Ahmed Pr

Idbaih

Mohammed Ahmed

Maite Verreault

Xavier Declaeves

Ahmed Idbaih
email: ahmed.idbaih@psl.aphp.fr

LIST OF FIGURES ABBREVIATIONS Role of Multi-Drug Resistance in Glioblastoma Chemoresistance: Focus on ABC Transporters

Keywords: Glioblastoma, ABC transporters, multidrug resistance, chemotherapies, Pglycoprotein, the clinical role of ABC proteins Glioblastoma, immune system, microenvironment, immune checkpoint proteins

Glioblastoma (GBM) is the most frequent and the most aggressive primary cancer of the brain in adults. Despite aggressive therapeutic interventions, the median overall survival is below 18 months after initial diagnosis. The current standard of care of newly diagnosed GBM includes concurrent administration of temozolomide (TMZ) and radiotherapy followed by adjuvant TMZ. Since 2005, TMZ remained the first-line chemotherapy in treating GBM patients with its ability to cross the blood-brain barrier.

However, after initial efficacy, GBM cells acquire resistance to TMZ and other chemotherapeutic agents via multiple mechanisms, including the expression of ATPbinding cassette (ABC) efflux proteins. These transporters are involved in normal physiological functions, i.e., physiological cholesterol transport and elimination of toxins, but also it plays a role in pathological conditions, i.e., chemotherapies drug resistance. In humans, each ABC protein has specific tissue's locations and specific functions. In this review, we highlight the role of ABC proteins superfamily members ABCB1, ABCC1 and ABCG2 in the resistance of GBM cells to chemotherapy.

List of Tables

 Table 1:
List of Figures

During the flow cytometry experimental design, I had the chance to work with a great collaborator Dr. Coralie Guerin, the head of the cytometry platform in Institute Curie. She provided us with enough support and efficient planning for our flow cytometry results.

I am grateful to everyone who supported me in the GLIOTEX team Nolwenn LEMAIRE, Emie QUISSAC. Thank you for your continuous help. Many thanks to my Ph.D. colleagues in the office: Yanis, Quentin, Isaias, Irma, and Alexa, for all the fun that we had and all the unforgettable moments that we had.

For the women who treated me and raised me to be a man, my Mum "Rabaa" no words express my deep love for you, may Allah bless you. My sisters Manal, Rana, Faten, Doaa, my brother Fady, my brother-in-law Muneeb, and all family members. Finally, I would like to express my gratitude to my family-in-law and the deepest gratitude for my wife "Sara" who supported me in every step during my thesis. You were with me in the darkest times and the happiest ones, and you are the angel behind all this work.

Resume

Le glioblastome (GBM) est le cancer cérébral primitif le plus fréquent chez l'adulte avec une incidence estimée entre 2 et 5 nouveaux cas par an et pour 100000 habitants en Amérique du Nord et en Europe. Le GBM représente plus de 50% des tumeurs cérébrales primitives malignes. Le pronostic des patients souffrant de GBM est globalement sombre.

Le traitement standard de première ligne chez les patients souffrant d'un GBM nouvellement diagnostiqué repose sur une résection neurochirurgicale aussi complète que possible suivie d'une radiochimiothérapie concomitante et d'une chimiothérapie adjuvante.

Malgré cette stratégie thérapeutique lourde, la majorité des GBM récidivent. Depuis 2015, le témozolomide est la chimiothérapie standard de première ligne. Au cours des dernières décennies, des efforts importants ont été menés pour développer de nouveaux traitements plus efficaces et mieux tolérés, notamment des thérapies moléculaires ciblées, des immunothérapies et des dispositifs médicaux.

Tout d'abord, dans le cadre de cette thèse nous avons synthétisé le rôle des membres de la superfamille des protéines ABC exprimées au sein de la barrière hématoencéphalique (BHE) et leur implication dans la résistance des GBM à la chimiothérapie.

Nous avons résumé les stratégies développées pour contourner la résistance à la chimiothérapie médiée par les transporteurs ABC dans le GBM. Ces stratégies correspondent soit à une inhibition partielle ou complète, chimique ou physique, des transporteurs ABC soit à un contournement des pompes d'efflux ABC : (i) des nanotransporteurs, (ii) des anticorps conjugués et (iii) des ultrasons. L'inhibition des transporteurs ABC empêche l'efflux des agents thérapeutiques des cellules endothéliales vers la circulation sanguine et augmente leur pénétration dans le cerveau sain et le cerveau tumoral. En plus des inhibitions chimique ou pharmacologique des protéines ABC, des approches physiques permettent également de contourner la BHE. En effet, récemment, il a été montré que les ultrasons étaient capables d'inhiber l'expression des transporteurs ABC. Les ultrasons peuvent réduire l'expression de la protéine ABCB1 dans les vaisseaux cérébraux sans affecter l'intégrité des autres protéines. Des études complémentaires sont nécessaires pour préciser le rôle des ultrasons dans le contournement de la BHE et dans l'augmentation de l'efficacité des chimiothérapies contre le GBM.

Ensuite nous avons étudié l'expression des protéines des checkpoints immunitaires CD80 et CD86 et leur valeur pronostique chez les patients présentant un GBM nouvellement diagnostiqué et traité selon le standard de soins. CD80 et CD86 sont exprimés dans les cellules tumorales des GBM mais également dans les cellules du microenvironnement. L'expression CD80 et CD86 semblent prédominer dans les cellules tumorales de GBM. Néanmoins des études complémentaires sont nécessaires pour apprécier précisément l'expression de CD80 et CD86 dans les différentes populations cellulaires composant le GBM (i.e., cellules tumorales et cellules du microenvironnement).

Une faible expression de CD80 et une faible expression de CD86 sont associées à un meilleur pronostic en termes de survie sans progression chez les patients souffrant d'un GBM nouvellement diagnostiqué. CD80 et CD86 sont des inhibiteurs des lymphocytes T.

Nous supposons que les GBM exprimant faiblement CD80 et CD86 limitent moins l'action anti-tumorale dans lymphocytes T cytotoxiques. Parallèlement, les GBM exprimant fortement CD80 et CD86 pourraient répondre de manière plus importante aux anticorps immunothérapeutiques anti-CTLA-4. Bien que nous ne soyons pas parvenu à démontrer, probablement en raison d'un manque de puissance statistique de notre cohorte d'entrainement, une valeur pronostique indépendante de l'expression de CD80 et CD86 chez les patients souffrant d'un GBM nouvellement diagnostiqué, une tendance est observée. Notre étude encourage à l'étude du microenvironnement tumoral à la recherche de biomarqueurs, pertinents, pronostiques et/ou prédictifs de la réponse aux traitements.

En effet, des biomarqueurs composites associant données cliniques, données tumorales et données du microenvironnement pourraient être très puissant pour guider l'évaluation pronostique et le traitement des patients souffrant de GBM.

Enfin, deux des obstacles à l'efficacité des traitements anti-tumoraux conventionnels contre le GBM est la BHE et le microenvironnement tumoral immunodéprimé. Notre thèse s'est focalisée sur le contournement de la BHE pour moduler le microenvironnement tumoral pour une meilleure efficacité thérapeutique.

L'ouverture, médiée par les ultrasons, de la BHE (OBMU) a été évaluée dans des modèles précliniques pour contourner la BHE et augmenter la pénétration intracérébrale de plusieurs types d'agents thérapeutiques. Les ultrasons pulsés de faible intensité peuvent être délivrés en direction du cerveau afin d'induire une cavitation de microbulles délivrées quant à elles par voie intraveineuse. Cette mise en cavitation des microbulles permet d'ouvrir les jonctions serrées localisées entre les cellules endothéliales de la BHE.

Plusieurs médicaments ont été testés en combinaison avec l'OBMU pour le traitement des GBM, notamment le témozolomide, la carmustine, l'irinotecan, le carboplatine, la doxorubicine et, les liposomes chargés de médicaments. Récemment, l'OBMU a été évaluée dans le cadre d'essais cliniques qui ont permis de confirmer sa sécurité et sa tolérance chez les patients et plus particulièrement les patients souffrant d'un GBM en récidive.

Les immunothérapies et notamment les inhibiteurs des checkpoints immunitaires (ICI) et les thérapies cellulaires ont révolutionné le traitement de plusieurs types de tumeurs solides via une stimulation de l'immunité anti-tumorale. Les premiers essais cliniques évaluant le nivolumab en monothérapie ou en combinaison avec l'ipilimumab ont été décevants avec une efficacité limitée et une certaine toxicité. L'essai clinique de phase III CheckMate-143 a comparé le nivolumab au bevacizumab. Malheureusement, le nivolumab ne s'est pas montré supérieur en termes d'efficacité par rapport au bevacizumab chez les patients souffrant de GBM. Plusieurs raisons peuvent expliquer la faible efficacité des ICI dans le GBM : (i) la faible charge mutationnelle des cellules de GBM, (ii) l'absence de biomarqueurs prédictifs de réponse au ICI guidant la prescription, (iii) la faible pénétration des ICI dans le parenchyme cérébral, (iv) la faible activation immunitaire périphérique dans les ganglions lymphatiques, (v) l'immunosuppression locale et, (vi) la faible pénétration des lymphocytes dans le cerveau normal et tumoral.

Notre travail a confirmé l'efficacité modeste des ICI chez des souris souffrant de GBM GL261 et Nfpp10. Nous avons donc étudié l'efficacité des ICI combinés à l'OBMU dans des modèles précliniques murins de GBM.

Les paramètres des ultrasons (i.e. la pression acoustique, les temps, la quantité de microbulles, la fréquence des ultrasons) influent la sécurité et l'efficacité de l'OBMU. La sécurité et l'efficacité de l'OBMU ont été évaluées chez des souris C57BL/6 greffées orthotopiquement avec des cellules de GBM GL261. L'OBMU a été optimisée pour cibler l'hémisphère cérébral droit dans lequel sont greffées les cellules tumorales GL261.

L'optimisation a porté sur les paramètres des ultrasons et la fréquence de l'ouverture de la BHE.

La concentration du nivolumab dans le cerveau est augmentée de 28 fois lorsqu'il est combiné à l'OBMU versus lorsqu'il est utilisé sans ouverture de la BHE. Dans l'autre sens, l'OBMU permet également le passage de molécules tumorales de la tumeur vers la circulation sanguine. En effet, une quantité significative d'ADN tumoral a été identifié dans le sang des souris souffrant de GBM et traitées par OBMU. De plus, nous avons montré que la BHE limite l'efficacité dans anticorps immunothérapeutiques anti-PD-L1 chez les souris souffrant de GBM GL261. De manière intéressante, l'efficacité des anti-PD-L1 est nettement augmentée quand ils sont combinés à une OBMU avec 76% de souris souffrant de GBM-GL261 survivantes à plus dans 100 jours versus seulement 26% (4/15) pour souris traitées par anti-PD-L1 seul. Enfin, nous avons montré une plus grande activation des cellules microgliales chez les souris traitées par OBMU versus les souris traitées par anti-PD-L1 seul.

Abbreviations

Full name Abbreviations

Chromosome The definition and the classification of primary brain tumors advanced over the last decades. The classification of primary brain tumors was initially based on histomorphological features with three major categories: (i) gliomas (including glioblastomas, GBM), (ii) meningiomas and, (iii) rare primary brain tumors (Figure 1).

The most common primary malignant brain tumors are diffuse gliomas. Diffuse gliomas account for almost 30% of all primary central nervous system (CNS) tumors and 80% of all malignant primary CNS tumors. The heterogeneity of diffuse gliomas was an obstacle to define specifically subcategories of diffuse gliomas with clinical relevance [START_REF] Finch | Advances in Research of Adult Gliomas[END_REF]. During the 20 th century, several researchers described a classification of diffuse gliomas. [START_REF] Kernohan | A simplified classification of the gliomas[END_REF] suggested that diffuse gliomas are originated from adult glial cells that mutate and acquire malignancy. In 1979, the World Health Organization (WHO) established a classification for diffuse glioma based on tumor cell phenotype (i.e., astrocytic, oligodendroglial or oligoastrocytic) and grade of malignancy (from II to IV). The grade of malignancy integrates tumor cell density, tumor cell differentiation, necrosis, mitosis and endotheliocapillary proliferation.

WHO classification was revised in 1993, 2000, 2007, and recently in 2016[START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF][START_REF] Stoyanov | On the Concepts and History of Glioblastoma Multiforme -Morphology, Genetics and Epigenetics[END_REF]. The novelty of the WHO 2016 classification was the implementation of molecular features for classification of diffuse gliomas [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF]. In addition to the phenotype and the grade of malignancy, molecular markers are now required for accurate and comprehensive diagnosis: (i) Isocitrate dehydrogenase (IDH) mutational status, (ii) chromosome arms 1p/19q status and, (iii) histone H3 mutational status.

