-5 spheroids, suspensions of each cell type were prepared in DMEM complete medium and then 200 mL of their opportune mixture was transferred into each well. The number of PANC-1 cells was fixed at 500 cells per well. Four PANC-1:MRC-5 ratios have been studied (i.e., 1:1; 1:2; 1:4 and 1:9).

For the construction of the hetero-type PANC-1:MRC-5:HUVEC spheroids, suspensions of each cell type were prepared in DMEM complete medium and then 200 mL of their opportune mixture was transferred into each well. The number of PANC-1 was fixed at 500 cells per well. A ratio of 1:9:4 among PANC-1:MRC-5: HUVEC has been studied. Optimization of the experimental protocol included (i) construction of a nanofilm of fibronectin-gelatin (FN-G) at the MRC-5 surface before seeding (see Section 2.3.2) and (ii) addition of human VEGF (50 ng.mL À1 ) (Thermo Fisher Scientific, France) to the culture medium.

Cell dispersions at different densities were prepared after automatic cell counting (Countess II, Thermo Fisher Scientific, France), according to manufacturer instructions. After cell seeding, plates were centrifuged (1100 rpm, 5 min, 20 °C) and then incubated in a humidified atmosphere with 5% CO 2 at 37 °C for a minimum of 48 h. For long term culture, half of the medium was replaced every 3 days.

MCTS constructed with luciferase-expressing cells were prepared according to the same protocols except for the use of phenol red-free DMEM (Sigma Aldrich, France) supplemented with 10% heat-inactivated FBS, 1% of 200 mM L-Glutamine solution, 1% of 100 mM sodium pyruvate solution (Sigma Aldrich, France), penicillin (50 U.mL -1 ) and streptomycin (0.05 mg.mL -1 ). Fluorescently-labelled spheroids were prepared using GFPexpressing MRC-5 fibroblasts and RFP-expressing HUVEC cells.

Fibronectin-gelatin coating of MRC-5

A fibronectin-gelatin nanometer sized film was constructed at the MRC-5 surface according to the previously validated Layerby-Layer (LbL) method [43]. Briefly, after trypsinization, fibroblasts were suspended in 1 mL of Tris-HCl buffer (50 mM, pH 7.4) (Sigma Aldrich, France), centrifuged (2500 rpm, 1 min, room temperature) and then alternatively incubated for 1 min using a Microtube Rotator (VWR, France) in 1 mL of 0.04 mg.mL À1 Human fibronectin or gelatin (Sigma Aldrich, France) Tris-HCl solutions (50 mM, pH 7.4). Each incubation was followed by a centrifugation (2500 rpm, 1 min, room temperature) and a washing step with Tris-HCl buffer (1 mL, 50 mM, pH 7.4). A total of 9 coating steps were performed. Then, fibroblasts were suspended in cell culture medium and used to construct the triple co-culture, as above described.

Spheroid characterization

Optical imaging

Routine spheroid monitoring was performed using the AxioObserver Z1 (Carl Zeiss, Germany) inverted microscope equipped with a Peltier cooled (À40 °C) CoolSnap HQ2 CCD camera (Photometrics, Tucson, USA) and a XL incubator thermostated at 37 °C providing 5% CO 2 . Transmitted light images of spheroids were collected directly from the poly-HEMA coated plates with a Plan-Apochromat 2.5x dry objective lens, a halogen lamp and a motorized stage used on an automated mode (AxioVision software/high content acquisitions).

By using an image-processing macro, specifically created with the Image-J Ò software, morphometric parameters such as spheroid area, perimeter, minimum diameter, major diameter, aspect ratio and circularity were obtained. Spheroid volume (V) was calculated according to the formula V = [(a 2 ) Â (b)]/2, in which a and b represent the minor and major diameter, respectively. For the growth profile characterization, images of 128 spheroids per condition have been collected at each time point.

Spheroid dissociation and single cell counting

To assess the number of cells per spheroid over time, six-toeight MCTS were harvested using a p5000 pipette to preserve their integrity, and then pooled in a microtube. Culture medium was carefully removed and spheroids were washed with 200 mL of phosphate buffered saline (PBS, Sigma Aldrich, France). After addition of 50 mL of trypsin (Sigma Aldrich, France), samples were incubated at 37 °C for 30 min and pipetted every 5 min with a p200 pipette to facilitate the dissociation of spheroids into a single-cell suspension. The enzyme action was stopped by the addition of 50 mL of FBS-containing culture medium. The total cell number per sample was measured using the Countess II Automated Cell Counter (Thermo Fisher Scientific, France) and the average cell number per spheroid was calculated dividing it by the number of used spheroids. Experiments have been performed in triplicate.

Adenosine triphosphate (ATP) cell viability assay

Individual MCTS (n = 6) were harvested from the round bottom wells and transferred in a microtube using a p5000 pipette. Culture medium was carefully removed, spheroids were washed with PBS (200 mL) to remove any extracellular ATP and then redispersed in 50 mL of phenol red-free DMEM. The spheroid suspension (50 mL) was then transferred into white-opaque 96-well plates (Corning Ò , Sigma Aldrich, France) and 50 mL of CellTiter-Glo Ò 3D reagent (Promega, France) was added into each well. Plates were protected from light and gently shaken for 10 min (room temperature) to induce spheroid lysis. Samples were incubated for additional 20 min in the dark, at room-temperature, to stabilize the bioluminescent signal, which was then recorded using a benchtop plate reader (EnSpire Alpha 2390; Perkin-Elmer, USA). The ATP concentration in each sample was quantified by using a calibration curve based on rATP standards (Promega, France) according to the manufacturer instructions.

Assessment of cell viability after exposure to cytotoxic drugs was carried out according to the same protocol with a slight mod-ification. Briefly, spheroids were cultured in 96 round-bottom well plates in 200 mL of medium. At determined time points, 150 mL of medium was carefully removed and 50 mL of CellTiter-Glo Ò 3D Reagent was added to each well (total final volume 100 mL). Plates were gently shaken in the dark for 10 min to lyse the samples and incubated for additional 20 min at room-temperature to stabilize the bioluminescent signal. Then, samples were individually transferred into white-opaque 96-well plates and the signal was measured using a benchtop plate reader (EnSpire Alpha 2390; Perkin-Elmer, USA) (8 replicates per condition, n = 2).

In vitro luciferase assay

The bioluminescence signal produced by luciferase-expressing PANC-1 or BxPC-3 cells was measured using the Neolite Reporter Gene Assay System (Perkin-Elmer, France) according to manufacturer instructions. Briefly, spheroids were cultured in 96 roundbottom well plates in 200 mL of medium. At determined time points, 150 mL of medium was carefully removed and 50 mL of Neolite reagent was added to each well (total final volume 100 mL). Plates were protected from light and gently shaken for 10 min (room temperature) to induce spheroid lysis. Samples were incubated for additional 20 min in the dark at room-temperature to stabilize the bioluminescent signal. Samples (100 mL) were individually transferred into white-opaque 96-well plates and the signal was measured using a benchtop plate reader (EnSpire Alpha 2390; Perkin-Elmer, USA) (6 or 8 replicates per condition, at least n = 3).

In another set of experiments, samples (100 mL) were individually transferred to a black-opaque 96-well plate (Corning Ò , Sigma Aldrich, France) and plate images were acquired using the IVIS Lumina Ò LT Series III (PerkinElmer, USA) (at least 3 replicates per condition, n = 3). Images were processed using the Living Image software (PerkinElmer, USA).

Histology and immunohistochemistry

MCTS were fixed for 2 h in 500 mL of 4% paraformaldehyde (Roti Ò -Histofix 4%, Roth Sochiel EURL, France) at room temperature. After inclusion in 4% low-melting agarose (Thermo Fisher Scientific, France), spheroids were embedded in paraffin and sectioned (Plate-Forme HistIM, Institut Cochin, Paris France). Sections (5 mm) were stained with haematoxylin and eosin (H&E) according to standard protocols. For immunohistochemical staining, the heat-mediated antigen retrieval of antibodies was carried out in citrate buffer at different pH (according to manufacturer protocols) and sections (5 mm) were then incubated with monoclonal antibody to cytokeratin AE1/AE3 (mouse, dil 1:50; M351501-2 Dako, France), fibronectin (rabbit, dil 1:250; ab2413 Abcam, UK) or CD-31 (mouse, dil 1:20; M082301-2 Dako, France). Primary antibodies were detected with peroxidase-conjugated secondary antibodies by using diaminobenzidine (DAB) as chromogen and haematoxylin as counterstain.

Selective Plane Illumination Microscopy (SPIM)

4-days old fluorescently-labelled spheroids were fixed in 500 mL of 4% paraformaldehyde (Roti Ò -Histofix 4%) (1 h, room temperature) and then permeabilized with 500 mL of 0.1% Triton X-100 (Sigma Aldrich, France) in PBS (1 h, room temperature). Then, Triton solution was replaced with 200 mL of PBS and cell nuclei were stained overnight with Hoechst 33342 (NucBlue TM Reagent, Thermo Fisher Scientific, France) in the dark at room temperature. MCTS were imaged with the Lightsheet Z.1 Microscope (Carl Zeiss, Germany) equipped with a Plan-Apochromat 20x 1NA water-immersion objective lens with left and right illumination. Hoechst, GFP and RFP fluorescent signals were recorded after sample excitation at 405, 488 and 561 nm, respectively. Samples were scanned using the Zen 2014 SP1 Black-Edition software (Carl Zeiss, Germany) and optical slices of 1 mm thickness were processed for the 3D reconstruction of each sample with the Image-J Ò software.

MCTS embedding in fibrin matrix

PANC 1:MRC-5:HUVEC (ratio 1:9:4, LbL, VEGF) spheroids were prepared by using RFP-expressing HUVEC cells and cultured for 4 days before the embedding in a fibrin matrix. Individual MCTS were harvested from the round bottom wells and transferred in a microtube using a p5000 pipette. Culture medium was carefully removed and spheroids were redispersed in 250 mL of bovine fibrinogen (Sigma Aldrich, France) solution (2.5 mg.mL À1 ) in PBS with aprotinin (0.15 U.mL À1 Sigma Aldrich, France) and type I collagen (0.2 mg.mL À1 , Gibco, France) [44]. After addition of 2.5 mL of thrombin (final concentration 0.5 U.mL À1 , Sigma, France), single spheroids were transferred into the wells of a Lab-Tek Ò II Chamber SlideTM (Thermo Fisher Scientific, France). The chamber slide was placed at room temperature for 5 min to allow fibrin polymerization and then 400 mL of EGM-2 medium was added. Embedded MCTS were maintained in a humid atmosphere at 37 °C with 5% CO 2 for three days before confocal imaging. Samples were imaged with an inverted Leica TCS SP8 microscope gated-STED (Leica, Germany) using a HC PL Fluotar CS2 10x/0.30 dry objective lens. The instrument was equipped with a WLL Laser (587 nm excitation wavelength for RFP). Red fluorescence emission was collected with a 597-800 nm wide emission slits. Transmission images were acquired with the same laser line and a PMT-trans detector. The pinhole was set at 1.0 Airy unit giving 4.29 mm optical slice thickness. 12 bit numerical images were done with the Leica SP8 LAS X software (Version 2.0.1; Leica, Germany).