H3K27M mutation NOS Grade IV Glioblastoma X X X Diffuse midline glioma X Grade III Astrocytoma X X X X Oligodendroglioma X Oligoastrocytoma X Grade II Astrocytoma X X X Oligodendroglioma X X

Glioblastoma IDH wildtype

Epidemiology

GBM incidence ranges from 2 to 5 cases per 100,000 people in North America and Europe, accounting for more than 50% of primary malignant CNS tumors cases. The number of new cases of GBM per year is estimated around 250,000 worldwide. GBM, the most common primary CNS malignancy, is characterized by high morbidity and mortality.

Since 2020, the cIMPACT-NOW consortium (the consortium to inform molecular and practical approaches to CNS tumor taxonomy) categorizes GBM as grade IV IDH wildtype CNS tumors [START_REF] Louis | cIMPACT-NOW update 6: new entity and diagnostic principle recommendations of the cIMPACT-Utrecht meeting on future CNS tumor classification and grading[END_REF].

Clinical presentation and diagnosis of GBM

The clinical presentation of GBM is variable, rapidly progressive, and can present up to a couple of months depending on the size of the tumor and location. The most common clinical presentation is headache that arises from an elevation of the intracranial pressure.

Other symptoms such as blurred vision, progressive focal neurological deficits, seizures, and/or cognitive disturbances are frequent [START_REF] Rajaratnam | Glioblastoma: Pathogenesis and Current Status of Chemotherapy and Other Novel Treatments[END_REF].

The The area colored in green corresponds to an area of vasogenic edema in which tumor cells are present [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF].

Standard treatment of GBM

I. In newly diagnosed GBM

The standard first-line treatment for newly diagnosed GBM includes major cancer treatment modalities [START_REF] Elham | Cancer and Treatment Modalities[END_REF]. As other solid tumors, a safe maximum surgical resection, radiotherapy, and chemotherapy are usually applied in the treatment of GBM.

Irradiation induces both direct and indirect effects on tumor growth. Direct effects of irradiations manifest as DNA damage. DNA damage can appear as cellular replication errors and mutations, leading to an episode of mitotic catastrophe and tumor cell's death.

Indirect effect of radiotherapy is linked to the host's antitumor immune responses.

Damage-associated molecular patterns from radiated cells alert antigen presenting cells and naïve T-lymphocytes. Furthermore, radiation induces blood brain barrier (BBB) disruption and allows drugs to reach CNS tumors [START_REF] Sia | Molecular Mechanisms of Radiation-Induced Cancer Cell Death: A Primer[END_REF]. Finally, chemotherapy aims to eradicate tumor cells with cytotoxic agents that target crucial stages of the cell cycle.

The standard first-line protocol was developed in 2005 [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF] for the treatment of newly diagnosed GBM, and it includes the following specific modalities:

• A maximum safe surgical resection is performed depending on the tumor location and the surrounding functional brain regions. In GBM, a total surgical resection is nearly impossible due to the invasive nature of GBM tumors.

• Radio-chemotherapy is then used to treat marginal cells surrounding the resection area and reach the invasive GBM cells. Fractional x-ray radiation therapy at 2 gray (Gy)

daily is applied five days a week for a total of 6 weeks (60 Gy). Radiotherapy is accompanied by concomitant chemotherapeutic alkylating agent, temozolomide

(TMZ) at a dose of 75 mg per square meter of body surface area (mg/m 2 ).

• Adjuvant chemotherapy with TMZ is then continued with six cycles at 150-200 mg/m 2 daily for five days per 28-day cycle.

In early 1980, TMZ has been synthesized as a novel derivative of imidazotetrazinones.

Mitozolomide, a prodrug with a higher antitumor activity than TMZ, was initially developed but clinical trials reported severe thrombocytopenia as an adverse drug reaction for mitozolomide limiting its usefulness in clinical practice. TMZ, a 3-methyl derivative of mitozolomide, showed a more acceptable toxicity profile with similar antitumor activity. TMZ is an alkylating agent with a small molecular weight (194.15 g/mol)

that readily passes the BBB. A high systemic bioavailability of TMZ was reported following an oral administration with about 20-30% drug concentration crossing the BBB to reach GBM cells [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF]. Overall, these therapeutic characters nominated TMZ for further clinical trials until was approved in 2005 [START_REF] Friedman | Temozolomide and Treatment of Malignant Glioma[END_REF], Stupp et al., 2005).

Recently, tumor treating fields (TTF) is a new method to eradicate cancer cells by using mild electrical field travelling through the skin and disrupting the ability of cancer cells to replicate. TTF disrupt cell division and induce GBM cell apoptosis and was shown to improve prognosis of newly diagnosed GBM patients in good clinical conditions after the surgical and the radio-chemotherapy concurrent steps [START_REF] Stupp | Effect of Tumor-Treating Fields Plus Maintenance Temozolomide vs Maintenance Temozolomide Alone on Survival in Patients With Glioblastoma: A Randomized Clinical Trial[END_REF].

II. At tumor recurrence

The majority of GBM will relapse regardless of the first line treatments used (described above). Surgical intervention can be considered as a second-line treatment plan when possible. In the same line, a limited number of patients are candidates for second irradiation. Few chemotherapies agents can be used in recurrent GBM cases, i.e., Lomustine with or without Bevacizumab (anti-vascular endothelial growth factor A -VEGF-A-therapeutic antibody) [START_REF] Wick | Lomustine and Bevacizumab in Progressive Glioblastoma[END_REF]) (Table 2). 110 mg/m2 orally every six weeks [START_REF] Jakobsen | Toxicity and efficacy of lomustine and bevacizumab in recurrent glioblastoma patients[END_REF] III. Innovative therapies TMZ remains today the standard first-line chemotherapeutic agent in GBM treatment [START_REF] Brat | cIMPACT-NOW update 5: recommended grading criteria and terminologies for IDH-mutant astrocytomas[END_REF]. For over five decades, significant efforts have been put into the development of new anti-cancer therapies for GBM including anti-neoplastic agents [START_REF] Atiq | Anti-neoplastic Potential of Flavonoids and Polysaccharide Phytochemicals in Glioblastoma[END_REF], molecular targeted drugs [START_REF] Touat | Glioblastoma targeted therapy: updated approaches from recent biological insights[END_REF], immunotherapeutic approaches [START_REF] Weenink | Immunotherapy in Glioblastoma: Current Shortcomings and Future Perspectives[END_REF], and medical devices [START_REF] Idbaih | Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed Ultrasound in Patients with Recurrent Glioblastoma[END_REF]. Indeed, multiple of these innovative therapies are currently under investigations in the setting of clinical trials (Table 3).

Prognostic factors in GBM patients

Patients with GBM have a dismal outcome with a median overall survival below 18 months with the current standard of care [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF]. In the past few years, several clinical and molecular biomarkers have been identified as reliable prognostic factors in GBM, as shown in (Table 4)

A standard evaluation of a patient's capacities to perform an ordinary activity is referred to as a Karnofsky Performance Status (KPS) score. The KPS score ranges from 0-100, where 100 is the highest capacity to perform ordinary activities. A KPS score of 70 (i.e., the patient can perform daily life activities at home but is unable to work) or above was associated with a better prognosis in GBM patients. The KPS score is therefore used as a tool to evaluate the eligibility and stratification of GBM patients in clinical trials.

It has been noted that age is also a significant predictor of GBM patient prognosis.

Indeed, Li et al. discovered that "≤ 70 years of age" was an independent beneficial factor in GBM patients [START_REF] Li | Prognostic factors influencing clinical outcomes of glioblastoma multiforme[END_REF][START_REF] Kudulaiti | A nomogram for individualized prediction of overall survival in patients with newly diagnosed glioblastoma: a real-world retrospective cohort study[END_REF].

Epigenetics can be defined as heritable gene expression changes that are not due to any alteration in the DNA sequence. These are reversible changes that can be induced and reversed through different environmental factors and are implicated in multiple diseases including cancer. The most widely studied epigenetic changes are DNA methylations, histone modification, and chromatin remodeling. Abnormal promoter sequence methylations are common in tumor cells and lead to gene stable transcriptional repression.

The most critical epigenetic change with clinical relevance in GBM affects O6-Methylguanine-DNA Methyltransferase (MGMT) expression. This enzyme is involved in DNA repair following the alkylation of guanine bases. In GBM, hypermethylation of the promoter of MGMT is frequently observed, leading to inhibition of the enzyme expression, preventing it from fulfilling its role as DNA damage protector. MGMT promoter methylation status is widely recognized as a predictive marker of response to alkylating agents such as TMZ and as independent prognostic factor regardless the treatment prescribed (Figure 3) [START_REF] Verhaak | Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1[END_REF]. However, this knowledge has led to minimal changes in how GBM patients are treated due to a lack of alternative therapy options and the variability in MGMT promoter methylation testing [START_REF] Malmström | Do we really know who has an MGMT methylated glioma? Results of an international survey regarding use of MGMT analyses for glioma[END_REF].

The identification of a mutation in the gene encoding IDH1 influenced prognosis of GBM patients. IDH1 and IDH2 belong to the IDH enzyme family located in the cytoplasm (IDH1) and mitochondria (IDH2). Both enzymes are involved in a certain number of cellular processes, including oxidative phosphorylation, glutamine metabolism, lipogenesis, and the redox status regulations. IDH2 mutations are rarely reported in GBM. The aberrant function of mutated IDH1 is the conversion of alpha-ketoglutarate to the novel oncometabolite 2-hydroxyglutarate which leads to genome-wide epigenetic changes in human gliomas. Tumors with mutated IDH1 and corresponding epigenetic changes demonstrated better prognosis than gliomas with wild-type IDH1 (Li et al., 2021b).

Overall, these observations highlight the importance of IDH1 mutations in the diagnosis, prognosis, and GBM treatment and have led to their inclusion in the latest WHO classification. Indeed, the WHO classification that previously relied on histopathological criteria to classify brain tumors was revised in 2016 to include vital molecular biomarkers such as IDH status. Efforts to further stratify IDH-wildtype tumors are continuously evolving [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF]. The cIMPACT-NOW consortium objectives is identifying new clinically relevant biomarkers to be incorporated to the upcoming WHO classification of primary CNS tumors [START_REF] Louis | cIMPACT-NOW update 6: new entity and diagnostic principle recommendations of the cIMPACT-Utrecht meeting on future CNS tumor classification and grading[END_REF].

Table 4: Prognostic factors in GBM patients

Marker Outcome

The extent of surgical resection Compared with sub-total resection, gross total resection (substantially) improves OS and PFS [START_REF] Smrdel | Glioblastoma in Patients over 70 Years of Age[END_REF]

Age

Prognosis in patients younger than 70 years of age is relatively favourable, with a significant portion of these patients living for more than two years.

KPS

A KPS > 70 is a marker for a better prognosis [START_REF] Lamborn | Prognostic factors for survival of patients with glioblastoma: recursive partitioning analysis[END_REF])

MGMT promoter status

Better OS and PFS in GBM treated with radio-and chemotherapy when MGMT promoter is methylated (Li et al., 2021b) Abbreviations: PFS: progression free survival; OS: overall survival Figure 3: MGMT promoter methylation as a prognostic factor in GBM [START_REF] Hegi | MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma[END_REF].

GBM cell biology and their microenvironment

GBM cells and heterogeneity

Tumor cells origin, and intratumor cell heterogeneity

Virchow made the first description of intratumoral heterogeneity at the beginning of the 19th century [START_REF] Balkwill | Inflammation and cancer: back to Virchow[END_REF]. Since then, technological advances have brought to light different cell populations with distinct molecular alterations within the same tumor bulk. The acquisition and accumulation of oncogenic molecular alterations in a normal cell will result in obtaining neoplastic characters. Three main models have been proposed to explain intratumoral heterogeneity:

• The clonal evolution model that was described by [START_REF] Nowell | The clonal evolution of tumor cell populations[END_REF]. He proposed that all tumor cells have originated from a single cell, and the acquisition of genetic and epigenetic alterations within a cell is responsible for the tumor progression. This model ignores the relevance of non-genetic variability and the potential functional interactions between the tumor cells and the tumor microenvironment (TME) [START_REF] Nowell | The clonal evolution of tumor cell populations[END_REF].

• Cancer stem cell (CSC) or GBM stem cell (GSC) model suggests a hierarchical organization whereby tumor growth is dependent on CSCs that are self-renewable and able to give differentiated tumor cells progenies. In GBM, cell identity responsible for carcinogenesis is not fully understood; however, accumulating evidence suggests that the origin could be an astrocyte, a glial progenitor, or a neural stem cell. Indeed, it was initially thought that GBM originates from astrocytes which differentiate into an immature progenitor state [START_REF] Nair | A cancer stem cell model as the point of origin of cancer-associated fibroblasts in tumor microenvironment[END_REF] however, there is controversy over this hypothesis. Recent studies suggest that transformed neural stem cells (NSC) into GSC is the origin of gliomagenesis. Since the 2000s, several teams have succeeded in isolating and cultivating GSC from cortical glial tumors, medulloblastoma and GBM.

This work made it possible to demonstrate the tumorigenic ability of GSC and to identify discriminating NSC-associated surface markers (e.g., CD133, A2B5, CD15, and CD44) or intracellular proteins (e.g., nestin and other the transcription factors SOX2, OLIG2, BMI1, and ASCL1) [START_REF] Lathia | Cancer stem cells in glioblastoma[END_REF].

• The third so-called "Big bang" model was proposed by [START_REF] Sottoriva | A Big Bang model of human colorectal tumor growth[END_REF]. This model suggests a hierarchical organization with the existence of different clonal subpopulations. In addition to the common mutations inherited from the original transformed/neoplastic cell, these clones are said to have shared and/or unique molecular alterations. These would appear silently and permissively during replications, accumulate, and be expressed at a given time, which varies according to the clones.

This model justifies several molecular subgroups, defined by Verhaak classification (Table 5) co-existing within the same tumor. Furthermore, it explains the variations in response to treatments observed within the same GBM due to heterogeneous subpopulation of clones with different genetic alterations and capacity to resist treatments [START_REF] Sun | Big Bang Tumor Growth and Clonal Evolution[END_REF].

Four signaling pathways are disrupted in GBM

Genetic alterations in GBM such as amplification, deletion, and/or mutation modulate the oncogenic pathways. Accumulation of such genetic alterations activates oncogenesis. The most extensively studied signaling pathways alterations in GBM are summarized in (Figure 4).

The Receptor Tyrosine Kinase (RTKs) are high affinity cell surface receptors for large number of hormones, growth factors and cytokines. RTK pathways is altered in 88% of GBMs, resulting in decreased apoptosis, and promoting invasion and proliferation of GBM cells. Mutations in RTKs activate a series of signaling pathways and cascades modulate gene and protein expression. In GBM, RTKs show molecular abnormalities, such as gene amplification in a wide spectrum of RTKs, such as EGFR (60%-70%), PDGFRA (12%-15%), and MET (5%). Furthermore, EGFRvIII variant is reported in 20% of GBM and is expressed heterogeneously across GBM cells part of EGFRvIII-positive tumors suggesting that its crucial contribution to gliomagenesis. It is possible that RTKs contribute to lead mitogenic cellular signaling pathways in GBM [START_REF] Crespo | Molecular and Genomic Alterations in Glioblastoma Multiforme[END_REF].

TP53 is a major protein involved in tumorigenesis. TP53 signaling is affected in 87% of cases and mainly leads to defects in processes controlling apoptosis, senescence, or cell cycle progression. In healthy cells, its activation will lead to senescence or apoptosis induction. TP53 loss of function (i.e., mutation or deletion) is reported in 35% of GBM cases. Amplification of MDM2 and MDM4, TP53 inhibitor proteins, are reported in 14% and 7% of GBMs, respectively [START_REF] Pedrote | Oncogenic Gain of Function in Glioblastoma Is Linked to Mutant p53 Amyloid Oligomers[END_REF].

The tumor suppressor retinoblastoma (pRB) signaling pathway is altered in 77% of GBMs. Loss of pRB expression is detected in 11% of GBM cases. In the G1 phase of the cell cycle, cyclin D/CDK4-dependent phosphorylation releases pRB, allowing cell cycle progression. In GBM, CDKN2A and CDKN2B are inactivated in 46.4 % cases, and CDK4 and CDK6 amplification have also been found in 13.4% of GBM showing that CDK4 and CDK6 contribute to GBM development [START_REF] Cao | CDK4/6 inhibition suppresses tumour growth and enhances the effect of temozolomide in glioma cells[END_REF].

Mutations in the TERT gene promoter are reported in 80% of GBMs, allowing activation of telomerase expression. The process of extending telomeres participates in immortalization of tumor cells. These mutations are mutually exclusive with mutations in the Alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler (ATRX) ATRX gene. Alternative lengthening of telomeres (ALT) phenotype is positively correlated with IDH1 mutant protein, ATRX protein loss, strong TP53 expression and absence of EGFR amplification. [START_REF] Chen | Malignant glioma: lessons from genomics, mouse models, and stem cells[END_REF]. ` 34

Tumor cell microenvironment

Niches and stem cells of GBM

GBM stem cells (GSC) are grouped together in specialized niches that provide signals that are essential for maintaining their phenotype and tumorigenic capacities. The discovery of these niches has made the study of GSCs more complex, both on their role within the tumor and on their regulation. Three niches have been described in GBM: (i) the perivascular niche, (ii) the hypoxic niche, and (iii) the invasive niche.

I. The perivascular niche

The perivascular niche is multi-cellular structure composed of neoplastic and nonneoplastic cells. The neoplastic cells are here tumor cells with varying degree of differentiation state, including GSC. One the other hand, the non-neoplastic cells include endothelial cells, pericytes, macrophages, neutrophils, myeloid-derived suppressor cells, reactive astrocytes, and infiltrating neural progenitor cells. Pericytes interact with tumor cells to promote their growth and to contribute to the blood tumor barrier (BTB), which has been described as leaky compared to the normal BBB. In contrast to normal brain micro-vessels, both astrocytes and pericytes coverage is incomplete in GBM vessels [START_REF] Hambardzumyan | Glioblastoma: Defining Tumor Niches[END_REF].

II. The hypoxic niche

In GBM, oxygen deprivation seems to be an essential regulator of GSC survival and the maintenance of their stemness. Therefore, hypoxic niches contribute to tumor progression via the stabilization of the factor hypoxia-inducible factor-1 alpha (HIF-1α), which allows the expression of angiogenesis, invasion, and survival promoting genes.

These niches are located near necrotic areas, where oxygenation is sufficient to allow cell survival. The onset of hypoxia is highly dependent on the altered state of the microvasculature, suggesting a relationship between the perivascular niches and the hypoxic niches. Instead of slowing down tumor development, impaired oxygen delivery act as a strong growth activator. Over the last years, accumulating evidence has pointed to hypoxia as a critical regulator of tumor cell survival, stemness, and immune surveillance in these niches.

III. The invasive niche

The invasive properties are thought to promote tumor cell aggressiveness; invading glioma cells make surgical resection incomplete and are partially responsible for tumor recurrence [START_REF] Hambardzumyan | Glioblastoma: Defining Tumor Niches[END_REF] [START_REF] Hambardzumyan | Glioblastoma: Defining Tumor Niches[END_REF]Bergers, 2015, Broekman et al., 2018).

Cellular components of TME

The CNS has long been considered an "immuno-privileged" organ due to the presence of the BBB limiting the exchanges between the brain and the blood vessels.

Leukocytes are the main cellular contributor in protection against tumors and infections.

Prior to their release to the blood stream, they are developed in hematopoietic organs such as the bone marrow or/and thymus. Leukocytes migrate to the specific infected tissues following an activation process. However, due to the BBB, their passage to the CNS is limited through few passage ways: (i) through the choroid plexus, (ii) across superficial leptomeningeal vessels into the subarachnoid space and (iii) through the perivascular space into the brain parenchyma [START_REF] Ratnam | Immunotherapy in CNS cancers: the role of immune cell trafficking[END_REF]. The passage of leukocytes to the brain parenchyma is partially restricted by the BBB. Indeed, endothelial cell (ECs) within the BBB and their tight junctions prevent the passage of cells to the brain. Furthermore, pericytes around the vascular structures of the parenchyma maintain and support the integrity of the BBB. In physiological conditions, leukocytes are not detected in the brain parenchyma. However, under pathological conditions i.e., GBM, the integrity of the BBB is compromised allowing lymphocytes to reach the GBM TME [START_REF] Weenink | Immunotherapy in Glioblastoma: Current Shortcomings and Future Perspectives[END_REF] Accumulating evidence have suggested that tumor development is not only due to the accumulation of intrinsic abnormalities but also to extrinsic signals from the TME. Indeed, the TME, which is defined as a cellular (i.e. blood vessels, immune cells and fibroblasts), molecular (i.e. intercellular signaling molecules, extracellular matrix -ECM-), and dynamic network surrounding tumor cells, plays a significant role in tumor biology [START_REF] Broekman | Multidimensional communication in the microenvirons of glioblastoma[END_REF]. The tumor and the TME are linked in a highly interactive manner (Figure 6). They influence each other through extracellular signals [START_REF] Huang | Current Immunotherapies for Glioblastoma Multiforme[END_REF].

I. Microglia and macrophages

Microglia, the brain-specific immune cells, ensure immunosurveillance of the brain parenchyma. Since the discovery of a cerebral lymphatic system [START_REF] De | Myeloid Cells in Glioblastoma Microenvironment[END_REF] and leukocyte extravasation to the GBM TME, microglia gained interest as an immune cellular component in the brain. Macrophages are the most abundant immune cells found amongst circulating leukocytes. Microglia corresponds to the resident macrophages in the CNS, while circulating macrophages originate from monocytes recruited from the blood.

Immunosuppression within GBM is characterized by enhancement of immunesuppressive cytokines and inhibition of T-lymphocytes proliferation. The presence of several immune-suppressive cytokines characterizes the GBM TME (e.g., Interleukins -IL-, IL-6 and IL-10, prostaglandin E2, IL-1, and transforming growth factor-beta -TGF-β). Each mediator affects the GBM immune TME in a specific matter. For example, TGF-β blocks the activation of T-lymphocytes, inhibits IL-2 production, and decreases NK-T lymphocytes activity. IL-2, a known immunosuppressive cytokine, is secreted mainly by macrophages and GBM cells within the TME. IL-2 enhances GBM cell growth and inhibits interferongamma (IFNγ) and tumor necrosis factor-alpha (TNF-). It is also associated with a downregulation of major histocompatibility complex (MHC) class II and enhancement of CD80/CD86 expression on the surface of infiltrating T-lymphocytes as well as on GBM cells [START_REF] Scheffel | Immunosuppression in Gliomas via PD-1/PD-L1 Axis and Adenosine Pathway[END_REF].

Figure 6: GBM microenvironment components. TME is defined as a cellular (blood vessels, immune cells, fibroblasts) and molecular (intercellular signalling molecules, extracellular matrix), and mechanical network surrounding tumor cells [START_REF] Broekman | Multidimensional communication in the microenvirons of glioblastoma[END_REF].

II. Antigen presentation and tumor-associated macrophages (TAM)

Antigens released from tumor cells are processed by antigen presenting cells (APC) on MHC class I and presented to cytotoxic T-lymphocytes. Microglia cells have been identified with the ability to present antigens to T-lymphocytes within the CNS. However, the downregulation of APCs within the TME decreases microglia's ability to exert this role.

In GBM, macrophages derived from monocyte precursors polarize into two distinct categories within the GBM TME. Exposure to INF-γ polarizes monocytes to M1 macrophages. The role of M1 macrophages are pro-inflammatory cytokines and chemokines secretion. Therefore, participate in the positive immune response and function as an immune monitor. On the other hand, M2 macrophages are involved in the anti-inflammatory cytokine's secretion therefore, reducing inflammation and contributing to immunosuppressive function and tumor growth [START_REF] Grégoire | Targeting Tumor Associated Macrophages to Overcome Conventional Treatment Resistance in Glioblastoma[END_REF].

M2 macrophages polarize through exposure to IL-4. TAMs are known to be capable of cross-presenting tumor antigens to T-lymphocytes and priming anti-tumor immune response (anti-inflammatory response). There is no definite answer about the importance of TAM in GBM antigen presentation. However, the presence of TAM is linked to GBM progression. Indeed, results from published articles reported that modulation of macrophage polarization has a regulatory effect on the GBM TME [START_REF] Saha | Macrophage Polarization Contributes to Glioblastoma Eradication by Combination Immunovirotherapy and Immune Checkpoint Blockade[END_REF]. The inhibitory effect of GBM TME rises from a regulatory link between M2 macrophages and tumor cells. Several factors such as colony-stimulating factor 1, TGF-1, macrophages inhibitory cytokines-1, and IL-10 can polarize TAMs to M2 phenotype and inhibiting their phagocytic capacity [START_REF] Grégoire | Targeting Tumor Associated Macrophages to Overcome Conventional Treatment Resistance in Glioblastoma[END_REF].

III. Regulatory T-lymphocytes

Glioma-associated neovascularization

Tumor vessels are structurally and functionally abnormal. The tumor vascularization is highly disorganized and shows several anomalies which are responsible for functional defects. These structural abnormalities include endothelial cell hyperplasia, a decrease in the number of pericytes in contact with endothelial cells, and tortuous vessel organization (Li et al., 2021a), all factors leading to increased vascular permeability. Glioma-associated neovascularization (GAN) is a complex and regulated process and is highly dependent on the balance between five separate pathways: (i) vascular co-option and (ii) angiogenesis, followed by (iii) vasculogenesis and (iv) vascular mimicry and finally (v) GBM-endothelial cell trans-differentiation.