Cytotoxicity evaluation

PANC-1 or BxPC-3 (seeding density 500 cells/well) spheroids (indicated as MCTS_#1 and MCTS_#1 BxPC-3 , respectively) and PANC-1:MRC-5:HUVEC or BxPC-3:MRC-5:HUVEC (ratio 1:9:4, LbL, VEGF) spheroids (indicated as MCTS_#3 and MCTS_#3 BxPC-3 , respectively) were prepared and cultured for 4 days before treatment with a series of concentrations of drugs (doxorubicin (doxo) and gemcitabine (gem)) in culture medium. At the end of the incubation period (48 h and/or 72 h), cytotoxicity assessment was performed by (i) optical imaging (see Section 2.4.1); (ii) ATP quantification (see Section 2.4.3) and/or (iii) qualitative and quantitative measurements of the bioluminescent signal (see Section 2.4.4). Untreated spheroids were used as control. The viability was calculated according to the ratio of the signal of the well containing treated spheroids versus the average signal of control wells (i.e., untreated spheroids).

Statistical analysis

Data are presented as mean values with standard error of mean (s.e.m). Statistical analysis was performed with the Prism Graph-Pad 7.0 software and the significance was calculated using a twoway Anova method, with a Sidak's multiple comparison post-test in which p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.005, *** p < 0.0005, **** p < 0.0001).

Results and discussion

Construction and characterization of multicellular tumor spheroids

Mono-type multicellular tumor spheroids

MCTS consisting of cancer cells only have been constructed according to the liquid overlay technique. We have used the PANC-1 cell line, reported as aggressive and poorly differentiated [45,46], and whose capacity to form multicellular spheroids had been previously described [47-53]. However, to our knowledge no comprehensive characterization of the growth profile of these spheroids was available. Hence, we have prepared PANC-1 spheroids by seeding cancer cells in non-adherent poly-HEMA-coated round-bottom plates at different densities (corresponding to 500, 1000, 2500 and 5000 cells per well) and carried out a broad analysis. Optical imaging at day 4 post seeding revealed the formation of uniform and reproducible 3D structures, whose volume, ATP content and cell number have been monitored over time for a period of 17 days. Representative images of spheroids made with 500 cells (Fig. 1a), showed the spontaneous assembly of cancer cells in compact spheroids with only few surrounding scattered isolated cells (day 4). Spheroid size increased over time and a dark central region was observed at days 13 and 17 post seeding. Similar behaviour was observed for spheroids prepared by seeding higher cell numbers (Fig. S1).

The follow up of the spheroid volume clearly revealed a variation rate inversely proportional to the number of seeded cells per well (Fig. 1b). The lowest cell density (500 cells/well) resulted in the formation of spheroids characterized by the fastest proliferation rate with the major diameter that rapidly increased from 400-500 lm (day 4) to 800-900 lm (day 17), corresponding to a 10-times increase of the volume (from 0.03 to 0.3 mm 3 ). On the contrary, only a 2-fold increase was observed for spheroids prepared by seeding 5000 cells/well.

The number of cells per spheroid, representative of the proliferation capacity of PANC-1 cell in 3D conditions, followed a similar profile (Fig. 1c). Seeding of 500 and 5000 cells/well led to spheroids containing at day 4, an average of 2000 (increment by a factor of 4) and 14,000 cells (increment by a factor of 2.7), respectively. The delayed proliferation of spheroids prepared at the highest cells density was also confirmed by a less rapid variation of the ATP content over time (Fig. 1d).

On the basis of these results, we have chosen for further studies spheroids made by seeding 500 cells/well. These spheroids will be from now indicated as MCTS_#1. Histological analysis of these spheroids by H&E and cytokeratin staining, at day 4 from seeding, revealed a uniform cell distribution and a compact core in which cells tightly adhered to each other (Fig. 1e,f). However, MCTS_#1 did not stain for fibronectin (Fig. 1g), clearly revealing the lack of deposition of this ECM component. The histological analysis at day 17 post seeding was in agreement with these results and no fibronectin staining was detectable (Fig. S2). According to the upregulation of FN in PDAC stroma and its crucial involvement in cancer cells survival, invasion and drug resistance [54-56], it was evident that these spheroids made of PANC-1 cells only did not allow to appropriately mimic the in vivo complexity of the PDAC.

Hetero-type multicellular spheroids: co-culture of cancer cells and fibroblasts

To mimic the tumor-stroma interactions we further gradually increased the complexity of our model and we moved to the construction of an intermediate hetero-type MCTS consisting of cancer cells and fibroblasts. We used MRC-5 fibroblasts, previously reported as a valid alternative to pancreatic stromal cells thanks to their capacity to promote the invasive phenotype of pancreatic cancer cells and to modulate the expression of cell adhesion markers as efficiently as PDAC patient-derived fibroblasts and pancreatic stellate cells [57,58]. Noteworthy is that this cell line has already been used in different multicellular pancreatic tumor models and it offered the possibility to study the tumor microenvironment and investigate the cross-talk between cancer cells and stromal ones [40,59-61]. In addition, successful reconstruction of the interaction between cancer cells and the microenvironment using fibroblasts derived from healthy tissues has also been described for breast cancer 3D models [37,62,63].

By keeping constant the number of PANC-1 (i.e., 500 cells per spheroid at day 0), we investigated co-cultures with fibroblasts at different ratios (1:1, 1:2, 1:4 and 1:9). In all tested conditions the two cell types spontaneously self-assembled without the need of supporting material and the size of spheroids progressively increased over time (from day 4 to day 17) (Fig. 2a, b and Fig. S3). Whatever the tested ratio, the number of cells per spheroid at day 4 was lower than expected according to the seeding density, thus clearly showing that a loss of cells occurred during the first steps of the co-culture assembly (Fig. 2c). Nevertheless, once formed, all spheroids continued to grow regularly as also confirmed by the evolution of the volume and the ATP content (Fig. 2b,d). Note that co-culture with fibroblasts enhanced the metabolic activity of cancer cells compared to spheroids made of PANC-1 only, resulting in a comparable effect to that induced by primary tumor-associated fibroblasts in Transwell Ò co-cultures [35]. The ratio PANC-1:MRC-5 1:9, which allowed, at day 4, to construct spheroids with the highest cell number was selected for further studies. These spheroids will be from now indicated as MCTS_#2.

H&E staining at day 4 of MCTS_#2 showed the assembly of the two cell types and the predominant localization in the core of elongated cells surrounded by a population of round cells (Fig. 2e). The latter was positively stained for the cytokeratin AE1/AE3 marker (specific for the detection of epithelial cells of neoplastic origin) thus revealing the peripheral localization of the PANC-1 cells. On the contrary, the core was characterized by cytokeratin-negative cells and was positively stained for the fibronectin, thus suggesting the accumulation in this area of fibroblasts responsible for the secretion of the ECM component (Fig. 2f,g).

Nevertheless, at day 7 post seeding (Fig. 2h-l) all cells positively stained for the cytokeratin marker revealing the infiltration of cancer cells in the core of the spheroid previously filled by fibroblasts. The disappearance of the MRC-5 cells well correlated with the negligible fibronectin staining (Fig. 2i,l). Same results were obtained at day 17 post seeding. (Fig. S4).

Cell spatial distribution at day 4 was assessed also by Selective Plane Illumination Microscopy (SPIM) imaging of MCTS_#2 constructed using GFP-expressing MRC-5 fibroblasts (Fig. S5a-c). SPIM allowed to observe the spheroid mass in the whole and to collect high resolution images bypassing the limitations in terms of depth of laser penetration of traditional imaging methods such as the confocal microscopy [64-66]. The blue staining of nuclei (Hoechst 33342) showed the distribution of cells in the 3D structure (Fig. S5b) and the green fluorescence in the core confirmed the presence of fibroblasts in this region at day 4 post seeding (Fig. S5c). However, in agreement with the histological characterization, at day 7 post seeding (Fig. S5d-f), the reduction of the green signal indicated the progressive loss of fibroblasts and their gradual replacement by rapidly proliferating cancer cells (Fig. S5f).

These results showed that the tumor microenvironment was not static and a progressive transformation occurred over time leading to the integration of cancer cells and components of the microenvironment (i.e., fibroblasts and fibronectin) for up to 4 days. A progressive loss of fibroblasts had been previously observed in lung [67] and prostate [68] MCTS as well as in spheroid co-cultures of fibroblasts and endothelial cells. [69,70] However, to the best of our knowledge, this is the first time that modifications of the composition of long-term cultured MCTS have been precisely assessed by histological and microscopic analysis in a hetero-type pancreatic tumor model.

Hetero-type multicellular spheroids: triple co-culture of cancer cells, fibroblasts and endothelial cells

The last step in the construction of a new model consisted in the introduction of HUVEC endothelial cells and the establishment of a triple co-culture. Note that hetero-type pancreatic tumor spheroids including fibroblast and endothelial components have not been previously reported. The choice of the HUVEC has been driven by their large availability and stable features compared to tumorderived endothelial cells, whose wide application in routine in vitro studies is still hindered by several obstacles. They include, for instance, the scarce availability of fresh tumor sample, the difficulty to isolate tumor endothelial cells with high degree of purity, the lack of well-defined culture conditions with consequent phenotype mutation, their short life span and thus the possibility to use them for a limited number of passages only [71].

Firstly, PANC-1 cancer cells (500 cells per spheroid at day 0) were mixed with MRC-5 fibroblasts and HUVEC at 1:9:4 ratio and seeded on poly-HEMA-coated plates according to the liquid overlay technique already used for MCTS_#1 and MCTS_#2. Cell assembly and spatial organization of the three cellular components were regularly monitored as early as 2 days after seeding. Also in this case, immunostaining revealed a peripheral localization of the cytokeratin-positive cells (i.e., PANC-1 cells) while the cytokeratin-negative ones and fibronectin were localised in the core of the spheroid (Fig. S6a,b). However, this assembly was characterized by weak hetero-type cell-to-cell contacts as attested by the frequent loss of PANC-1 cells during sample preparation. In addition, CD31 immunostaining, used as a marker of the endothelial cells, highlighted only a feeble presence of HUVEC cells (Fig. S6c). MCTS_#1 and MCTS_#2 spheroids at day 4 post seeding were used as negative controls (Fig. S7). Two days after (day 4), spheroids were more compact (Fig. S6d-f) but the CD31 staining was negligible (Fig. S6f). As we evidenced above, a reorganisation of the cell composition with time was observed also in these spheroids, with a progressive accumulation of PANC-1 cells in the core of the spheroids together with a decrease of the fibronectin content (Fig. S6d,e).