Vascular co-option was reported for the first time in 1999 and was described as the first process involved in the organization of tumor cells around normal tissue vasculature. [START_REF] Holash | Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF[END_REF] was the first person to report vascular co-option in a rat model of glioma. Early tumors were well vascularized, and it took at least four weeks for an angiogenic response to be observed at the tumor's edge. [START_REF] Winkler | Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis[END_REF] discussed the invasive potential of glioma cells after being in close contact with the surrounding micro-vessels. Vascularization occurred via vascular co-option (Figure 7-A) but not angiogenesis. [START_REF] Hardee | Mechanisms of glioma-associated neovascularization[END_REF].

Angiogenesis, a step following the vascular co-option, is known as new vessels developing from pre-existing micro-vessels. Angiogenesis processes were described in 1976 when [START_REF] Brem | The role of vascular proliferation in the growth of brain tumors[END_REF] observed a high neovascularization process in GBM animal models. Hypoxic glioma cells around necrosis release proangiogenic factors, and other hypoxia independent mechanisms shift the angiogenic balance toward proangiogenic phenotype (Figure 7-B). GBM angiogenic phase is characterized by the formation of an irregular vascular network, with dilated and distorted arteries, abnormal branching, and shunts, contributing to abnormal perfusion. GBMs have immature vasculature with excessive leakage. GAN is driven by many key pathways identified (e.g., erythropoietin and their receptor, macrophages migration inhibitory factor, basic fibroblast growth factor, and placental growth factor) [START_REF] Xue | Aberrant glioblastoma neovascularization patterns and their correlation with DCE-MRI-derived parameters following temozolomide and bevacizumab treatment[END_REF] Vasculogenesis has been identified to include mobilization, differentiation, and recruitment of marrow-derived cells known as endothelial progenitor cells (Figure 7-C).

Similarly, to the angiogenic process, vasculogenesis is induced by both hypoxiadependent and independent mechanisms. The most well-known factors are the SDF-1 and CXCR4 pathways [START_REF] Sun | Mesenchymal stem cells promote glioma neovascularization in vivo by fusing with cancer stem cells[END_REF] Vascular mimicry characterizes tumor cells that organize themselves with ECM to mimic the structure of a vessel (Figure 7-D). Thus, the cells forming these structures do not express endothelial cell markers (CD31, CD34) but may show gene alterations specific to GBM cells e.g., EGFR amplification. This vascular mimicry appears to be connected to functional blood vessels and, although permeable, would increase nutrient delivery to the tumor. However, it is accepted that the neovessels formed exhibit altered structures and functionalities.

The final step takes place as tumor cells align themselves to form ECs lining the vascular channels. The endothelial transformation happens at both the functional and the molecular level, and is characterized by typical endothelial-specific biomarkers (Figure 7-E) [START_REF] Hardee | Mechanisms of glioma-associated neovascularization[END_REF].

The blood-brain barrier

The essential and complex organ which is the human brain is separated from the blood by the BBB. The BBB is a specificity of the CNS blood vessels. The BBB isolates the brain from the blood for protection purposes [START_REF] Kadry | A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity[END_REF]. Indeed, it prevents potentially toxic molecules circulating in the bloodstream to access brain cells, while ensuring the supply of nutrients to maintain homeostasis [START_REF] Al Rihani | Disease-Induced Modulation of Drug Transporters at the Blood-Brain Barrier Level[END_REF]. Highly specialized brain capillary ECs form an important part of the BBB. In addition to ECs, various cells described in (Figure 8) contribute to the biophysical structure of BBB.

To date, five mechanisms are known to regulate the exchanges of molecules from blood to brain and vice versa and described in detail in (Role of Multi-Drug Resistance in Glioblastoma Chemoresistance: Focus on ABC Transporters) BTB is referred to as a biological and physiological altered version of the BBB that with increased permeability to chemotherapies and/or other molecules. The structural changes in the GBM TME are responsible for the irregular, disorganized, large, and leaky micro-vessels that constitute the BTB. Despite the altered barrier functions of BTB within the tumor body, it is not completely open. Therefore, the penetration of chemotherapy is increased, but not to a level that is detected in non-cerebral tissues in a total absence of BBB. Furthermore, tumor cells present outside the zone of altered BTB i.e., in the surrounding brain tissue, are protected as described previously in (Figure 2) Figure 8: BBB is formed of different types of cells tightly knit together. Highly specialized ECs surround blood vessels and form part of the BBB. In addition to brain ECs, various cells contribute to the structure of BBB. Pericytes (represented in green) are attached to endothelium cells via gap junctions whilst astrocytes end feet (represented in purple) surround ECs of the BBB, providing structural and functional support to these cells. Adapted from [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF].

Several factors influence the ability of a chemotherapeutic agents to cross the BBB.

The physicochemical properties of the chemical agents highly influence the ability of compounds to cross the BBB. The main physicochemical properties are (i) size, (ii) liposolubility, (iii) electrical charge, (iv) interactions with plasma proteins, and (v) interactions with ABC efflux pumps. Based on these properties, compounds can be predicted to cross the BBB. In silico models have been developed to allow a prediction of the compounds that cross the BBB. However, none of these models allows accurate and consistent data with the in vivo models.

One of the well-accepted models to predict compounds' abilities to cross the BBB has been developed by analyzing 2500 compounds and called the rule of 5 (RO5). The RO5 states that compounds with molecular weight (MW) >500 Daltons, Log P >5, >10 hydrogen bond acceptors, five hydrogen bond donors, and compounds that are substrates for efflux pumps are predicted to have poor absorption and can hardly cross the BBB [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF]. A more recent tool was developed to evaluate the potential of molecules to cross the BBB with more precision than the RO5. The CNS Multiparameter Optimization Desirability (CNS-MPO) tool depends on six fundamental physicochemical properties: (i) lipophilicity, (ii) calculated distribution coefficient at pH 7.4, (iii) MW, (iv) topological polar surface area, (v) number of hydrogen-bond donors, and (vi) most basic center [START_REF] Wager | Central Nervous System Multiparameter Optimization Desirability: Application in Drug Discovery[END_REF].

Therapeutic strategies to modulate the tumor microenvironment

Modulation of the immune system

History and concept of immune system's modulation

The relationship between immune functions and cancer cells was reported for the first time by Rudolf Virchow 150 years ago. He observed the presence of leukocytes within tumor tissue. Therefore, he suggested that the leukocyte infiltrate reflected that cancer's origin lies in chronic inflammation [START_REF] Balkwill | Inflammation and cancer: back to Virchow[END_REF]. William Coley hypothesized the concept that our immune system can effectively recognize and eliminate cancer cells. He injected living or inactivated bacteria in the intra-tumor regions. The idea of Coley's toxins generated several discussions between researchers and scientists. His hypothesis that activated phagocytes would kill both living bacteria and adjunctive tumor cells was accepted at that time, following evidence that injection of bacteria in the intratumor region led to cancer shrinkage. Although the concept showed an innovative idea regarding cancer treatment, the responses were heterogeneous, and the success rate was not promising [START_REF] Carlson | Talkin' Toxins: From Coley's to Modern Cancer Immunotherapy[END_REF]. Cancer is characterized by alterations in molecular pathways and cellular processes. These alterations result in diverse neoantigens presented by MHC class I on tumor cells' surface. These complexes can be recognized by CD8 + Tlymphocytes in cancer patients.

Although cancer progression involves a variety of methods to overcome the host's immunity, immunotherapy can restore and even improve the patient's immune system.

Many immunotherapeutic approaches have already shown efficacy in patients, while other new therapeutic approaches remain under development. Immune checkpoint blocking antibodies (ICBs) are currently under clinical investigation [START_REF] Persico | Checkpoint Inhibitors as High-Grade Gliomas Treatment: State of the Art and Future Perspectives[END_REF]. To date, 1 st of April 2021, 4 042 clinical trials of immunotherapy in all types of cancer are listed on ClinicalTrials.gov.

One of the most attractive immunotherapy features is its ability to target cancer cells and thus spare healthy tissue. This characteristic differentiates immunotherapy from other "traditional" therapies such as radiation therapy and chemotherapy. The efficacy of immunotherapy was first demonstrated in the treatment of melanoma and renal cell carcinoma with high doses of IL-2 and is now spreading to other haematological and solid cancers [START_REF] Ventola | Cancer Immunotherapy, Part 1: Current Strategies and Agents. P &amp[END_REF].

Antitumor immune response

Our knowledge of fundamental cellular and molecular mechanisms of the immune system's innate and adaptive components has evolved. Cells from the innate system have receptors that can detect foreign microorganisms and dying cells. Macrophages and neutrophils provide early defense against microorganisms, while dendritic cells (DC) provide a linkage to the immune system's adaptive components. Immunological reactions against a growing tumor require an integrated response between the innate and adaptive immune responses. Based on our current knowledge of immune responses, several distinct steps must be completed, endogenously or therapeutically, to produce an effective antitumor response (Figure 9). Oncogenesis processes in tumor cells generate neoantigens that start the initial step in antitumor immune response when DCs detect such neoantigens. Additionally, pro-inflammatory molecules, together with chemokines released by the tumor cells themselves, will recruit innate immune cells to this local source of "danger" [START_REF] Pio | Complementing the Cancer-Immunity Cycle[END_REF]. Initiation of the antitumor response occurs when innate

Tumor escape mechanisms

The term "cancer immunosurveillance" was discredited in 2003. This process was initially thought to be a protective function of the host immune system at the onset of malignant transformation of cells. However, it has been recognized that both the innate and adaptive immune compartments protect the host from tumor progression and edit the immunogenicity of tumors that might form. Therefore, the term "cancer immunoediting" has been proposed to emphasize the immune system's dual role in preventing tumor progression. This process is made up of three phases, called the "3Es": elimination, equilibrium, and escape. Once normal cells have been transformed to tumor cells by combining oncogenic processes and loss of intrinsic tumor suppressor mechanisms, the immune system acts as an extrinsic tumor suppressor mechanism by eliminating tumor cells or by preventing their growth [START_REF] Pearson | Immune Escape in Glioblastoma Multiforme and the Adaptation of Immunotherapies for Treatment[END_REF] In the elimination step, formerly known as immunosurveillance, innate and acquired immunity cells recognize cancerous cells and eliminate them, leading to a return to physiological tissue. However, if antitumor immunity is not able to eliminate tumor cells acquired immunity cells can modulate tumor growth without being able to eradicate it (equilibrium phase). Additional mutations are estimated at this point allowing tumor cells to escape recognition, destruction, and control by the immune system, leading to clinically detectable tumors (escape). The elimination phase represents the original concept of cancer immunosurveillance. If this phase makes it possible to completely eradicate the developing tumor, no progression occurs to the following phases. For this reason, cancer is more common in older people in whom immune function, and thus cancer immunosurveillance, begins to decline [START_REF] Razavi | Immune Evasion Strategies of Glioblastoma[END_REF].

During the equilibrium phase, the host's immune system and tumor cells that survived the elimination phase enter a dynamic equilibrium state. Lymphocytes and the IFN-γ factor exert a relentless selection pressure on tumor cells which may be sufficient to contain but not fully eradicate tumor cells. Although many original tumor cells are destroyed, new variants arise. These variants carry different mutations that allow tumor cells to resist and escape immune response. Equilibrium is probably the longest of the three phases and can last for several years in humans. For some solid tumors, it is estimated that 20 years may separate the initial exposure to the carcinogen and the tumor clinical detection. During this period, the heterogeneity and genetic instability of cancer cells that survive the elimination phase are probably the main factors that allow cancer to resist the host's immune system [START_REF] Pearson | Immune Escape in Glioblastoma Multiforme and the Adaptation of Immunotherapies for Treatment[END_REF]. Escape from the immune system now represents one of the characteristics common to cancer cells. During the escape phase, tumor cells selected during the equilibrium phase can grow. This breach of the host's immune defenses likely occurs when genetic and epigenetic changes within tumor cells confer resistance to detection and elimination by the immune system. Tumor cells must employ multiple strategies to escape from the immune system innate and adaptive antitumor responses. Much work has focused on defining the molecular and cellular mechanisms of tumor escape. They operate at many levels and involve the tumor, the TME, and the innate and adaptive immunity components [START_REF] Razavi | Immune Evasion Strategies of Glioblastoma[END_REF]. Some of the most well-determined escape mechanisms were described within the TME section and are summarized in (Table 6).

Immunotherapy

The choice of the most appropriate immunotherapy depends on many factors, i.e., the treatment's objectives (curative or palliative), the patient's status, the type of cancer, the rate of tumor progression, and the efficacy versus the adverse effects. Adverse effect reactions associated with immunotherapy may be moderate to severe and localized to systemic effects. Some immunotherapies activate the body's immune system in general, while others specifically target distinct tumor antigens. Another important consideration in using immunotherapy is the possibility of inducing a long-lasting antitumor immune response through the immune memory. While this effect can be beneficial, it could lead to long-lasting toxicities. The primary role of immunotherapy is to re-activate the host's deficient immune system to initiate and maintain immune reactions against tumor cells.

The majority immune evasion mechanism used by TME represents therapeutic target points to restore immune control. Several immunotherapeutic approaches can be combined in some patients [START_REF] Carlson | Talkin' Toxins: From Coley's to Modern Cancer Immunotherapy[END_REF].

Monoclonal antibodies

Monoclonal antibodies (MAbs) approved to be a significant strategy used in the treatment of solid tumors and hematological malignancies. They have a unique specificity for a specific antigen which allow them to bind to epitopes on the surface of cancer cells or immune cells. Therapeutic Abs are related to the immunoglobulin G family and composed of fragments that bind to their antigen. Furthermore, they are known as "naked," as they are not conjugated with another active principle such as chemotherapy or radiotherapeutic agent. The primary mechanisms of action of the majority of naked mAbs are antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Other mechanisms are also reported, such as the direct triggering of cell death or the blocking of angiogenesis and cell survival signaling pathways. All-human MAbs show lower immunogenicity compared to murine, chimeric, or humanized mAbs.

Therapeutic MAbs with a non-human sequence is more easily identified as foreign subjects and induce host immune responses. Reduced efficacy was observed in nonhuman MAbs mainly due to increased clearance and more adverse reactions, such as injection site reactions. The use of naked MAbs has significantly improved the treatment of certain solid tumors [START_REF] Zahavi | Monoclonal Antibodies in Cancer Therapy[END_REF].

Monoclonal antibodies against checkpoint proteins

MAbs that block immune checkpoints represent up-and-coming treatments for various cancer types as they have remarkable and long-lasting responses in some patients.

Unlike chemotherapies, MAbs are well tolerated and provide long-term benefits on patient survival. A notable example is pembrolizumab's success, an anti-PD-1 antibody combined with surgery and radiation therapy have eradicated all melanoma traces in former President Jimmy Carter. The mechanism of action of ICBs was a breakthrough in the conception of cancer treatment and led for a Nobel Prize in Physiology [START_REF] Huang | Immune checkpoint inhibitors win the 2018 Nobel Prize[END_REF]. Chemotherapy and radiation therapy are directed to destroy cancer cells, while ICBs target the tumor-induced immunosuppression. These MAbs block checkpoint proteins on the surface of T-lymphocytes that are responsible for the immune response, resulting in prolonged antitumor responses [START_REF] Desland | The CNS and the Brain Tumor Microenvironment: Implications for Glioblastoma Immunotherapy[END_REF].

Immunomodulatory antibodies can prevent checkpoint ligand/receptor interactions.

They bind either to immune checkpoint proteins on T-lymphocytes, such as : (i) cytotoxic

T-lymphocyte-associated antigen-4 (CTLA-4) and its ligands CD80/CD86 or (ii) PD-1 and its ligands programmed cell death ligand 1 (PD-L1). These ICBs have demonstrated clinical efficacy, but many other ICBs have been identified and under developments (Figure 11).

Stimulation of the immune system with ICBs i.e., Ipilimumab, anti-CTLA-4, and atezolizumab, anti-PD-L1, showed promising effects alone or with other chemotherapies on treating multiple cancers. Ipilimumab was the first humanized anti-CTLA-4 approved by the American federal drug administration (FDA) to treat inoperable melanoma [START_REF] Tarhini | Tremelimumab: a review of development to date in solid tumors[END_REF].

Five years later, atezolizumab was the first humanized anti-PDL1 to be approved by the FDA to treat advanced or metastatic urothelial carcinoma [START_REF] Hsu | A Comprehensive Review of US FDA-Approved Immune Checkpoint Inhibitors in Urothelial Carcinoma[END_REF]. However, the combination of nivolumab and ipilimumab with GBM in clinical trials ended with immune-related severe adverse effects and avelumab monotherapy, anti-PD-L1 has a small effect on progression-free survival. In preclinical settings, ICBs efficacy was enhanced when antibodies were delivered to brain tumors. [START_REF] Guo | Deliver anti-PD-L1 into brain by p-hydroxybenzoic acid to enhance immunotherapeutic effect for glioblastoma[END_REF]. PD-L1 proteins are expressed as surface molecules by cancerous cells as GBM cells [START_REF] Hao | PD-L1 Expression in Glioblastoma, the Clinical and Prognostic Significance: A Systematic Literature Review and Meta-Analysis[END_REF] and provide a tumor escape mechanism when binds to PD-1 proteins at the surface of activated T-lymphocytes leading to T lymphocytes exhaustion [START_REF] Azoury | Immune Checkpoint Inhibitors for Cancer Therapy: Clinical Efficacy and Safety[END_REF].

On average, clinical data show that approximately 15-20% of patients respond to ICBs [START_REF] Azoury | Immune Checkpoint Inhibitors for Cancer Therapy: Clinical Efficacy and Safety[END_REF]. Currently, the responses observed with ICBs are more often partial responses at rates comparable to other targeted therapies or chemotherapies. ICBs have drastically reduced the tumor's size or even eliminated it in some patients allowing a surgical removal of the tumor. Besides, the duration of possible responses with ICBs can be extended to longer periods and patients sometimes considered completely cured. Anti-CTLA-4 stimulate circulating T-lymphocytes and their response may take months to activate enough T-lymphocytes to produce a favorable clinical outcome. In contrast, anti-PD-1/PD-L1 produces a faster response as these antibodies act on activated T lymphocytes which are directed against tumors [START_REF] Wei | Fundamental Mechanisms of Immune Checkpoint Blockade Therapy[END_REF].

I. Anti-CTLA-4

Anti-CTLA-4 was the first ICB to be tested in the clinic. This receptor is exclusively expressed on T-lymphocytes' surface, and its primary function is to regulate the amplitude of early activation of T-lymphocytes. B7-1 and B7-2 proteins bind to CD28 at the surface of T-lymphocytes and promote the activation of T-lymphocytes by amplifying T-cell receptors (TCR) signals resulting in co-activation signal. On the other hand, CTLA-4 has a higher affinity for B7-1/2 than CD28 and inhibits T lymphocytes activation by providing inhibitory signals and competing with CD28 for binding to B7-1/2 [START_REF] Rowshanravan | CTLA-4: a moving target in immunotherapy[END_REF].

The exact molecular signaling pathway amplified by CTLA-4 binding remains unclear, however, studies reported a kinase signals' disruption triggered by CD28 and TCR.

Furthermore, CTLA-4 appears to activate (i.e., src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) and protein phosphatase 2A), which oppose the phosphorylation cascade initiated by the activation of TCR and CD28, therefore, leading to the opposing of T-lymphocytes activations. Activation of CTLA-4 also increases the immunosuppressive action of Tregs while decreasing the production of IL-2 and the expression of its receptor. It is proposed that anti-CTLA-4 reduces the ability of Tregs to control the anticancer immune response and autoimmunity. Overall, the mechanism of action of anti-CTLA-4 involves both the elevation of T lymphocytes activity and the inhibition of Treg activity.

II. Anti PD-1 and anti PD-L1

The programmed cell death (PD-1) protein is a type 1 transmembrane receptor that was identified in 1992 [START_REF] Ishida | PD-1: Its Discovery, Involvement in Cancer Immunotherapy, and Beyond[END_REF]. A negative regulatory function for PD-1 was first proposed when mice deficient of PD-1 developed spontaneous autoimmune phenotype.

PD-1 receptor, present on the surface of activated CD8 + T lymphocytes. The primary role of PD-1 is to regulate peripheral T-lymphocytes activity and to prevent autoimmunity during an inflammatory response. PD-1 binding with its ligands PD-L1 and PD-L2 results in a decreased proliferation, apoptosis, and decreased cytokine production. Like CTLA-4, PD-1 is strongly expressed on Tregs' surface and induces their proliferation, therefore suppressing the functions of CD4 + and CD8 + T-lymphocytes.

The PD-1 pathway may also regulate T-lymphocytes activation to a state of immune tolerance in secondary lymphoid tissues during the early immune response. The expression of PD-1 is broader than that of CTLA-4 because it is not limited to Tlymphocytes and affects other activated cells such as B lymphocytes and NK lymphocytes, limiting their lytic activity. PD-1 regulates the activation of T-lymphocytes through the inhibition of kinases. When PD-1 binds to its ligand, the SHP2 phosphatase is inhibited. It can, therefore, no longer dephosphorylate the TCR signaling molecules. Unlike CTLA-4 ligands, PD-L1 and PD-L2 are overexpressed on cancer cells and in the TME. Therefore, the expression of PD-L1 is described in many types of cancer, including solid tumors. PD-L1 is also commonly expressed on myeloid cells of the TME. As a result, blocking PD-1/PD-L1 leads to a more active and prolonged antitumor immune response. Blocking PD-1 with ICBs induces and increases the activation, expansion, and the effector functions of Tlymphocytes. Furthermore, it may increase antitumor responses by reducing the number and the immunosuppressive activity of Tregs in the TME.

Overcoming, disrupting, or bypassing the BBB

The BBB limits drug penetration into normal and tumor tissue

It is estimated that 20% of small molecules crosses the BBB while none of the large therapeutic agents (i.e., antibodies) crosses it in physiological conditions. In neurooncology, many studies have measured the capacity of therapeutic compounds to cross the BBB. For examples, studies show that ~25% of TMZ reaches the brain following oral administration. Furthermore, nitrosoureas have a similar percentage of brain penetration (~25%). On the other hand, compounds such as etoposide and platinum derivatives are less likely to reach the brain [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF]. Therefore, several innovative strategies are continuously evolving to overcome the BBB by increasing drug delivery (i.e., systemic, or local administration of chemotherapy) or by increasing the drug penetration either by drug chemical modification or BBB modulation.

I. Intra-tumor injection

A local delivery of chemotherapy is possible through catheter insertion during MRI imaging to allow a proper insertion procedure. The main advantage of the cathetermediated local delivery is to reduce systemic toxicity of chemotherapies. Almost all chemotherapies can be delivered by this method. However, some adverse neurotoxic effects as seizure may be observed, therefore limiting the use of certain medications (i.e., taxanes and platinum derivatives). A clinical trial administering DTI-015 (BCNU in 100% ethanol) directly to patients with recurrent malignant gliomas showed that BCNU stabilized the tumor growth in 72% of patients. However, the reported three deaths in the clinical trial limited the success of this method [START_REF] Hassenbusch | Stereotactic injection of DTI-015 into recurrent malignant gliomas: phase I/II trial[END_REF].

II. Convection enhanced delivery

Edward Oldfield's group developed convection enhanced delivery (CED) in 1990. CED is based on the insertion of a catheter during neurosurgical procedure. The catheter is placed with a positively pressured pump that allow the drug to be delivered slowly over a specific time. Several antineoplastic agents were tested using CED (e.g., cisplatin, methotrexate, paclitaxel, nimustine, topotecan and carboplatin). The success of CED is highly dependent on several factors but mainly the drug itself and the tumor site became the major concern in this technique. A recent clinical trial reporting the efficacy of carboplatin delivered by CED showed that it reduced recurrent GBM tumor size in 58% of patients and enhanced their clinical conditions [START_REF] Barua | A novel implantable catheter system with transcutaneous port for intermittent convection-enhanced delivery of carboplatin for recurrent glioblastoma[END_REF].

III. In situ biodegradable wafers

Drug-loaded polymer wafers (e.g., Gliadel ® ) have been developed and used for the direct delivery of antineoplastic agents to brain tumors. One of the disadvantages of this method is an effect called the sink effect in which the concentration of the drug declines rapidly after their release from the polymer. A systematic review that analyzed all clinical trials on the efficacy of Gliadel ® wafers combined with systemic administration of TMZ suggested a positive additive effect on survival without increased toxicity in GBM patients [START_REF] Ashby | Gliadel wafer implantation combined with standard radiotherapy and concurrent followed by adjuvant temozolomide for treatment of newly diagnosed high-grade glioma: a systematic literature review[END_REF].

IV. BBB and efflux pumps modulations

Using partial or complete inhibitors of ABC transporters can be combined with their substrates to enhance their CNS penetration and anticancer activity. A variety of modulators were tested to suppress activity of efflux proteins, mainly (ABCB1 and ABCG2) and few were successful enough to reach clinical trials. In GBM, a limited number of clinical trials were initiated to modulate ABC proteins. To date, these trials failed to show significant clinical benefit, which could be related to a few reasons. Firstly, the study design was not optimal i.e., in the early clinical trials, the patients were not stratified based on high expression of ABC proteins, and a precise evaluation of the role of ABCB1 and ABCG2 transporters in patients could not be conducted. Secondly, modulators of ABC proteins could change the pharmacokinetics of other drugs reducing their anti-tumor properties.

Thirdly, the dose that was selected to inhibit the ABC proteins was not sufficient or a higher dose could not be applied safely in patients.

V.