To promote integration and residence of endothelial cells within the spheroid, the preparation method was slightly modified. Briefly, before mixing with PANC-1 and HUVEC, a Layer-by-Layer (LbL) nanofilm of fibronectin and gelatin (FN-G) was constructed at the surface of the MRC-5 fibroblasts, according to a previously validated technique [72]. The FN-G coating on the cell membrane, which mimicked the natural ECM [73-75], had already been successfully applied to the construction of stable multilayer multicellular tissues on Transwell Ò inserts [43,76-80]. Such coating was capable of promoting cell adhesion into the spheroids, facilitating the triple co-culture with the HUVEC cells. Compared to the traditional protocol, this approach allowed to increase the number of CD31-positive cells, which after two days of culture formed a discontinuous layer at the fibroblasts/cancer cells interface. However, the latter were only loosely attached to the external spheroid boundaries (Fig. S8a-c). At day 4, spheroids were more compact, but also in this case the presence of endothelial cells was strongly reduced (Fig. S8d-f). As expected, the FN-G nanofilm supported a better assembly of the three cell types in the spheroid, but it was not enough to ensure their prolonged 3D co-culture. Of note, because of the presence of the adhesive FN-G layer, these spheroids were less fragile and could be more easily handled, thus facilitating their routinely use at day 4. Accordingly, this preparation method was maintained for all the successive experiences. Although collagen, rather than gelatin, is the main component of the pancreatic tumor ECM, the latter has been chosen for the cell coating due to its solubility in neutral medium, which ensures a higher cytocompatibility. On the contrary, the acidic solution of collagen is not well suitable for the culture condition due to the low pH value. Moreover, being the gelatin obtained by collagen denaturation, it maintains a chemical composition very similar to that of the parent molecule.

Significant improvement was then obtained by culturing the LbL-assembled spheroids in medium supplemented with VEGF (50 ng.mL À1 ), a well-known endothelial survival factor [81] and angiogenesis inducer [82]. In addition, VEGF already showed a beneficial effect on the viability and sprouting of HUVEC spheroids [34,83,84]. In these experimental conditions, at day 2, a thick layer of endothelial cells was observed around the fibronectin-rich cytokeratin-negative core (Fig. S9c). On the contrary, no stable adhesion of cancer cells occurred, and they could be easily lost during sample preparation (Fig. S9). A more compact spheroid was, instead, obtained after a culture period of 4 days (Fig. 3a). PANC-1 cancer cells, that are cytokeratin-positive cells, mostly organized in an external layer surrounding cytokeratin-negative cells with which cell-to-cell hetero contacts were established (Fig. 3b). Fibronectin staining revealed the homogenous distribution of this ECM protein in the core (Fig. 3c) in which a network of CD31-positive endothelial cells was also well visible (Fig. 3d). The latter closely resembled to the collapsed vessels observed in PDAC [31,33]. Thus, after opportune optimization of the experimental conditions (that is, LbL coating and VEGF supplement) we successfully constructed a hetero-type pancreatic MCTS based on a triple co-culture of cancer cells, fibroblasts and endothelial cells. These spheroids (PANC-1:MRC-5:HUVEC, LbL, VEGF) will be from now indicated as MCTS_#3.

Histological analysis showed that the optimal triple co-culture was achieved at day 4. At day 7, all cells were positively stained for the cytokeratin (Fig. 4a), as previously observed also for MCTS_#2 (Fig. 2h-l), and no more fibronectin and HUVEC were detectable (Fig. 4b,c).

The spatial distribution of the three cell types at day 4 was assessed in detail by SPIM imaging of MCTS_#3 constructed using GFP-expressing MRC-5 fibroblasts and RFP-expressing HUVEC cells (Fig. 5). The uniform blue staining revealed a homogeneous distribution of cells in the 3D structure (Fig. 5b). The green fluorescence confirmed the presence of fibroblasts in the core (Fig. 5c), which correlated with histology images showing a core made of cytokeratin-negative cells and rich in fibronectin (Fig. 3b,c). Redfluorescent HUVECs were also visible in the center of the spheroid confirming their strong association with the fibroblasts (Fig. 5d).

The slightly elongated shape of HUVEC suggested their potential capacity, in opportune conditions to develop vessel-like networks (Fig. S10), as previously observed by Boutin and co-workers in their imaging analysis of rat cortical spheroids [85].

In agreement with the histological characterization, SPIM imaging of MCTS_#3 spheroids at day 7 showed the progressive loss of the stromal cells. (Fig. 5e-h).

According to the stability of the triple co-culture, characterization of MCTS_#3 was carried out for up to 7 days from seeding. Morphology and growth kinetic were microscopically assessed and optical-images showed spheroids with a dense core and a ruf-fled peripheral cell layer whose compactness increased over time (Fig. 6a). Since day 2, the triple co-culture led to the formation of larger spheroids as compared to MCTS_#1 and MCTS_#2 (Fig. S11) and they reached a mean volume of 0.09 mm 3 ($600 lm in diameter) at day 4 (Fig. 6b). At this time point, MCTS_#1 and MCTS_#2 showed a mean volume of 0.03 and 0.04 mm 3 , respectively (Figs. 1 and2). Variation of the number of cells over time revealed the same trend (Fig. 6c). Also in this case, single cell counting in MCTS_#3 evidenced that not all cells assembled to form the spheroids but that a certain loss occurred, and the number of cells per spheroid at day 2 was lower than the seeded one. This corroborated the hypothesis of the existence of a critical initial number of cells for establishing heterotypic interactions and stable 3D assembly. The strong influence of the microenvironment on the spheroid metabolic activity was confirmed by the ATP content (Fig. 6d) which, at day 4, was up to two times higher than in spheroids made of only cancer cells (0.54 and 1.2 mM for MCTS_#1 and MCTS_#3). This influence, likely mediated by the direct 3D cell-to-cell contact as well as by the production of growth factors and cytokines [60,86] persisted also at day 7 when spheroids where mainly composed of cancer cells (Fig. 4). Among the secreted soluble factors, the hepatocyte growth factor (HGF) might play a key role. It has been demonstrated that by binding to its transmembrane receptor c-Met, the HGF may activate several signaling pathways involved in pancreatic cancer cells growth and invasion, tumor progression, desmoplastic reaction and resistance to treatment [87-89]. In addition, previous studies have reported a high secretion of HGF by MRC-5 fibroblasts, which was responsible of an enhanced proliferation and migration of several types of cancer cells [35,90-92]. Thus, although further investigation would be required to confirm this hypothesis, it is possible that the proliferation of PANC-1 might be mediated by the HGF secreted by the MRC-5 cells. Overall, these results demonstrated that the structural and functional interactions between cancer cells and several stromal components have been successfully recreated in MCTS_#3.

Cytotoxic studies: effect of the tumor microenvironment

It is well known that the tumor microenvironment strongly influences the drug sensitivity of cancer cells and may affect the efficacy of treatments [29]. Therefore, we aimed to verify whether the MCTS_#3 model was capable or not to reproduce such influence. Firstly, as proof of concept we assessed the cell viability in MCTS_#3 and MCTS_#1 after a 48 h and 72 h exposure to a single dose of doxorubicin (0.5 mM, IC 50 for PANC-1 cells grown in 2D monolayer). Because of their stability and 3D compact structure, spheroids at day 4 were used for the onset of the treatment. Spheroids were constructed using luciferase-expressing PANC-1 cells, enabling to specifically monitor the cytotoxicity of doxorubicin on cancer cells by quantification of the luciferase activity. After 48 h of incubation, the survival of cancer cells was similar in both types of spheroids and only a general decrease of $10% could be measured in comparison to their respective untreated controls (Fig. 7a). However, after longer exposure (72 h) higher survival was observed in MCTS_#3. Sensitivity of cancer cells was influenced by the presence of the tumor microenvironment (Fig. 7a). Bioluminescence images of these spheroids were in agreement with the quantitative results. Only a weak difference of the pseudocolor was observed between control (untreated) and doxo-treated MCTS_#3. On the contrary, for MCTS_#1 the colour changed from green to light blue (lowest values) after treatment, which underlined the higher sensitivity to doxorubicin (Fig. 7b). Accordingly, the overall spheroid viability, as measured by the ATP content, was also significantly different at 72 h and confirmed the key role of the tumor microenvironment on the sensitivity of the spheroids to the treatment (Fig. 7c). In addition, the anti-proliferative effect of the drug has been also microscopically assessed by collecting bright-field images of spheroids (n = 64 per condition). Quantitative information of the growth inhibition was provided by the variation of the spheroid volume after treatment [93]. Once again data revealed the lowest anticancer efficacy of the drug on MCTS_#3 and further corroborated the influence of the microenvironment on the proliferation of the tumor mass (Fig. 6d). The higher sensitivity of MCTS_#1 compared to MCTS_#3 was observed also after exposure of spheroids, constructed using luciferase-expressing PANC-1 cells, to a series of doxorubicin concentrations (Fig. S12a). Similar results were obtained after incubation with gemcitabine in spite of the different mechanism of actions of the two anticancer drugs (Fig. S12b). Indeed, doxorubicin exerts its cytotoxic activity by intercalation between DNA base pairs blocking DNA replication and transcription, blockade of topoisomerase II activity as well as generation of free radicals, [94] while, on the other hand, gemcitabine acts as nucleoside analogue [95]. Noteworthy is that, after exposure to doxorubicin and gemcitabine, MCTS_#3 displayed higher resistance to treatments also when compared to MCTS_#2, thus confirming the crucial role of a complex tumor microenvironment consisting of multiple cell types (Fig. S13).

We have then modified the culture condition to assess whether similar results could be obtained by using another pancreatic cell line. Thus, both mono-type and hetero-type (i.e., triple coculture) multicellular spheroids, have been constructed replacing the PANC-1 cell line with the BxPC-3 one (indicated as MCTS_#1 BxPC-3 and MCTS_#3 BxPC-3, respectively). However, exposure of these spheroids to growing concentrations of doxorubicin and gemcitabine did not induce a dose/effect response. In addition, for each concentration tested, we observed a higher viability of mono-type spheroids compared to the hetero-type triple coculture (Fig. S14). This was attributed to the more compact structure of MCTS_#1 BxPC-3 (Fig. S15), which artefactually opposed to the diffusion/penetration of the drug through the tumor mass. These results clearly highlight that accurate selection of the cell types used for the construction of the spheroids and characterization of the resulting cell assembly are a key requisite to develop valuable tools for accurate preclinical screening of therapeutic strategies for pancreatic cancer treatment.