Drug modification Drug modification strategies using nanocarriers (nanocapsules, liposomes, micelles, or dendrimers) were developed to allow drugs to enter the BBB via endocytosis, and/or to improve drug half-live and protection from clearance mechanisms [START_REF] Zhao | Nanocarrier-based drug combination therapy for glioblastoma[END_REF]. One clinical study investigated pegylated liposomal doxorubicin's efficacy when administered with TMZ and radiotherapy in newly diagnosed GBM patients. This study showed that the pegylated liposomal doxorubicin form is safe and tolerable. However, no meaningful efficacy was observed from the addition of liposomal doxorubicin to TMZ treatment (NCT00944801) [START_REF] Beier | RNOP-09: pegylated liposomal doxorubicine and prolonged temozolomide in addition to radiotherapy in newly diagnosed glioblastoma--a phase II study[END_REF]. Another study evaluated the safety and the pharmacokinetics of a liposomal form of irinotecan, the study confirmed the safety of this formulation. However, the efficacy is still under investigation [START_REF] Clarke | A phase I trial of intravenous liposomal irinotecan in patients with recurrent highgrade gliomas[END_REF].

Antibody-drug conjugates (ADCs) were also evaluated against a variety of targets in GBM and this approach is currently under investigation [START_REF] Gan | Antibody-drug conjugates in glioblastoma therapy: the right drugs to the right cells[END_REF]. ADCs are a newly developed biopharmaceutical compounds that allow the targeting of tumor cells while sparing healthy cells. This method is based on the use of an antibody to carry the substrate when binding to its ligand. In 2017, a published clinical trial showed a promising efficacy of depatuxizumab mafodotin (ABT-414), an ADC targeting wild-type or mutant forms of EGFR to selectively deliver a cytotoxic agent, in EGFR-amplified recurrent GBM patients with manageable adverse effects [START_REF] Reardon | Efficacy and safety results of ABT-414 in combination with radiation and temozolomide in newly diagnosed glioblastoma[END_REF].

VI. Ultrasound-mediated BBB opening (UMBO)

UMBO was used in pre-clinical models to bypass BBB and to increase brain penetration of a wide variety of therapeutics. Low-intensity pulsed ultrasound can be delivered to the brain to induce a safe oscillation of intravenously injected microbubbles within blood vessels [START_REF] Hynynen | Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits[END_REF]. Oscillation of these microbubbles opens the BBB by reversibly disrupting the tight junctions between ECs. A range of drugs has been tested for use with UMBO for treating gliomas and include temozolomide, carmustine, irinotecan, carboplatin, doxorubicin, and drug-loaded liposomes [START_REF] Beccaria | Ultrasound-induced opening of the blood-brain barrier to enhance temozolomide and irinotecan delivery: an experimental study in rabbits[END_REF], Goldwirt et al., 2016). UMBO has recently moved to clinical trials where its clinical safety was confirmed: Sonocloud-induced UMBO was found to be safe and tolerable among recurrent GBM patients [START_REF] Carpentier | Clinical trial of blood-brain barrier disruption by pulsed ultrasound[END_REF]. Two other phase 1 and phase 2 clinical trials are currently in progress to evaluate UMBO's efficacy in combination with carboplatin in patients with recurrent GBM (NCT03744026).

Role of ultrasound-mediated BBB opening

I. Ultrasound mediated BBB opening

In 2001, Hynynen laboratory developed a method of UMBO which has proven to be safe and effective. UMBO relies on a mechanism termed cavitation, where ultrasound propagate through the tissue and encounter micron-size bubbles (listed in Table 7),

making them expand at low acoustic pressure and contract at higher pressure. Indeed, UMBO is highly dependent on the emission of low intensity ultrasound waves that alter the conformation of microbubbles. The interaction between the microbubbles and the ultrasound waves results in a microbubbles expansion and contraction within the capillaries. The expansion of the microbubbles fills the capillary lumen resulting in a mechanical stretching on the micro-vessels wall. This results in the opening of the tight junctions. Furthermore, the microbubbles may decrease the blood flow, allowing a compensatory mechanism of BBB opening due to ischemia. It is believed that UMBO can enhance the main four known mechanisms that regulate the exchanges of molecules from blood to brain and vice versa. Transcytosis, trans-endothelial openings, opening of the tight junctions and the free passage of molecules through the permeable endothelium can be all be enhanced by UMBO and be responsible for the increased delivery of chemotherapies and molecules into the brain parenchyma (Figure 12).

The sub-harmonic (SH) acoustic emission leads to the oscillation regime of microbubbles called "stable cavitation", which is generally associated with a safe BBB disruption. On the other hand, broad-band (BB) acoustic emission is linked with the cavitation regime called "inertial cavitation", where the bubbles collapse. This type of cavitation is often associated with side-effects (Figure 14) [START_REF] Dauba | Recent Advances on Ultrasound Contrast Agents for Blood-Brain Barrier Opening with Focused Ultrasound[END_REF]. Therefore, this value should remain low during the UMBO protocol to maintain a safe and effective UMBO (Figure 12). Half-life (minutes) 2 1.9 1.3 The extent of UMBO is usually assessed through the injection of optical or fluorescent dyes that cannot cross the BBB (e.g. Evans blue dye is ~70 KDa when bound to albumin in vivo ) [START_REF] Yao | Evans Blue Dye: A Revisit of Its Applications in Biomedicine[END_REF]. When UMBO is applied, Evans's blue will cross the BBB and is detected in the brain parenchyma. Other methods include post-gadolinium MRI acquisition and two-photon live microscopy (Figure 13). Furthermore, in vivo imaging using two-photon microscopy in combination with a dye (dextran-conjugated 10 KDa) was used as a method to evaluate the passage of a dye following UMBO . Gadolinium does not cross the BBB in physiological conditions (500-900 Daltons). Following UMBO, post gadolinium MRI images show a significant enhancement of the contrast agents across the brain [START_REF] Idbaih | Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed Ultrasound in Patients with Recurrent Glioblastoma[END_REF].

Prior to clinical application, the safety of UMBO protocols must be established,

showing no adverse effects or tissue damages with a successful BBB opening (Figure 14) Type of ultrasound (focused or unfocused), pulse frequency, repetition and duration, the amplitude, the total exposure time, the microbubbles used (Table 7) and their dose must be optimized. These parameters will define the effectiveness and the safety of the opening.

Possible side-effects that must be avoided include vasogenic edema, extravasation of red blood cells leading to fatal hemorrhages or tissue damages.

UMBO and antibodies delivery to the brain

The first two studies that showed the possibilities to enhance antibodies delivery by focused ultrasound to the brain were published in 2006. Both studies were conducted in healthy rodents, and they aimed to prove the concept of antibody delivery using UMBO.

Anti-D4 receptor antibody was successfully detected using immunohistochemistry (IHC).

Furthermore, IHC detection of the delivered antibody was consistent with Evans blue passage following UMBO (Kinoshita et al., 2006a). In 2013, another published study reported a reduction in glioma tumor size following antibody loaded microbubbles and UMBO targeting VEGF [START_REF] Fan | Antiangiogenic-targeting drug-loaded microbubbles combined with focused ultrasound for glioma treatment[END_REF].

Interestingly, results were published in 2016 reporting that bevacizumab concentration was increased up to 57-fold with UMBO. UMBO at 0.8 MPa facilitates the passage of bevacizumab for up to 57-fold. However, this acoustic pressure is not safe. At a safer UMBO parameter, up to 5-fold bevacizumab concentration was reported [START_REF] Liu | Focused Ultrasound Enhances Central Nervous System Delivery of Bevacizumab for Malignant Glioma Treatment[END_REF]. A very recently published study reported enhancing delivery of high molecular weight dextran's (70 and 500 KDa) with UMBO at different acoustic pressure [START_REF] Valdez | Distribution and Diffusion of Macromolecule Delivery to the Brain via Focused Ultrasound using Magnetic Resonance and Multispectral Fluorescence Imaging[END_REF]. Recent articles that evaluated UMBO in combination with antibodies and large molecules are summarized in (Table 8). The SonoCloud-9 UMBO device, developed by CarThera ® , can be implanted after resection surgery, limiting the number of eligible patients. It can also be implanted during a dedicated surgery, but it can become a limitation compared with less invasive approaches. The SonoCloud ® approach is very well integrated with the standard oncology procedures, as it only requires five minutes of activation before or after the drug administration. The volume treated with this device is significant for large infiltrative GBM tumors as in the current clinical trial (NCT03744026).

Thesis Objectives

This thesis aims to evaluate the modulation of GBM TME, including the BBB and the immune system, to increase efficacy of anti-tumor therapeutic strategies in both GBM patients and preclinical mouse models. Therefore, the study objectives are divided into In this review, I used publicly available data obtained from PubMed and Google Scholar. Original published articles were selected using the keywords: ABC transporters, glioblastoma, temozolomide, and chemotherapy. A total of 36 selected articles was used for further reviewing. I described the most-commonly investigated ABC proteins members (ABCB1, ABCC1 and ABCG2) and their role in the resistance of GBM cells to chemotherapy.

The review article was submitted for publication and is listed in the results section.

B. Exploring the expression of immune checkpoint proteins, mainly CD80 and CD86, in

GBM TME and their role as a prognostic factor in newly diagnosed GBM patients GBM patients have a dismal outcome with a median OS of 15 months after initial diagnosis with known prognostic factors such as age, KPS and MGMT . Immunotherapies have dramatically improved the prognosis of multiple non-neurological cancers. In the setting of primary brain cancers, the results of clinical trials are still disappointing.

Nonetheless, specific GBM patients respond to these therapies, supporting the need for identifying biomarkers to guide the prescription of immunotherapies.

Checkpoint proteins such as the Cluster of Differentiation CD80 (also known as B7-1) and CD86 (known as B7-2) are expressed on the surface of tumor cells [START_REF] Ville | Co-Stimulatory Blockade of the CD28/CD80-86/CTLA-4 Balance in Transplantation: Impact on Memory T Cells[END_REF].

Furthermore, CD80 protein expression was observed in infiltrative tumor lymphocytes in melanoma [START_REF] Hersey | Expression of the co-stimulatory molecule B7 on melanoma cells[END_REF]. CTLA-4 and the Cluster of Differentiation CD28 are located on the surface T-lymphocytes. CTLA-4 has a higher affinity to CD80 and CD86, and when bound to these ligands, T-lymphocytes remain inactive and exhausted [START_REF] Rowshanravan | CTLA-4: a moving target in immunotherapy[END_REF]. Antibodies targeting CTLA-4 were used preclinical studies in multiple solid tumors, followed by several ongoing clinical trials [START_REF] Letendre | Ipilimumab: from preclinical development to future clinical perspectives in melanoma[END_REF]. Ipilimumab (anti-CTLA-4) has also shown responses in patients with brain metastases, highlighting efficacy within the CNS [START_REF] Amaral | Combined immunotherapy with nivolumab and ipilimumab with and without local therapy in patients with melanoma brain metastasis: a DeCOG* study in 380 patients[END_REF].

The expression of PD-L1 was inversely correlated with OS in GBM patients [START_REF] Nduom | PD-L1 expression and prognostic impact in glioblastoma[END_REF]. Here, I investigated whether the expression of CD80 and CD86 in GBM tissues could be used as a biomarker to predict the efficacy of ipilimumab among GBM patients.

I studied the RNA and protein expression of checkpoint proteins CD80 and CD86 in GBM patient samples and their possible correlation with clinical outcome in newly diagnosed GBM patients aged below 70 and with good performance status and treated with the standard of care.

C. Evaluating the role of UMBO and its potential to be combined with immune checkpoint blockers, i.e., anti-PD-L1, to overcome GBM resistance to ICB

The existence of BBB as a specificity of the CNS blood vessels prevents most systemic therapeutics from reaching the brain [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF]. In GBM, structural and functional changes of the BBB are frequent and lead to the generation of the BTB, allowing larger chemotherapies to reach the tumor. Although BTB enhances the delivery of some chemotherapeutic agents, large therapeutic antibodies have no chance to reach the brain at their therapeutic levels [START_REF] Veldhuijzen | Brain Access of Monoclonal Antibodies as Imaged and Quantified by <sup>89</sup>Zr-Antibody PET[END_REF] Several innovative strategies are known to enhance the delivery of chemotherapeutic agents and therapeutic antibodies [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF].

UMBO using low-intensity pulsed ultrasound (LIPU) is one of the safe and effective methods to enhance the delivery of chemotherapeutic agents in both animals and patients. In clinical trials, UMBO showed adequate safety and efficacy among recurrent GBM patients [START_REF] Idbaih | Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed Ultrasound in Patients with Recurrent Glioblastoma[END_REF]. LIPU is delivered to the brain simultaneously with an intravenous injection of micron-sized bubbles for a few minutes, allowing microbubbles oscillation. Microbubbles oscillation produces a mechanical stretching on the vessel walls that allow the opening of tight junctions (Chen et al., 2019a).

The choice of therapeutic agents to deliver after UMBO is crucial and remains a point of discussion among researchers and regulators. Stimulation of the immune system with anti-CTLA-4 and anti-PD-L1 showed promising effects in multiple cancers. However, the combination of anti-PD-1 and anti-PD-L1 in GBM clinical trials ended with severe immunerelated adverse effects. Therefore, there is a need to explore strategies to improve the efficacy of these immune modulators in GBM.

The third objective of my thesis is to study biological and therapeutic impacts of ICBs delivered with UMBO in GBM preclinical mouse models.

Results

•

Introduction

Glioblastoma (GBM) is the most common and the deadliest primary brain cancer in adults with a median overall below 18 months after initial diagnosis [START_REF] Ostrom | CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014[END_REF].

The current standard of care in newly diagnosed GBM patients, established in 2005, relies on concurrent administration of temozolomide (TMZ) and radiotherapy regimen followed by adjuvant TMZ alone [START_REF] Pace | European Association for Neuro-Oncology (EANO) guidelines for palliative care in adults with glioma[END_REF]. Despite remarkable efforts in the neurooncology field to develop new treatments, TMZ remains today the standard first-line chemotherapy in GBM patients 'treatment [START_REF] Ostrom | CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2010-2014[END_REF][START_REF] Pace | European Association for Neuro-Oncology (EANO) guidelines for palliative care in adults with glioma[END_REF]. TMZ is an alkylating agent with a small molecular weight (194.15 g/mol) that readily passes the blood-brain barrier (BBB) [START_REF] Information | Temozolomide [Online[END_REF].

The essential and complex organ, which is the human brain, is separated from the BBB's blood. The BBB is a specificity of the central nervous system (CNS) blood vessels.

The BBB isolates the brain from the blood for protection purposes [START_REF] Saunders | The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of history[END_REF].

Indeed, it prevents potentially toxic molecules circulating in the bloodstream to access brain cells while ensuring the supply of nutrients to maintain homeostasis [START_REF] Shen | ABC transporters and drug efflux at the blood-brain barrier[END_REF]. Highly specialized brain capillary endothelial cells (ECs) form an essential part of the BBB. In addition to ECs, various cells contribute to the biophysical structure of BBB described in (Figure 15). To date, five mechanisms are known to regulate the exchanges of molecules from blood to brain and vice versa. Passive diffusion of molecules through the BBB can occur paracellularly for very low molecular weight molecules (e.g., inorganic ions, water, gazes) and transcellularly for lipophilic compounds. An active transport can also occur either by : (i) transcytosis for some proteins (e.g., leptin, insulin, transferrin) or (ii) carrier-mediated proteins belonging to two major transporter superfamilies for small molecules. The solute carrier (SLC) superfamily contains more than 400 transporters that allow exchanges of small molecules through the BBB while ATPbinding cassette (ABC) transporters limit CNS penetration of small molecules by effluxing substrates from the brain ECs directly into the bloodstream (Figure 15) [START_REF] Saunders | The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of history[END_REF][START_REF] Chaves | Human ABC transporters at blood-CNS interfaces as determinants of CNS drug penetration[END_REF][START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF].

GBM cells acquire resistance to anticancer drugs via multiple mechanisms without being exhaustive: (i) acquisition of mutation in DNA repair genes, (ii) activation of alternative signaling pathways, (iii) immune escape, (iv) invasive switch from angiogenic growth and, (v) multidrug resistance mechanism (MDR) [START_REF] Gillet | Mechanisms of multidrug resistance in cancer[END_REF][START_REF] Ramirez | Glioblastoma multiforme therapy and mechanisms of resistance[END_REF][START_REF] Fomichov | Temozolomide rechallenge in recurrent glioblastoma: when is it useful?[END_REF]. MDR phenotype is observed in many types of cancers and induces : (i) increased efflux of drugs outside tumor cells and, (ii) reduced influx of drugs inside tumor cells [START_REF] Turk | Identification of compounds selectively killing multidrug-resistant cancer cells[END_REF]. ABC transporters are efflux pump proteins involved in MDR. To date, 49 members of ABC proteins have been identified to be involved in different biological mechanisms within the human body, and are classified in seven subfamilies; ABCA (12 proteins), ABCB (11 proteins), ABCC (13 proteins), ABCD (4 proteins), ABCE (1 protein), ABCF (3 proteins), and ABCG (5 proteins) (Drean et al., 2018b).

ABC transporters are expressed in various tissues such as the liver and the intestine and have a distinct role in absorption, distribution, and excretion of drugs. Some ABC transporters are predominantly expressed in ECs of the BBB [START_REF] Mahringer | ABC transporters at the blood-brain barrier[END_REF].

Indeed, ABCB1 and ABCG2 are expressed in ECs of the BBB, while others (ABCC, ABCG2) can be found in other cells such as astrocytes and neurons [START_REF] Linton | Structure and function of ABC transporters: the ATP switch provides flexible control[END_REF][START_REF] Shen | ABC transporters and drug efflux at the blood-brain barrier[END_REF][START_REF] Mahringer | ABC transporters at the blood-brain barrier[END_REF]. In humans, each ABC protein is expressed in specific locations and exhibit specific functions. i.e. ABCA subfamily members are expressed mainly in the CNS while ABCB subfamily members are mainly expressed in the BBB and liver [START_REF] Lockhart | Pharmacogenetics of ATP-binding cassette transporters in cancer and chemotherapy[END_REF]. ABC proteins were studied in several types of cancers [START_REF] Schinkel | Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview[END_REF]. In GBM, three proteins (ABCB1, ABCC1 and ABCG2) were extensively studied and were shown to impact GBM cells biology. In this review, we highlight the role of ABC protein family mainly (ABCB1, ABCC1 and ABCG2) in resistance of GBM cells to chemotherapy.

Structure and Functions of ABC Proteins

Structure of ABC Proteins

The BBB was described for the first time in the 20 th century when an intravenous injection of Evans blue significantly stained all tissue except the CNS while a direct intrathecal injection could stain only the brain tissue. This staining pattern highlighted the possibility of a barrier, termed the BBB, that prevents the dye to reach the brain tissue [START_REF] Saunders | The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of history[END_REF]. The BBB's integrity is preserved throughout life to maintain homeostasis and regulate the influx and efflux of nutrients/metabolites between the blood and the brain [START_REF] Pulgar | Transcytosis to Cross the Blood Brain Barrier, New Advancements and Challenges[END_REF]. ABC transporters, within the BBB, play a pivotal role in brain protection by eliminating harmful agents.

The ABC transporters' primary function is to actively transport their substrates, ranging from low molecular weight molecules to polypeptides, outside cells. Despite the large number of ABC transporters, they share structural similarities. In general, a typical ABC protein includes two functional units called transmembrane domain (TMD) and nucleotide-binding domains (NBD) (Figure 16). The NBDs are the ATPs binding units, and These helices determine the direction of transport of the ABC transporters i.e. importer or exporter [START_REF] Zolnerciks | Structure of ABC transporters[END_REF][START_REF] Linton | Structure and function of ABC transporters: the ATP switch provides flexible control[END_REF].

Functions of ABC proteins

Several physiological functions are reported for each ABC subfamilies. However, ABC proteins' main function is to actively transport cytotoxic xenobiotics and endobiotics against their concentration gradient [START_REF] Zolnerciks | Structure of ABC transporters[END_REF]. According to their location, they transport many substrates including anions, metal ions, peptides and lipophilic compounds [START_REF] Mahringer | ABC transporters at the blood-brain barrier[END_REF]. ABCA subfamily is mainly responsible for lipids and cholesterol transport while ABCB, ABCC and ABCG subfamily members are mainly associated with drug resistance and elimination of xenobiotics. Genetic variants in ABC proteins are linked to genetic disorders e.g. a pathogenic variant in the ABCD2 is responsible for 95% of cases of X-linked adrenoleukodystrophy (ALD) disorder. (Table 9)

summarizes the physiological functions of each ABC subfamilies. 17) [START_REF] Mahringer | ABC transporters at the blood-brain barrier[END_REF][START_REF] Vasiliou | Human ATP-binding cassette (ABC) transporter family[END_REF][START_REF] Tivnan | Inhibition of multidrug resistance protein 1 (MRP1) improves chemotherapy drug response in primary and recurrent glioblastoma multiforme[END_REF][START_REF] Linton | Structure and function of ABC transporters: the ATP switch provides flexible control[END_REF][START_REF] Zolnerciks | Structure of ABC transporters[END_REF].

Mechanisms of action of ABC transporters

ABC transporters in glioblastoma

Our review used public data obtained from PubMed and Google Scholar. Original published articles were obtained using the keywords (ABC transporters, glioblastoma, TMZ, and chemotherapy). 151 articles appeared in the results from the search engines.

Another step was carried out to exclude the duplicated and review articles. Following the removal of duplicated article and review articles, 91 abstracts were reviewed (abstract review), and only articles that studied ABC transporters in glioblastoma were selected. Full texts were obtained for all 36 selected articles using access from https://universiteparissud.focus.universite-paris-saclay.fr/ and https://insermbiblio.inist.fr/ using personal access. (Figure 18) summarizes the methodology used in the reviewing processes.

ABCB1 (MDR1, P-Glycoprotein)

ABCB1 which is also known as MDR1 and P-glycoprotein (P-GP) was identified by

Victor Ling in 1976 [START_REF] Juliano | A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants[END_REF]) making it the first studied protein among all ABC proteins. ABCB1 protein is a 170 KDa glycoprotein highly expressed in endocrine tissues, liver, gallbladder and brain, and it is usually co-expressed with the ABCG2 protein [START_REF] Atlas | [END_REF]. The physiological impact of ABCB1 protein was accidentally identified in 1994 by [START_REF] Schinkel | Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs[END_REF], who found that a homozygous knockout of ABCB1 in laboratory mice induced a 100-fold increase in susceptibility to antiparasitic medications [START_REF] Borst | P-glycoprotein ABCB1: a major player in drug handling by mammals[END_REF].

In humans, ABCB1 protein is encoded by the ABCB1 gene. An update was published in 2011 to illustrate the role of ABCB1 genetic polymorphisms, which accounts for more than 65 exon related single-nucleotide polymorphisms (SNPs) [START_REF] Wolf | An update on ABCB1 pharmacogenetics: insights from a 3D model into the location and evolutionary conservation of residues corresponding to SNPs associated with drug pharmacokinetics[END_REF]. These SNPs could be responsible for the differences in drug response and toxicity in several types of cancers [START_REF] Lockhart | Pharmacogenetics of ATP-binding cassette transporters in cancer and chemotherapy[END_REF]. In brain, ABCB1 is localized in the luminal membrane of ECs of the BBB [START_REF] Mahringer | ABC transporters at the blood-brain barrier[END_REF]. It has an essential role protecting the brain from a possible brain uptake of toxic molecules or metabolic substances with a wide range of known substrates including TMZ (de Gooijer et al., 2018b).

Schaich et al., investigated the role of three different SNPs of ABCB1 in GBM patients treated with TMZ. He showed that the rs1128503 SNP in MDR1 exon 12 is an independent predictive biomarker of response to TMZ. Patients with GBM exhibiting the homozygous allele (C/C allele) have better survival compared to their heterozygous variant counterparts [START_REF] Schaich | A MDR1 (ABCB1) gene single nucleotide polymorphism predicts outcome of temozolomide treatment in glioblastoma patients[END_REF]. However, more recently, another large clinical cohort analyzed the clinical impact of four SNPs variants (rs2229109, rs1128503, rs2032582 and rs1045642) in patients with newly diagnosed GBM patients treated with the standard of care. They did not find any clinical value of the SNPs investigated in a large Swedish cohort, hence could not validate the results obtained from Schaich study [START_REF] Malmstrom | ABCB1 single-nucleotide variants and survival in patients with glioblastoma treated with radiotherapy concomitant with temozolomide[END_REF]. One pilot clinical trial tried to evaluate ABCB1 protein among glioma patients. They measured the uptake of ( 11 C) N-desmethyl-loperamide (( 11 C)dLop) using positron emission tomography (PET) imaging as a marker of ABCB1 activity. The clinical study aimed to recruit ten patients, however, only two registered patients are available in the clinical trial database, suggesting that early termination of the trial was due to the lack of patients that fits the inclusion criteria of the study.

Several studies have investigated the role of ABCB1 in the context of TMZ treatment in GBM preclinical models. ABCB1 downregulation was associated with increased efficacy of TMZ in U87 cell lines [START_REF] Zhang | EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of Pglycoprotein inhibition[END_REF], Munoz et al., 2015b). Two recent studies showed that that downregulation of ABCB1 also increases efficacy of TMZ in vitro and in vivo in GBM preclinical models [START_REF] Tso | Bone morphogenetic protein 7 sensitizes O6-methylguanine methyltransferase expressing-glioblastoma stem cells to clinically relevant dose of temozolomide[END_REF]Chen, 2018). Furthermore, an in vivo study reported a higher concentration of irinotecan in the brain of Mdr1a (-/-) mice versus wild-type when both exposed to the same dose of irinotecan [START_REF] Goldwirt | Irinotecan and temozolomide brain distribution: a focus on ABCB1[END_REF].