Conclusions

The failure of treatments and the poor prognosis of pancreatic ductal adenocarcinoma are related to the extremely complex physio-pathology of this tumor, combining abundant fibrosis and collapsed vasculature, which strongly limits drugs accessibility. In this study, we reported on the construction and detailed characterization of a scaffold-free 3D model capable to integrate in a hetero-type multicellular spheroid the cancerous component of the pancreatic tumor as well as its microenvironment made of fibroblasts, endothelial cells and extracellular matrix. To our knowledge, no analogous model combining the fibrotic tissue and a collapsed vessel-like structure, both hallmark of the PDAC, has been previously constructed. Evaluation of spheroid growth and response to treatment confirmed the influence of the microenvironment on drug sensitivity of pancreatic cancer cells, and showed the capacity of this model to replicate the resistance to treatments often observed in vivo. Overall, we believe that this easy to handle spheroid model, reproducing crucial features of the PDAC, could find application in standardized drug screening protocols as well as in the assessment of cancer cells-stroma interactions and tumor angiogenesis. Title : Shedding lights on cancer cells and their microenvironment: development of 3D in vitro tumor models to shorten the translation of nanomedicines from the bench to the bedside Keywords : 3D tumor models; spheroids; drug delivery; nanomedicines Abstract: A discrepancy between promising preclinical in vitro results and the in vivo performances is often observed in the early stage of drug development and might be ascribed to the lack of capacity of the models commonly used for in vitro studies to faithfully reproduce the pathophysiology of solid tumors. These models mainly consist of cancer cells cultured as 2D monolayers or assembled to form 3D multicellular tumor spheroids (MCTS). However, being composed exclusively of one cell type, these models are too simplistic. They do not allow to reproduce the heterogeneous cellular composition, as well as, the complex architecture of the tumor and its surrounding microenvironment. Thus, they fail to replicate the multiple biological barriers that drugs have to cross in order to reach the target cells. The aim of this PhD thesis was to construct a heterotype MCTS for an appropriate in vitro evaluation of the therapeutic potential of nanomedicines and other chemotherapies.

Attention has been focused on the pancreatic ductal adenocarcinoma (PDAC) and a novel hetero-type MCTS based on a triple co-culture of pancreatic cancer cells, fibroblasts and endothelial cells was created. This 3D model demonstrated the capacity to reproduce in vitro the influence of the microenvironment on the sensitivity of cancer cells to chemotherapy. In addition, by combining the 3D model and the innovative Light Sheet Fluorescence Microscopy (LSFM), we investigated the penetration of the anticancer drug doxorubicin (in a free form and loaded into nanoparticles (NPs)) in a high informative manner. Then, in order to acquire a better understanding on how nanomedicines and other anticancer chemotherapies interact with the tumor, we sought to combine the hetero-type 3D culture with controlled flow conditions in a microfluidic device. To reach this goal we designed and fabricated a tailor-made chip suitable to host both a MCTS and a perfusable microvascular network (i.e., MCTS-on-a-chip).

Fig. 2

 2 Fig. 2 Schematic representation of the main techniques used for spheroid construction: (a) liquid overlay; (b) hanging drop method; (c) spinner flask; (d) NASA bioreactor; (e) micromolding microwells. Adapted with permission from ref. 50. Copyright 2013 American Chemical Society.

Fig. 3

 3 Fig. 3 Tissue penetration and antitumor efficacy on 4T1 MCTS of Taxol, ∼70 nm Ptx nanocrystals (NPs), RGD-grafted ∼70 nm Ptx nanocrystals (iNPs), ∼10nm Ptx nanocrystals (NDs) and RGDgrafted ∼10nm Ptx nanocrystals (iNDs). (a) CLSM images after 24 h incubation with Cy5-labeled Taxol/NDs/NPs/iNDs/iNPs, (b) CLSM images of TUNEL analysis after 24 h treatment with the different Ptx formulations; dead cells are red and live cells appear in green. (c) Growth inhibitory effect of the different formulations. Reproduced with permission from ref. 56. Copyright 2015 WILEY-VCH.

Fig. 4

 4 Fig. 4 (a) Structure of PEG-b-PAEMA-PAMAM/Pt and schematic illustration showing the selfassembly into the pH-sensitive cluster nanobombs (SCNs/Pt) at neutral pH and the disintegration of into small particles at tumor acidic pH. (b) CLSM images showing in vitro penetration of fluorescently-labeled SCNs/Cy5 and ICNs/Cy5 in BxPC-3 multicellular spheroids. Scale bar: 100 µm. (c) In vivo real-time microdistribution of SCNs/Cy5 and ICNs/Cy5 in BxPC-3 xenografts after intravenous administration. Scale bar: 100 µm. Reproduced with permission from ref. 133.

Fig. 5

 5 Fig. 5 Uptake and penetration of shape specific particles in spheroid. Scanning Electron Microscope (SEM) images of (a) 220 nm × 100 nm disks (H/D ~0.45), (b) 325 nm × 100 nm disks (H/D ~0.3), (c) 400 nm × 100 nm × 100 nm rods and (d) 800 nm × 100 nm × 100 nm rods. (e-h) Two-Photon pictures of spheroids incubated with the disks or rods in the correspondent upper panel. (i) Normalized radial intensity distribution as a function of distance from the center of the spheroid. Reproduced with permission from ref. 85. Copyright 2015 Wiley-VCH.

Fig. 6

 6 Fig. 6 (a) Accumulation and permeation behaviors of G2, G4, and G7 PAMAM dendrimers in MCF-7 MCTS as function of the surface charge. (b) Tumor penetration of amine-terminated G2, G4, and G7 PAMAM dendrimers. Reproduced with permission from ref. 72. Copyright 2016 American Chemical Society.

Fig. 7

 7 Fig. 7 (a) Absorbance values (mean ± standard error) corresponding to the DNA content in LNCaP spheroids treated with curcumin-loaded nanoparticles for 7 days. (Curcumin concentration: 30 µM). ***p < 0.001. ****, p < 0.0001. (b) LNCaP spheroids treated with free curcumin and curcuminloaded nanoparticles for 7 days. (Curcumin concentration: 150 µM). Scale bars: 200 µm. Reproduced with permission from ref. 91. Copyright 2016 Royal Society of Chemistry.

Fig. 8

 8 Fig. 8 (a) Schematic representation of the synthesis of block copolymer and formation of UCM and CKM. (b) Schematic hypothesis of penetration differences for Doxo-loaded CKM and UCM. (c) Doxo delivery into pancreatic MCTS by CKM and UCM revealed by CLSM. (d) Inhibition of the pancreatic MCTS growth by Doxo-loaded micelles. Microphotographs of pancreatic spheroids before and after treatment with Doxo-loaded micelles, free drug (Doxo-h) or untreated (Cont). Reproduced with permission from ref. 90. Copyright 2015 Royal Society of Chemistry.

Fig. 9

 9 Fig. 9 CLSM images of HEK293T multicellular spheroids 22 h after transfection with (a) PME-(PEG 3.5k ) 1.69 ; (b) PME-(PEG 3.4k -FA 1 ) 1.66 , or (c) PME-(PEG 3.4k -FA 2 ) 1.72 complexes. (d) Relative fluorescence intensity (treated group/blank) of HEK293T multicellular spheroids treated with PME-(PEG 3.5k ) 1.69 , PME-(PEG 3.4k -FA 1 ) 1.66 , or PME-(PEG 3.4k -FA 2 ) 1.72 complexes for 4 h and for further 18 h culture. Reproduced with permission from ref. 94. Copyright 2015 American Chemical Society.

  Penetration observed in vitro by Confocal Laser Scanning Microscopy (CLSM) was confirmed in vivo in A549-tumor bearing mice by threedimensional near-infrared fluorescence (NIRF) imaging and resulted in significant suppression of tumor development.

Fig. 10

 10 Fig. 10 Structured Illumination Microscopy (Confocal-like) images of specific binding between chitosan-decorated doxorubicin-loaded nanoparticles (nDOXO) and mammosphere cells: (a) colocalization of nDOXO and CD44 receptors; (b) Binding between nDOXO and free Doxo (fDOXO) with mammosphere cells. (c) Tumor volume as a function of time for four different treatments and image of tumors collected on day 80 after initial drug administration. Reproduced with permission from ref.122. Copyright 2015 American Chemical Society.

Fig. 11 (

 11 Fig. 11 (a) CLSM images of SH-SY5Y MCTS incubated with free Doxo, Doxo-loaded CS-PAPBA NPs and Doxo-loaded iRGD-CS-PAPBA NPs for 2 h, 4 h and 8 h, respectively. (b) In vivo tumor growth curves of H22 tumor-bearing mice that received different treatments. Data are represented as mean ± SD (n = 10). * Represents P < 0.05 since the 7 th day and ** represents P < 0.01 since the 11 th day. Reproduced with permission from ref. 117. Copyright 2013 American Chemical Society

  108

Fig. 12 (

 12 Fig. 12 (a) Optical images of MDA-MB-231 MCTS treated with fresh medium (control), naked pTRAIL (pTRAIL), poly(ethylene imine)/pTRAIL complex (PEI/pTRAIL) and G4-F735/pTRAIL complex (G4-F735/pTRAIL) at different time points. (b) Time-elapsed evolution of tumor sizes in vivo. Reproduced with permission from ref. 92. Copyright 2016 Royal Society of Chemistry.

Fig. 13

 13 Fig. 13 3D images of MCF-7 spheroids after incubation with (a) free Doxo and (b) Doxo-loaded IONP@P(HBA)-b-P(OEGA) for 17 h. The representative confocal images (left) were taken every 5 µm section from the top to bottom in the middle of an intact spheroid, whereas the 3D image (right) was reconstructed using Imaris software. Reproduced with permission from ref. 81. Copyright 2013 American Chemical Society.

Fig. 14 (

 14 Fig. 14 (a) Schematic of the PDMS microfluidic device on a microscope stage. (b) Schematic (left) and image (right) of 40 nm fluorescent PEG-NPs administered for 1 h at 50 µL/h entering the spheroid and accumulating in the interstitial spaces (arrows). Scale bar: 100 µm. (c) Schematic (left) and image (right) of 110 nm fluorescent PEG-NPs administered for 1 h at 50 µL/h being excluded from the spheroid. Scale bar: 100 µm. (d) Representative images of tumor fluorescence from mice injected with NPs in the tail vein at 48 h post-injection. (e) Quantification of animal fluorescence at 2 and 48 h using whole animal images. Reproduced with permission from ref. 185. Copyright 2013 Nature Publishing group.

Fig. 15 (

 15 Fig. 15 (a) Bright field image of the microfluidic channel. The black arrow shows the direction of medium flow. Scale bar: 300 µm. (b) Bright-field image of the MCTS in the microfluidic channel. (top) The MCTS in matrigel prior to Doxo-HCl treatment. (bottom) Real-time, enlarged views of the MCTS during 24 h of treatment with Doxo-HCl. Scale bar: 50 µm. Reproduced with permission from ref. 187. Copyright 2013 American Chemical Society.

Chapter I 86 2

 86 .4.8. Cytotoxicity evaluation PANC-1 or BxPC-3 (seeding density 500 cells/well) spheroids (indicated as MCTS_#1 and MCTS_#1 BxPC-3 , respectively) and PANC-1:MRC-5:HUVEC or BxPC-3:MRC-5:HUVEC (ratio 1:9:4, LbL, VEGF) spheroids (indicated as MCTS_#3 and MCTS_#3 BxPC-3 , respectively)

Fig. 1 .

 1 Fig. 1. Mono-type PANC-1 spheroid characterization. (a) Representative optical imaging of MCTS_#1 (seeding density of 500 cells per well) at days 4, 7, 13 and 17 post seeding. Scale bars: 200 µm. Evolution over time of (b) volume, (c) cell number and (d) ATP content per spheroid as function of the initial seeding density. Values represent mean ± s.e.m. *p < 0.05 **p < 0.005, *** p < 0.0005 **** p < 0.0001 by a two-way Anova method, with a Sidak's post-test at days 4, 7 and 17 post seeding, chosen as representative time points. Histological analysis of 5 µm sections of MCTS_#1 at day 4 post seeding: (e) haematoxylin eosin staining, (f) cytokeratin AE1/AE3 and (g) fibronectin immunostaining. Scale bars: 50 µm.

Fig. 2 .

 2 Fig. 2. Hetero-type PANC-1: MRC-5 spheroid characterization. (a) Representative optical imaging of MCTS_#2 (PANC-1:MRC-5 seeding ratio 1:9) at days 4, 7, 13 and 17 post seeding. Scale bars: 200 µm. Evolution over time of (b) volume, (c) cell number and (d) ATP content per spheroid as function of the initial seeding density (PANC-1:MRC-5 ratio 1:1, 1:2, 1:4 and 1:9). Values represent mean ± s.e.m. Histological analysis of 5 µm sections of MCTS_#2 at day 4 (e, f, g) and day 7 (h, i, l) post seeding: (e, h) haematoxylin eosin staining, (f, i) cytokeratin AE1/AE3 and (g, l) fibronectin immunostaining. Scale bars: 50 µm.

Fig. 3 .

 3 Fig. 3. Hetero-type PANC-1:MRC-5:HUVEC spheroid characterization. Histological analysis of 5 µm sections of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at day 4 post seeding: (a) haematoxylin eosin staining, (b) cytokeratin AE1/AE3, (c) fibronectin and (d) CD31 immunostaining. Scale bars: 50 µm.

Fig. 4 .

 4 Fig. 4. Hetero-type PANC-1:MRC-5:HUVEC spheroid characterization. Histological analysis of 5 µm sections of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at day 7 post seeding: (e) cytokeratin AE1/AE3, (f) fibronectin and (g) CD31 immunostaining. Scale bars: 50 µm.

Fig. 5 .

 5 Fig. 5. SPIM 3D topography of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at day 4 and day 7. (a, e) Overlay of blue (Hoechst 33342, nuclei), green (GFP-expressing MRC-5 fibroblasts) and red (RFP-expressing HUVECs) fluorescence; (b, f) single blue channel (Excitation/emission 405/440 nm) showing all cell nuclei; (c, g) single green channel (Excitation/emission 488/525 nm) showing GFP-expressing MRC-5 fibroblasts; (d, h) single red channel (Excitation/emission 561/605 nm) with RFP-expressing HUVEC cells. Scale bars: 100 µm.

Fig. 6 .

 6 Fig. 6. Hetero-type PANC-1:MRC-5:HUVEC spheroid characterization. (a) Representative optical imaging of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at days 2, 3, 4 and 7 post seeding. Scale bars: 200 µm. Evolution over time of (b) volume, (c) cell number and (d) ATP content per spheroid in MCTS_#3. Values represent mean ± s.e.m. **** p < 0.0001 by a two-way Anova method, with a Sidak's post-test.

Chapter I 102 Fig. 7 .

 1027 Fig. 7. (a) PANC-1 cell viability (luciferase activity) in MCTS_#1 and MCTS_#3 following exposure to doxorubicin (0.5 µM) for 48 h and 72 h (n=3). Spheroids have been constructed using luciferase-expressing PANC-1 cells. (b) Representative bioluminescence images of control and doxo-treated MCTS_#1 and MCTS_#3. Incubation time 72 h. (c) Spheroid viability (ATP quantification) following exposure to doxorubicin (0.5 µM) for 48 h and 72 h (n=2). (d) Spheroid growth inhibition (volume measurement) following exposure to doxorubicin (0.5 µM) for 48 h and 72 h. Data represents mean ± s.e.m. **** p < 0.0001 by a two-way Anova method, with a Sidak's post-test.

Fig. S5 .

 S5 Fig. S5. SPIM 3D topography of MCTS_#2 at day 4 and day 7 post seeding. (a,d) Overlay of blue (Hoechst 33342, nuclei) and green (GFP-expressing MRC-5 fibroblasts) fluorescence; (b,e) single blue channel (Excitation/emission 405/440 nm) showing all cell nuclei; (c,f) single green channel (Excitation/emission 488/525 nm) for detection of GFP-expressing MRC-5 fibroblasts. Scale bars: 100 µm.

Fig. S7 .

 S7 Fig. S7. CD31 immunostaining (negative controls). Histological analysis of 5 µm sections at day 4 post seeding: (a) MCTS_#1 and (b) MCTS_#2. Scale bars: 50 µm.

Fig. S9 .

 S9 Fig. S9. Histological analysis of 5 µm sections of hetero-type PANC-1:MRC-5:HUVEC MCTS_#3 (ratio 1:9:4) at day 2 post seeding: (a) cytokeratin AE1/AE3, (b) fibronectin and (c) CD31 immunostaining. Scale bars: 50 µm.

Fig. S11 .

 S11 Fig. S11. Representative optical imaging of (a) MCTS_#1 and (b) MCTS_#2 at days 2 and 3 post seeding. Scale bars: 200 µm.

Fig. S13 .

 S13 Fig. S13. Spheroid viability (ATP quantification) following exposure to doxorubicin (doxo, 0.5 µM) or gemcitabine (gem, 50 µM) for 72 h (n=2). Data represents mean ± s.e.m. **** p < 0.0001 by a two-way Anova method, with a Sidak's post-test.

Fig. S15 .

 S15 Fig. S15. Representative optical imaging of (a) MCTS_#1 BxPC-3 and (b) MCTS_#3 BxPC-3 at day 4 post seeding, before exposure to cytotoxic drugs. Scale bars: 200 µm.

Fig. 1 .

 1 Fig. 1. (a) Schematic illustration of the LSFM detection and illuminations lenses; (b) the mounting of a gel embedded-sample that allows the physical rotation for the multi-view imaging. Adapted with permission from ref. [40]. Copyright 2014 Springer Nature.

Fig. 2 .

 2 Fig. 2. Schematization of the image processing that combines the multi-view imaging and the z-stack fusion. (a) the original fluorescent sample; (b) z-stack collected from the detection at 0°; (c) z-stack collected from the detection at 180°; (d) optical fusion of (b) and (c) in a single data set with higher isotropic resolution. Arrows indicate the axis along which the light is detected. Adapted with permission from ref. [57]. Copyright 2007 OSA Publishing.

Fig. 3 .

 3 Fig. 3. Confocal imaging of MCTS after incubation with doxorubicin (20 µM) for 3 h at 37 °C. (a) maximum intensity projection (MIP) of the z-stack; (b) optical section n° 5 at ~ 5 µm depth; (c) optical section n° 50 at ~ 50 µm depth; (d) optical section n° 100 at ~ 100 µm depth. Scale bars: 100 µm.

Fig. 4 .

 4 Fig. 4. LSFM imaging of MCTS after incubation with doxorubicin (20 µM, 3 h, 37 °C). Images from the z-stack recorded at 0°: (a) maximum intensity projection (MIP), (b) optical section n° 80 at ~ 58 µm depth, (c) optical section n° 150 at ~ 110 µm depth, (d) optical section from the middle (n° 260) at ~ 190 µm depth. Images from the z-stack recorded after 180° rotation of the sample: (e) maximum intensity projection (MIP), (f) optical section n° 80 at ~ 58 µm depth, (g) optical section n° 150 at ~ 110 µm depth, (h) optical section from the middle (n° 267) at ~ 195 µm depth. Scale bars: 100 µm

Fig. 5 .

 5 Fig. 5. Representative optical image of large MCTS (5000 PANC-1 + 45000 MRC-5 + 20000 HUVEC cells/well) at days 4 post seeding. Scale bar: 200 µm.

Fig. 6 .

 6 Fig. 6. 3D tomography with LSFM of large MCTS after incubation with 20 µM of doxo for 4 h at 37 °C. Overlays of blue (nuclei, Hoechst 33342 staining) and red (doxo) channels. (a) 0° rotation, (b) 90° rotation, (c) 180° rotation. Localization of (d) section 1, (e) section 2 and (f) section 3 in relation to the entire spheroid. Scale bars: 200 µm. (g) section 1 at ~ 113 µm depth, (h) section 2 at ~ 403 µm and (i) section 3 at ~ 861 µm depth. Scale bars: 100 µm.

Fig. 7 .

 7 Fig. 7. (a) Structure of the heterobifunctional Gem-PI-Dox polymer prodrug. PI = polyisoprene, Gem = gemcitabine and Dox = doxorubicin. (b) representative Cryo-TEM image of Gem-PI-Dox nanoparticles. Scale bar: 1 µm.

Fig. 8 .

 8 Fig. 8. 3D tomography with LSFM of large MCTS after incubation with doxo-loaded NPs (20µM equiv. doxo, 4 h, 37 °C). Overlays of blue (nuclei, Hoechst 33342 staining) and red (doxo) channels. (a) 0° rotation, (b) 90° rotation, (c) 180° rotation. Scale bars: 200 µm.

Fig. 2 .

 2 Fig. 2. Schematic representation (not in scale) of the angiogenesis configuration (pattern A) of the MCTS-on-a-chip. M1, M2 and M3: medium channels (■); V1 and V2: vessel channels filled with hydrogel (■); TS: tumor-stroma chamber filled with hydrogel + fibroblasts (■) + a hetero-type MCTS (■); R: reservoir; I: hydrogel injection port (for sake of clarity only one of each has been indicated in the figure). The seeding point of the endothelial cells and the growth direction of the vessels are indicated by red arrows (►).

Fig. 3 .

 3 Fig. 3. Schematic representation (not in scale) of the vasculogenesis configuration (pattern V) of the MCTS-on-a-chip. M1, M2 and M3: medium channels (■); V1 and V2: vessel channels filled with hydrogel + endothelial cells (■); S1 and S2: stroma channels filled with hydrogel + fibroblasts (■); TS: tumor-stroma chamber filled with hydrogel + fibroblasts (■) + a heterotype MCTS (■). R: reservoirs; I: hydrogel injection port (for sake of clarity only one of each has been indicated in the figure). The seeding point of the endothelial cells and the growth direction of the vessels are indicated by red arrows (►).

Fig. 4 .

 4 Fig. 4. The optical mask with pattern A for the angiogenesis configuration. (a) exported image of the mask design bearing the microscale pattern A generated with Tanner L-Edit IC Layout software (Mentor, Germany); (b) pattern details of the area in the red square.

Fig. 5 .

 5 Fig. 5. The optical mask with pattern V for the vasculogenesis configuration. (a) exported image of the mask design bearing the microscale pattern V generated with Tanner L-Edit IC Layout software (Mentor, Germany); (b) pattern details of the area in the red square.

Fig. 6 .

 6 Fig. 6. Chemical structures and reactions of the SU-8 crosslinking. (a) SU-8 molecule; (b) UV activation of the triphenyl-sulfonium herafluoroantimonate and formation of the photoacid initiator (hexafluoroantimonic acid); (c) polymerization of the SU-8 monomers. Adapted with permission from ref. [50]. Copyright 2015 Springer Nature.

Fig. 7 .

 7 Fig. 7. Representative optical imaging of the developed SU-8 angiogenesis pattern A deposed on Si wafer. (a) detail of a large channel; (b) medium reservoir; (c) details of interspaces between adjacent channels; (d) trapezoidal and hexagonal openings. Scale bars: 500 µm.

Fig. 8 .

 8 Fig. 8. Representative optical imaging of the developed SU-8 patterns deposed on Si wafer: (a) angiogenesis configuration and (b) vasculogenesis configuration. (i) detail of a large channel; (ii) medium reservoir; (iii) details of interspaces between adjacent channels; (iv) trapezoidal and hexagonal openings. Scale bars: 500 µm.

Fig. 9 .

 9 Fig. 9. The new optical mask with pattern A for the angiogenesis configuration. (a) exported image of the mask design bearing the microscale pattern A generated with Tanner L-Edit IC Layout software (Mentor, Germany); (b) pattern details of the area in the red square. The orange arrow indicates an example of modified (more regular and thicker) channel boundary (◄).

Fig. 10 .

 10 Fig. 10. The new optical mask with pattern V for the vasculogenesis configuration. (a) exported image of the mask design bearing the microscale pattern V generated with Tanner L-Edit IC Layout software (Mentor, Germany); (b) pattern details of the area in the red square. The orange arrow indicates an example of modified (more regular and thicker) channel boundary (◄).

Fig. 11 .

 11 Fig. 11. Representative optical imaging of the developed SU-8 pattern V (vasculogenesis) deposed on Si wafer. (a) detail of a large channel; (b) medium reservoir; (c) details of interspaces between adjacent channels; (d) rectangular openings. Scale bars: 500 µm.

For the construction of

  this 3D model we have worked on the assembly of cells in the form of multicellular tumor spheroids (MCTS) and we have constructed a hetero-type model integrating the cancerous component of pancreatic tumor (PANC-1 cells) and its microenvironment made of fibroblasts (MRC-5), endothelial cells (HUVEC) and components of the extracellular matrix. As detailed in Chapter I, we started from a simple mono-type MCTS made only of PANC-1 cancer cells and then the complexity of the 3D model has been stepwise increased with the addition of the stromal components. First, a double co-culture MCTS has been constructed as intermediate model by the combination of pancreatic cancer cells and MRC-5 fibroblasts. The latter, although of non-pancreatic origin and derived from a healthy tissue, have been previously validated as representative pancreatic stromal cells. Indeed, these fibroblasts have demonstrated the capacity to secrete soluble factors capable to enhance the invasiveness of pancreatic cancer cells similarly to what was observed with pancreatic primary fibroblasts [3].

  PhD research project has been carried out in the framework of the European project NABBA (Design and Development of advanced NAnomedicines to overcome Biological BArriers and to treat severe diseases) whose global aim was to synthesize new drug delivery systems and to assess their ability to cross multiple biological barriers to ensure superior therapeutic benefits. In this project, we have focused our attention on the pancreatic ductal adenocarcinoma (PDAC), for which no curative options are currently available. The excessive dense fibrotic reaction (i.e., desmoplasia), hallmark of this tumor, strongly limits the bioavailability of therapeutic agents to cancer cells making the PDAC a non-responder to traditional chemotherapy. During my PhD, I have faced the lack of reliable models able to reproduce in vitro the complex physiology of the PDAC for an efficient evaluation of any novel therapeutic strategy.This research work has led to the successful development of a 3D multicellular tumor spheroid of pancreatic cancer based on a novel triple co-culture in which cancer cells have been integrated with microenvironment components including fibroblasts, endothelial cells and extracellular matrix. The experimental set up has been carefully optimized to allow the co-existence of cancer cells, fibrotic tissue and collapsed vessel-like structures, which was well achieved at day 4 post seeding. We have demonstrated that such complex microenvironment, made of different cell types, affected the response of cancer cells to the drug treatment. Overall, this easy to handle spheroid model could find application in standardized drug screening protocols, as well as, in the assessment of cancer cells-stroma interactions. Of note, in a future perspective, the construction of such hetero-type spheroids by using patient-derived cells could pave the road toward the personalized medicine. Such approach would allow to overcome interpatient variability and to personalize treatment protocols thus enabling a better management of the disease and optimizing the effectiveness of the therapeutic intervention.

  Multicellular tumor spheroids are scaffold-free spherical selfassembled aggregates of cancer cells displaying an intermedi-Simona MuraDr Simona Mura gained her degree in Pharmaceutical Chemistry and Technology in 2005. In 2009, she was awarded her PhD in Chemistry and Technology of Drugs at the University of Cagliari, Italy, working on the design and in vitro evaluation of novel vesicular systems for the topical delivery of drugs. In 2008 she joined the group of Prof. Elias Fattal (UMR CNRS 8612) at the University Paris-Sud, Chatenay-Malabry, France, as a post-doctoral research assistant to study the lung toxicity of biodegradable nanoparticles designed for pulmonary administration of drugs. In 2011 she was appointed Associate Professor within the framework of the "CNRS-Higher Education chairs (Chaire d'excellence)" programme, in the group of Prof. Patrick Couvreur (UMR CNRS 8612) at the University Paris-Sud, Chatenay-Malabry, France. Her research focuses on the design of novel lipid and biopolymer-based nanoscale drug delivery systems for the treatment of severe neoplastic diseases and on the development of valuable 3D in vitro models for the assessment of their therapeutic efficacy.

Fig. 1

 1 Fig. 1 Schematic representation of cell organization and gradients in tumor spheroids. Adapted with permission from ref. 44. Copyright 2008 Wiley-VCH.

Fig. 2

 2 Fig. 2 Schematic representation of the main techniques used for spheroid construction: (a) liquid overlay; (b) hanging drop method; (c) spinner flask; (d) NASA bioreactor; (e) micromolding microwells. Adapted with permission from ref. 50. Copyright 2013 American Chemical Society.

  MTT tetrazolium salt into an insoluble formazan product by the mitochondrial NADPH dehydrogenases Absorbance measurement at 570 nm 2-(2-Methoxy-4-nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2tetrazolium (WST-1) a,b Evaluation of intracellular metabolic activity 122 and 123 Reduction of the WST-1 into a water soluble formazan product by NADH dehydrogenase and plasma membrane electron transport Absorbance measurement at 450 nm 3-(4,5-Dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) a,b Evaluation of intracellular metabolic activity 65 and 88 Reduction of the MTS tetrazolium salt into an water soluble formazan product by the mitochondrial NADPH dehydrogenases Absorbance measurement at 490 nm AlamarBlue® a,b Evaluation of intracellular metabolic activity 110 and 146 Reduction of resazurin to resorufin Fluorescence measurement. Ex 540-570 nm/Em 580-610 nm

Fig. 4

 4 Fig. 4 (a) Structure of PEG-b-PAEMA-PAMAM/Pt and schematic illustration showing the self-assembly into the pH-sensitive cluster nanobombs (SCNs/Pt) at neutral pH and the disintegration into small particles at tumor acidic pH. (b) CLSM images showing in vitro penetration of fluorescently-labeled SCNs/Cy5 and ICNs/Cy5 in BxPC-3 multicellular spheroids. Scale bar: 100 μm. (c) In vivo real-time microdistribution of SCNs/Cy5 and ICNs/Cy5 in BxPC-3 xenografts after intravenous administration. Scale bar: 100 μm. Reproduced with permission from ref. 133. Copyright 2016 American Chemical Society.

Fig. 3

 3 Fig. 3 Tissue penetration and antitumor efficacy in 4T1 MCTS of Taxol, ∼70 nm Ptx nanocrystals (NPs), RGD-grafted ∼70 nm Ptx nanocrystals (iNPs), ∼10 nm Ptx nanocrystals (NDs) and RGD-grafted ∼10 nm Ptx nanocrystals (iNDs). (a) CLSM images after 24 h incubation with Cy5labeled Taxol/NDs/NPs/iNDs/iNPs, (b) CLSM images of TUNEL analysis after 24 h treatment with the different Ptx formulations; dead cells are shown in red and live cells appear in green. (c) Growth inhibitory effect of the different formulations. Reproduced with permission from ref. 56. Copyright 2015 Wiley-VCH.

Fig. 5

 5 Fig. 5 Uptake and penetration of shape specific particles in spheroids. Scanning Electron Microscopy (SEM) images of (a) 220 nm × 100 nm discs (H/D ∼ 0.45), (b) 325 nm × 100 nm discs (H/D ∼ 0.3), (c) 400 nm × 100 nm × 100 nm rods and (d) 800 nm × 100 nm × 100 nm rods. (e-h) Twophoton pictures of spheroids incubated with the discs or rods in the correspondent upper panel. (i) Normalized radial intensity distribution as a function of distance from the center of the spheroid. Reproduced with permission from ref. 85. Copyright 2015 Wiley-VCH.

Fig. 6

 6 Fig. 6 (a) Accumulation and permeation behaviors of G2, G4, and G7 PAMAM dendrimers in MCF-7 MCTS as a function of the surface charge. (b) Tumor penetration of amine-terminated G2, G4, and G7 PAMAM dendrimers. Reproduced with permission from ref. 72. Copyright 2016 American Chemical Society.

Fig. 7

 7 Fig. 7 (a) Absorbance values (mean ± standard error) corresponding to the DNA content in LNCaP spheroids treated with curcumin-loaded nanoparticles for 7 days. (Curcumin concentration: 30 µM.) ***p < 0.001. ****p < 0.0001. (b) LNCaP spheroids treated with free curcumin and curcuminloaded nanoparticles for 7 days. (Curcumin concentration: 150 µM.) Scale bars: 200 μm. Reproduced with permission from ref. 91. Copyright 2016 Royal Society of Chemistry.

Fig. 8

 8 Fig. 8 (a) Schematic representation of the synthesis of block copolymer and formation of UCM and CKM. (b) Schematic hypothesis of penetration differences for Doxo-loaded CKM and UCM. (c) Doxo delivery into pancreatic MCTS by CKM and UCM revealed by CLSM. (d) Inhibition of the pancreatic MCTS growth by Doxo-loaded micelles. Microphotographs of pancreatic spheroids before and after treatment with Doxo-loaded micelles, free drug (Doxo-h) or untreated (Cont). Reproduced with permission from ref. 90. Copyright 2015 Royal Society of Chemistry.

Fig. 9

 9 Fig. 9 CLSM images of HEK293T multicellular spheroids 22 h after transfection with (a) PME-(PEG 3.5k ) 1.69 ; (b) PME-(PEG 3.4k -FA 1 ) 1.66 , or (c) PME-(PEG 3.4k -FA 2 ) 1.72 complexes. (d) Relative fluorescence intensity (treated group/blank) of HEK293T multicellular spheroids treated with PME-(PEG 3.5k ) 1.69 , PME-(PEG 3.4k -FA 1 ) 1.66 , or PME-(PEG 3.4k -FA 2 ) 1.72 complexes for 4 h and for a further 18 h culture. Reproduced with permission from ref. 94. Copyright 2015 American Chemical Society.

Fig. 10

 10 Fig. 10 Structured illumination microscopy (confocal-like) images of the specific binding between chitosan-decorated doxorubicin-loaded nanoparticles (nDOXO) and mammosphere cells: (a) co-localization of nDOXO and CD44 receptors; (b) binding between nDOXO and free Doxo (fDOXO) with mammosphere cells. (c) Tumor volume as a function of time for four different treatments and image of tumors collected on day 80 after the initial drug administration. Reproduced with permission from ref. 122. Copyright 2015 American Chemical Society.

  Fig. 11 (a) CLSM images of SH-SY5Y MCTS incubated with free Doxo, Doxo-loaded CS-PAPBA NPs and Doxo-loaded iRGD-CS-PAPBA NPs for 2 h, 4 h and 8 h, respectively. (b) In vivo tumor growth curves of H22 tumor-bearing mice that received different treatments. Data are presented as the mean ± SD (n = 10). * represents P < 0.05 since the 7 th day and ** represents P < 0.01 since the 11 th day. Reproduced with permission from ref. 117. Copyright 2013 American Chemical Society.

Fig. 12 (

 12 Fig. 12 (a) Optical images of MDA-MB-231 MCTS treated with a fresh medium (control), naked pTRAIL ( pTRAIL), poly(ethylene imine)/pTRAIL complex (PEI/pTRAIL) and G4-F735/pTRAIL complex (G4-F735/pTRAIL) at different time points. (b) Time-elapsed evolution of tumor sizes in vivo. Reproduced with permission from ref. 92. Copyright 2016 Royal Society of Chemistry.

Fig. 13

 13 Fig. 13 3D images of MCF-7 spheroids after incubation with (a) free Doxo and (b) Doxo-loaded IONP@P(HBA)-b-P(OEGA) for 17 h. The representative confocal images (left) were taken every 5 μm section from the top to bottom in the middle of an intact spheroid, whereas the 3D image (right) was reconstructed using Imaris software. Reproduced with permission from ref. 81. Copyright 2013 American Chemical Society.

Fig. 14 (

 14 Fig. 14 (a) Schematic of the PDMS microfluidic device on a microscope stage. (b) Schematic (left) and image (right) of 40 nm fluorescent PEG-NPs administered for 1 h at 50 μL h -1 entering the spheroid and accumulating in the interstitial spaces (arrows). Scale bar: 100 μm. (c) Schematic (left) and image (right) of 110 nm fluorescent PEG-NPs administered for 1 h at 50 μL h -1 being excluded from the spheroid. Scale bar: 100 μm. (d) Representative images of tumor fluorescence from mice injected with NPs in the tail vein at 48 h post-injection. (e) Quantification of animal fluorescence at 2 and 48 h using whole animal images. Reproduced with permission from ref. 185. Copyright 2013 Nature Publishing Group.

Fig. 15 (

 15 Fig. 15 (a) Bright field image of the microfluidic channel. The black arrow shows the direction of medium flow. Scale bar: 300 μm. (b) Bright-field image of the MCTS in the microfluidic channel. (top) The MCTS in Matrigel prior to Doxo-HCl treatment. (bottom) Real-time, enlarged views of the MCTS during 24 h of treatment with Doxo-HCl. Scale bar: 50 μm. Reproduced with permission from ref. 187. Copyright 2013 American Chemical Society.

Fig. 1 .

 1 Fig. 1. Mono-type PANC-1 spheroid characterization. (a) Representative optical imaging of MCTS_#1 (seeding density of 500 cells per well) at days 4, 7, 13 and 17 post seeding. Scale bars: 200 mm. Evolution over time of (b) volume, (c) cell number and (d) ATP content per spheroid as function of the initial seeding density. Values represent mean ± s.e.m. *p < 0.05 **p < 0.005, ***p < 0.0005 ****p < 0.0001 by a two-way Anova method, with a Sidak's post-test at days 4, 7 and 17 post seeding, chosen as representative time points. Histological analysis of 5 mm sections of MCTS_#1 at day 4 post seeding: (e) haematoxylin eosin staining, (f) cytokeratin AE1/AE3 and (g) fibronectin immunostaining. Scale bars: 50 mm.

Fig. 2 .

 2 Fig. 2. Hetero-type PANC 1: MRC-5 spheroid characterization. (a) Representative optical imaging of MCTS_#2 (PANC-1:MRC-5 seeding ratio 1:9) at days 4, 7, 13 and 17 post seeding. Scale bars: 200 mm. Evolution over time of (b) volume, (c) cell number and (d) ATP content per spheroid as function of the initial seeding density (PANC-1:MRC-5 ratio 1:1, 1:2, 1:4 and 1:9). Values represent mean ± s.e.m. Histological analysis of 5 mm sections of MCTS_#2 at day 4 (e, f, g) and day 7 (h, i, l) post seeding: (e, h) haematoxylin eosin staining, (f, i) cytokeratin AE1/AE3 and (g, l) fibronectin immunostaining. Scale bars: 50 mm.

Fig. 3 .

 3 Fig. 3. Hetero-type PANC-1:MRC-5:HUVEC spheroid characterization. Histological analysis of 5 mm sections of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at day 4 post seeding: (a) haematoxylin eosin staining, (b) cytokeratin AE1/AE3, (c) fibronectin and (d) CD31 immunostaining. Scale bars: 50 mm.

Fig. 4 .

 4 Fig. 4. Hetero-type PANC-1:MRC-5:HUVEC spheroid characterization. Histological analysis of 5 mm sections of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at day 7 post seeding: (a) cytokeratin AE1/AE3, (b) fibronectin and (c) CD31 immunostaining. Scale bars: 50 mm.

Fig. 5 .

 5 Fig. 5. SPIM 3D topography of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at day 4 and day 7. (a, e) Overlay of blue (Hoechst 33342, nuclei), green (GFP-expressing MRC-5 fibroblasts) and red (RFP-expressing HUVECs) fluorescence; (b, f) single blue channel (Excitation/emission 405/440 nm) showing all cell nuclei; (c, g) single green channel (Excitation/emission 488/525 nm) showing GFP-expressing MRC-5 fibroblasts; (d, h) single red channel (Excitation/emission 561/605 nm) with RFP-expressing HUVEC cells. Scale bars: 100 mm.

Fig. 6 .

 6 Fig. 6. Hetero-type PANC-1:MRC-5:HUVEC spheroid characterization. (a) Representative optical imaging of MCTS_#3 (PANC-1:MRC-5:HUVEC, LbL, VEGF) at days 2, 3, 4 and 7 post seeding. Scale bars: 200 mm. Evolution over time of (b) volume, (c) cell number and (d) ATP content per spheroid in MCTS_#3. Values represent mean ± s.e.m. ****p < 0.0001 by a two-way Anova method, with a Sidak's post-test.

  technological development and demonstration under grant agreement no. 642028 H2020-MSCA-ITN-2014. The CNRS and the French Ministry of Research are also warmly acknowledged for financial support. Pr Christian Poüs and Mr Patrick Lêchene are gratefully acknowledged for the creation of the image-processing macro with Image-J Ò . Miss Meriem Garfa-Traoré and Mr Nicolas Goudin of the Cell Imaging Platform (SFR Neker, Paris France) are warmly acknowledged for the technical assistance and the fruitful discussions on the result interpretation during the imaging with SPIM. Dr Stéphanie Denis is gratefully acknowledged for the assistance in cell lines maintenance. Dr Delphine Courilleau, technical manager of the CIBLOT Plate-Forme (Institut Paris-Saclay d'Innovation Thérapeutique, Châtenay Malabry France), is warmly

Fig. 7 .

 7 Fig. 7. (a) PANC-1 cell viability (luciferase activity) in MCTS_#1 and MCTS_#3 following exposure to doxorubicin (0.5 mM) for 48 h and 72 h (n = 3). Spheroids have been constructed using luciferase-expressing PANC-1 cells. (b) Representative bioluminescence images of control and doxo-treated MCTS_#1 and MCTS_#3. Incubation time 72 h. (c) Spheroid viability (ATP quantification) following exposure to doxorubicin (0.5 mM) for 48 h and 72 h (n = 2). (d) Spheroid growth inhibition (volume measurement) following exposure to doxorubicin (0.5 mM) for 48 h and 72 h. Data represents mean ± s.e.m. ****p < 0.0001 by a two-way Anova method, with a Sidak's post-test.

Table 1 .

 1 Methods used for the evaluation of polymer-based nanomedicines on 3D MCTS

	Analyzed	Assay/Detection	Description/Principle
	Parameter	methods

Table 1 .

 1 Tested protocols for the optimization of the SU-8 photo-lithography process.

	s = speed
	a = acceleration
	t = time
	* = step repeated for each layer

Table 1

 1 Methods used for the evaluation of polymer-based nanomedicines in 3D MCTS

Table 1 (

 1 Contd.)

block copolymer,95 led to results in clear contrast to those previously published by the same group of Stenzel.90,124 Indeed, the micelles with the highest level of crosslinking were capable of the deepest penetration in LNCaP prostate multicellular tumor spheroids, but displayed the lowest cytotoxicity. This behavior could be explained by a too compact structure that hindered intracellular reductive agents (e.g., glutathione) to diffuse in the core of the micelles thus slowing down crosslinked micelles disassembly and release of the loaded drug.95 Compared to the 2D monolayer cultures, the 3D spheroid model would provide a reliable correlation between micelles penetration and cytotoxicity, thus allowing figuring out the real contribution of the crosslinking in terms of drug delivery efficiency.

obtained in vitro clearly highlighted a limit of the 3D models, which cannot fully reproduce the complexity of living organisms and the behavior of NPs after intravenous administration. Thus, although evident that 3D models would allow a more relevant preclinical screening of nanomedicines compared to 2D cultures, at present they cannot completely replace the in vivo experimentation.Another proposed approach consisted in the loading of matrigel-embedded preformed spheroids in the central channel of a microfluidic device, while continuous medium addition in the lateral channels recreated some blood flow conditions. This allowed to assess in dynamic conditions treatment responses to doxorubicin in free from and loaded into micelles (Fig.15).187

This journal is © The Royal Society of Chemistry 2017Polym. Chem.

G. Lazzari et al. / Acta Biomaterialia 78 (2018) 296-307

Acknowledgements 1 Acknowledgements Acknowledgements Acknowledgements Acknowledgements The authors acknowledge the financial support of the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska Curie grant agreement N o 642028, the CNRS and the French Ministry of Research. Acknowledgements The research leading these results has received funding from the European Union's Horizon 2020 research and innovation program under Marie Skłodowska Curie grant agreement no. 642028. The CNRS and the French Ministry of Research are also warmly acknowledged for financial support. Pr Christian Poüs and Mr Patrick Lêchene are gratefully acknowledged for the creation of the image-processing macro with Image-J ® . Miss Meriem Garfa-Traoré and Mr Nicolas Goudin of the Cell Imaging Platform (SFR Neker, Paris France) are warmly acknowledged for the technical assistance and the fruitful discussions on the result interpretation during the imaging with SPIM. Dr Stéphanie Denis is gratefully acknowledged for the assistance in cell lines maintenance. Dr Delphine Courilleau, technical manager of the CIBLOT Plate-Forme (Institut Paris-Saclay d'Innovation Thérapeutique, Châtenay Malabry France), is warmly acknowledged for the assistance with the bioluminescence measurements. Chapter I 104 Miss Maryline Favier and Miss Fabiola Ely-Marius from HistIM Plate-Forme (Institut Cochin, Paris France) are warmly acknowledged for their expertise in the histological analysis of spheroid samples. Pr Andras Dinnyes, Dr Julianna Kobolak and Dr Anita Fehér from BioTalentum Ltd (Gödöllő, Hungary) are gratefully acknowledged for the technical assistance in the lentiviral transduction of the cell lines. Acknowledgements The research leading these results has received funding from the European Union's Horizon 2020 research and innovation program under Marie Skłodowska Curie grant agreement no. 642028. The CNRS and the French Ministry of Research are also warmly acknowledged for financial support. Acknowledgements The research leading these results has received funding from the European Union's Horizon 2020 research and innovation program under Marie Skłodowska Curie grant agreement no. 642028. The CNRS and the French Ministry of Research are also warmly acknowledged for financial support. Miss Julie Lachaux of the C2N (Centre for Nanosciences and Nanotechnology) is warmly acknowledged for the technical assistance in photo-lithography.

Acknowledgements

The authors acknowledge financial support from the European Union's Horizon 2020 research and innovation programme under Marie Skłodowska Curie grant agreement no. 642028, the CNRS and the French Ministry of Research.

Notes and references

Acknowledgements

The research leading these results has received funding from the European Union's HORIZON 2020 Programme for research, acknowledged for the assistance with the bioluminescence measurements. Miss Maryline Favier and Miss Fabiola Ely-Marius from HistIM Plate-Forme (Institut Cochin, Paris France) are warmly acknowledged for their expertise in the histological analysis of spheroid samples. Pr Andras Dinnyes, Dr Julianna Kobolak and Dr Anita Fehér from BioTalentum Ltd (Gödöll} o, Hungary) are gratefully acknowledged for the technical assistance in the lentiviral transduction of the cell lines.

This PhD is part of the European project NABBA (Design and Development of advanced NAnomedicines to overcome Biological BArriers and to treat severe diseases). The research leading these results has received funding from the European Union's HORIZON 2020 Programme for research, technological development and demonstration under grant agreement N° 642028 H2020-MSCA-ITN-2014. Cette thèse s'inscrit dans le cadre du projet européen NABBA (Design and Development of advanced NAnomedicines to overcome Biological BArriers and to treat severe diseases). Les recherches qui ont abouti à ces résultats ont été financées par le programme HORIZON 2020 de l'Union Européenne pour la recherche, le développement technologique et la démonstration dans le cadre de la convention de subvention N ° 642028 H2020-MSCA-ITN-2014.

Abstract

The preclinical drug screening of pancreatic cancer treatments suffers from the absence of appropriate models capable to reproduce in vitro the heterogeneous tumor microenvironment and its stiff desmoplasia. Driven by this pressing need, we describe in this paper the conception and the characterization of a novel 3D tumor model, consisting of a triple coculture of pancreatic cancer cells (PANC-1), fibroblasts (MRC-5) and endothelial cells (HUVEC), which assembled to form a hetero-type multicellular tumor spheroid (MCTS). By histological analyses and Selective Plain Illumination Microscopy (SPIM) we have monitored the spatial distribution of each cell type and the evolution of the spheroid composition.

Results revealed the presence of a core rich in fibroblasts and fibronectin in which endothelial cells were homogeneously distributed. The integration of the three cell types enabled to reproduce in vitro with fidelity the influence of the surrounding environment on the sensitivity of cancer cells to chemotherapy. To our knowledge, this is the first time that a scaffold-free pancreatic cancer spheroid model combining both tumor and multiple stromal components has been designed. It holds the possibility to become an advantageous tool for a pertinent assessment of the efficacy of various therapeutic strategies.

Keywords

Multicellular tumor spheroids; pancreatic cancer; 3D models; tumor microenvironment; layerby-layer coating Chapter I

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

Multicellular

Chapter I

Abstract

We have recently constructed a multicellular spheroid model of pancreatic tumor based on a triple co-culture of cancer cells, fibroblasts and endothelial cells and characterized by the presence of fibronectin, an important component of the tumor extracellular matrix. By combining cancer cells and stromal components, this model recreates in vitro the threedimensional (3D) architecture of solid tumors. In this study, we have used these hetero-type spheroids as a tool to assess in vitro the penetration of doxorubicin (used as a model drug), both in free from or loaded into nanoparticles, through the whole tumor mass and we have investigated whether microscopy images, acquired by Confocal Laser Scanning Microscopy (CLSM) and Light Sheet Fluorescence Microscopy (LSFM) would provide reliable information on this process. Results clearly demonstrated that CLSM was not suitable to completely monitor the diffusion even of small molecules such as the doxorubicin. Indeed, it allowed to scan only a layer of 100 µm depth and no information on the deeper layers could be available as a consequence of a progressive loss of the fluorescence signal. On the contrary, a complete 3D tomography of the hetero-type MCTS, obtained by LSFM and multi-view image fusion, revealed that the fluorescent molecule was able to reach the core of spheroids as large as 1 mm of diameter. No doxorubicin-loaded nanoparticles (NPs), instead, were detected in the spheroids, corroborating the challenge of nanomedicine delivery through the biological barriers in part recreated in our 3D tumor model. Overall the combination of hetero-type MCTS and LSFM allowed to carry out a highly informative microscopic assessment and represents a suitable approach to precisely follow up the drug penetration in the tumor. Accordingly, it would provide a useful support in the preclinical investigation and optimization of nanoscale systems for drug delivery to solid tumors.

Conflict of interest

The authors declare no conflict of interest.

Chapter III

Combining 3D cell culture and microfluidic technologies in a chip.

*Corresponding authors

E-mail address: simona.mura@u-psud.fr; anne-marie.haghiri@c2n.upsaclay.fr

Abstract

In recent years, multicellular tumor spheroids (MCTS) have been proposed as relevant threedimensional (3D) models in the preclinical screening of different drug delivery systems. Still, the in vitro studies performed in plate wells, in which tested compounds are added to the spheroids under static conditions, does not allow to capture entirely the complexity of the in vivo drug-cell interaction. The combination of MCTS and microfluidic technologies would offer the possibility to face this issue and therefore enable the evaluation of free drugs and nanomedicines under more pertinent dynamic conditions.

Herein, we describe the design and construction of a microfluidic device tailor made to enable the combination of a hetero-type pancreatic MCTS, embedded in a fibroblast-containing matrix, with perfusable microvessels (i.e., MCTS-on-a-chip). The optimization of the photolithography protocol allowed the fabrication of a patterned mold on silicon wafer by using the SU-8 negative photoresist. A tumor-stroma chamber was integrated with a system of channels, each endowed with specific geometry (i.e., shape and sizing of microchannels and pillars) to achieve a rigorous spatial control of the different cellular components. The fabrication of the final device in poly(dimethylsiloxane) (PDMS) was achieved by soft-lithography (replica molding) and we succeeded in the construction of an intact system of microchannels in which no fluid leakage was observed. The next step will consist in the introduction of the cellular components. Once assembled and entirely characterized, this MCTS-on-a-chip will represent a useful tool for a fast and cost-effective investigation of different drug delivery systems under controlled flow. It would provide more predictive information compared to the traditional static in vitro two dimensional (2D) and 3D models.

Conclusions

The optimization of the photo-lithography process herein described represented the first step in the construction of the MCTS-on-a-chip which will integrate the 3D hetero-type spheroid and a perfusable microvascular network. We fabricated SU-8 molds on silicon wafer and the well-defined pattern was transferred in the PDMS stamp by soft-lithography (replica molding). The verified absence of hydrogel leakage should ensure the spatial control of the various cellular components, whose integration will be assessed in further experiments.

Once assembled and characterized, the MCTS-on-a-chip will enable to monitor the effect of the flow conditions on the transport of drugs and nanomedicines. Their penetration and accumulation in the tumor mass will be investigated with a high degree of relevance in a microenvironment that would closely mimic the in vivo conditions.

Conflict of interest

The authors declare no conflict of interest.

We have demonstrated that the hetero-type 3D tumor model represents a more realistic system compared to conventional 2D monocultures of cancer cells, but to further enhance the predictivity of the preclinical in vitro studies, more biomimetic features are required. With this aim, as discussed in Chapter III, we decided to combine the 3D culture methodologies with the microfluidic technology to design a microfluidic device to host both the hetero-type spheroid, embedded in a fibroblast-containing matrix, and perfusable microvessels (i.e.,

MCTS-on-a-chip).

The first step in the construction of this device has been the careful optimization of the photolithography protocol for the fabrication of a suitable poly(dimethylsiloxane) (PDMS) chip.

We focused on the creation of molds with well-defined geometrical features able to ensure the spatial control of the different cellular components. In the attempt to generate molds with high aspect/ratio structures (height between 50 µm and 250 µm), we have compared two different photoresists, the SU-8 3050 and the SU-8 2050, and we have demonstrated the higher coating performance of the latter, which allowed to create a regular pattern with uniform surfaces. Moreover, we have improved the quality of the developed design through the optimization of the UV exposition by acting on two key parameters: i) the UV wavelength and ii) the light pass between the optical mask and the surface of the SU-8 coated wafer. While the manufacturer recommended to use selectively the 365 nm wavelength to activate the crosslinking of the SU-8 photoresist, we have observed that a better pattern transfer was obtained by removing the filter and exposing the sample to all UV wavelengths, thus maximizing the laser intensity. In addition, we have placed the optical mask in strong vacuum contact with the SU-8 surface to reduce the distance between the illumination lens and the sample. This trick limited the light diffraction and the subsequent variations in the pattern design, thus increasing its final resolution.

The optical mask design has been another critical issue. The first patterns were created

Conflict of interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.actbio.2018.08.008.