The antitumor efficacy of TMZ against three intracranial tumor GBM models was significantly enhanced when Abcb1a/b and Abcg2 were genetically deficient or pharmacologically inhibited (de Gooijer et al., 2018b, de Gooijer et al., 2018a). ABCB1

expression can be altered by several compounds including carbonic anhydrase XII (CAXII), Bone morphogenetic protein 7 (BMP7) and TMZ [START_REF] Riganti | Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/beta-catenin pathway[END_REF][START_REF] Salaroglio | Carbonic Anhydrase XII Inhibitors Overcome P-Glycoprotein-Mediated Resistance to Temozolomide in Glioblastoma[END_REF][START_REF] Tso | Bone morphogenetic protein 7 sensitizes O6-methylguanine methyltransferase expressing-glioblastoma stem cells to clinically relevant dose of temozolomide[END_REF]. Tso et al found that BMP7 sensitizes GBM stem cells to clinically relevant doses of TMZ [START_REF] Tso | Bone morphogenetic protein 7 sensitizes O6-methylguanine methyltransferase expressing-glioblastoma stem cells to clinically relevant dose of temozolomide[END_REF] while Riganti and Salaroglio have found that GBM exposure to TMZ downregulates the expression of ABCB1 [START_REF] Riganti | Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/beta-catenin pathway[END_REF]. A recent study showed that CAXII could also reduce ABCB1 activity and sensitize GBM cells to TMZ [START_REF] Salaroglio | Carbonic Anhydrase XII Inhibitors Overcome P-Glycoprotein-Mediated Resistance to Temozolomide in Glioblastoma[END_REF]. (Table 9) summarizes the xenobiotic that alters the function of ABCB1, ABCG2 and ABCC1 transporters in GBM.

ABCC1 (MRP1)

The multidrug resistance-associated protein 1 (MRP1) is encoded by the ABCC1 gene. It was described for the first time by [START_REF] Cole | Overexpression of a transporter gene in a multidrugresistant human lung cancer cell line[END_REF]. ABCC1 protein is a 180-190 KDa protein and is ubiquitously expressed in many tissues in humans. It is highly expressed in intestine, kidney and testis, while a lower expression is detected in the lung, colon and brain [START_REF] Uhlen | Proteomics. Tissue-based map of the human proteome[END_REF]. ABCC1 protein has a wide range of substrates including anticancer drugs tested in GBM cell lines. i.e., vinca alkaloids (vincristine and vinblastine)

and topoisomerase inhibitors (mitoxantrone) [START_REF] Yin | Multidrug resistance-associated protein 1 (MRP1/ABCC1) polymorphism: from discovery to clinical application[END_REF]Zhang, 2011, Peignan et al., 2011).

Many genetic alterations were detected in ABCC1 gene, and most of them are SNPs in non-coding sequences and introns. A complete list of all ABCC1 SNPs can be obtained from available public database accessible from the national center for biotechnology information.

In the literature, ABCC1 inhibitors including KIAP -an anti-apoptotic protein-reduce ABCC1 activity and sensitize U251 GBM cell line to TMZ (Liu et al., 2015b). Two in vitro studies found that ABCC1 inhibition sensitizes cells to vincristine and etoposide but not to TMZ [START_REF] Peignan | Combined use of anticancer drugs and an inhibitor of multiple drug resistance-associated protein-1 increases sensitivity and decreases survival of glioblastoma multiforme cells in vitro[END_REF]. Furthermore, a study showed that MK571, an inhibitor of ABCC1 and ABCC4, increased the anti-tumor efficacy of vincristine and etoposide in primary GBM cell lines [START_REF] Tivnan | Inhibition of multidrug resistance protein 1 (MRP1) improves chemotherapy drug response in primary and recurrent glioblastoma multiforme[END_REF]. On the other hand, the overexpression of both ABCB1 and ABCG1 in GBM cell lines (U87 and U251) is associated with resistance to TMZ [START_REF] Liang | Overexpression of ILK promotes temozolomide resistance in glioma cells[END_REF].

ABCG2, Breast Cancer Resistance Protein (BCRP)

The ABCG2 protein was the first MRP-associated protein to be discovered. This 72 KDa protein was first identified in 1998 after being cloned from a human breast cancer cell line, which led to its alias, BCRP [START_REF] Doyle | A multidrug resistance transporter from human MCF-7 breast cancer cells[END_REF]. It is highly expressed in the small intestine, colon, rectum, placenta, and smooth muscles in humans while a lower expression is detected in adrenal and thyroid glands, lung and cerebral cortex (Atlas). In isolated brain microvessels and cortex biopsies from 12 patients with epilepsy or glioma, the expression of ABCG2 protein was 1.6 folds the expression of ABCB1 [START_REF] Shawahna | Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels[END_REF]. ABCG2

protein is a ABC half transporter, therefore it requires the dimerization of two NBDs to function as a drug efflux pump [START_REF] Mao | Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update[END_REF]. Many SNPs were identified in the ABCG2 gene. The frequency of SNPs in ABCG2 gene is highly variable among ethnic groups, potentially associated with heterogeneous drug responses among these groups [START_REF] Hira | BCRP/ABCG2 and high-alert medications: Biochemical, pharmacokinetic, pharmacogenetic, and clinical implications[END_REF] In 2017, a study enrolling 50 caucasian GBM patients found a correlation between expression of 8 different proteins (ABCG2, XIAP, MGMT, MSH2, pATM, pTP53, pAKT, Nestin) including ABCG2 and they reported a correlation between ABCG2 and the poor prognosis among GBM patients treated with the TMZ [START_REF] Emery | Expression and function of ABCG2 and XIAP in glioblastomas[END_REF]. To study the role of ABCG2 protein in vitro, they used GBM stem cells (GSC) and noticed an enhanced efficacy of TMZ following the inhibition of ABCG2. Therefore, they considered ABCG2 a promising therapeutic target in GBMs [START_REF] Emery | Expression and function of ABCG2 and XIAP in glioblastomas[END_REF]. However, another study reported that ABCG2 knockdown results in the upregulation of other drug transporters (ABCB1 and ABCC3) when treated with TMZ [START_REF] Chua | Characterization of a side population of astrocytoma cells in response to temozolomide[END_REF], suggesting that there might compensate mechanisms between transporters.

Several studies reported the importance of ABCG2 in drug response in GBM. An in vivo study reported that ABCG2 knockout in mice is associated with a better overall survival compared to wild-type mice when treated with dasatinib, a Src inhibitor [START_REF] Agarwal | Active efflux of Dasatinib from the brain limits efficacy against murine glioblastoma: broad implications for the clinical use of molecularly targeted agents[END_REF]. Another study showed that melatonin enhanced ABCG2 promoter methylation and sensitized GBM cell lines to mitoxantrone, doxorubicin and TMZ [START_REF] Martin | Melatonin-induced methylation of the ABCG2/BCRP promoter as a novel mechanism to overcome multidrug resistance in brain tumour stem cells[END_REF]. Consistently with this study, the overexpression of ABCG2 in human GBM cell lines is found to be associated with mitoxantrone resistance [START_REF] Rao | Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells[END_REF]. It was also reported that dual knockout of ABCB1 and ABCG2 improves efficacy of TMZ therapy in spontaneous GBM mouse models [START_REF] Lin | ABCB1, ABCG2, and PTEN determine the response of glioblastoma to temozolomide and ABT-888 therapy[END_REF]. Finally, TMZ exposure, in U87 and T89G cells, was found to increase ABCB1 and ABCG2 mRNA expression. Therefore, exposure to TMZ itself could modulate the levels of ABC proteins and could induce TMZ resistance among patients [START_REF] Munoz | Temozolomide induces the production of epidermal growth factor to regulate MDR1 expression in glioblastoma cells[END_REF].

Clinical Value

To date, several strategies are developed to overcome the ABC transporters mediated MDR. These strategies are summarized in (Figure 19) and they vary from partial/complete inhibition to bypass approaches: (i) nanocarriers technologies, (ii)

antibody-drug conjugates -ADC-, and (iii) ultrasound-mediated BBB opening (UMBO).

Using partial or complete inhibitors of ABC transporters can be combined with their substrates to enhance their CNS penetration and anticancer activity. Variety of modulators were tested to suppress activity of efflux proteins mainly (ABCB1 and ABCG2) and few were successful enough to reach clinical trials. In GBM, a limited number of clinical trials were initiated to modulate ABC proteins. To date, these trials failed to show significant clinical benefit, which could be related to a few reasons. Firstly, the study design was not optimal i.e., in the early clinical trials, the patients were not stratified based on high expression of ABC proteins, and a precise evaluation of the role of ABCB1 and ABCG2 transporters in patients could not conducted. Secondly, modulators of ABC proteins could change the pharmacokinetics of other drugs reducing their anti-tumor properties. Thirdly, the dose that was selected to inhibit the ABC protein was not sufficient or a higher dose could not be applied safely in patients.

The selection of cancer cell line is a crucial step in in vitro studies dedicated to GBM.

Many commercial human cells lines are available for GBM. However, a study from our laboratory has tested ABC proteins expression in GBM patient-derived cell lines (PDCL)

and their parental tumors (Drean et al., 2018a). The study showed that PDCLs recapitulated better ABC gene expression pattern of human GBM compared to commercial cell lines and can thus be considered a better model to test the biology of ABC proteins in GBM. In addition, we found that fetal bovine serum that is usually added to cell culture medium for commercial GBM cell lines modulates resistance to TMZ. Recently, a study highlighted the importance of using low passage number PDCL and serum-free medium when studying the role of ABC transporters in vitro. The high passaging number of commercial GBM cell lines could change the expression level of ABC protein and could lead to conclusions irrelevant to newly diagnosed human tumor [START_REF] Tamaki | The controversial role of ABC transporters in clinical oncology[END_REF], Drean et al., 2018a[START_REF] Leonard | The role of ABC transporters in clinical practice[END_REF].

Furthermore, sophisticated strategies using nanocarriers (nanocapsules, liposomes, micelles, dendrimers) for ABC protein substrates were developed to allow these substrates to enter via endocytosis, improving drug half-live and drug protection [START_REF] Zhao | Nanocarrier-based drug combination therapy for glioblastoma[END_REF].

One clinical study investigated pegylated liposomal doxorubicin's efficacy when administered with TMZ and radiotherapy in newly diagnosed GBM patients. This study showed that the pegylated liposomal doxorubicin form is safe and tolerable however, no meaningful efficacy was observed from either the prolongation of TMZ therapy or the addition of liposomal doxorubicin (NCT00944801) [START_REF] Beier | RNOP-09: pegylated liposomal doxorubicine and prolonged temozolomide in addition to radiotherapy in newly diagnosed glioblastoma--a phase II study[END_REF]. Another study evaluated the safety and the pharmacokinetics of a liposomal form of irinotecan, the study confirmed the safety of liposomal form of irinotecan. However, the efficacy still under investigation [START_REF] Clarke | A phase I trial of intravenous liposomal irinotecan in patients with recurrent highgrade gliomas[END_REF]. Therefore, the nanocarrier forms' utilisation could be effective tools in future clinical studies [START_REF] Zhao | Nanocarrier-based drug combination therapy for glioblastoma[END_REF].

Another strategy consists in the use of antibody-drug conjugates (ADCs) against a variety of targets in GBM and this approach is currently under investigation [START_REF] Gan | Antibody-drug conjugates in glioblastoma therapy: the right drugs to the right cells[END_REF]. ADCs are a newly developed biopharmaceutical compounds that allow the targeting of tumour cells while sparing healthy cells. This method is based on the use of an antibody to carry the substrate when binding to its ligand. In 2017, a published clinical trial showed a promising efficacy of depatuxizumab mafodotin (ABT-414), an ADC specific for the activated form of EGFR to selectively deliver a cytotoxic, in epidermal growth factor receptor (EGFR)-amplified recurrent GBM patients with manageable adverse effects [START_REF] Reardon | Efficacy and safety results of ABT-414 in combination with radiation and temozolomide in newly diagnosed glioblastoma[END_REF]. Another clinical trial was designed to evaluate its efficacy in newly diagnosed GBM patients (NCT02573324).

Finally, UMBO was used in pre-clinical models to bypass BBB efflux transporters and increase the brain's penetration of a wide variety of therapeutics. Low-intensity pulsed ultrasound can be delivered to the brain to induce a safe oscillation of intravenously injected microbubbles within blood vessels [START_REF] Hynynen | Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits[END_REF]. Oscillation of these microbubbles opens the BBB by reversibly disrupting the tight junctions between ECs. A range of drugs have been tested for use with UMBO for treating gliomas and include TMZ, carmustine, irinotecan, carboplatin, doxorubicin, and drug-loaded liposomes (Beccaria et al., 2016[START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF]. A new study showed that UMBO could decrease the expression of ABCB1 protein in cerebral vessels without affecting the integrity of other proteins [START_REF] Choi | Diminished Expression of P-glycoprotein Using Focused Ultrasound Is Associated With JNK-Dependent Signaling Pathway in Cerebral Blood Vessels[END_REF]. UMBO has recently moved to clinical trials where its clinical safety was confirmed: Sonocloud-induced UMBO was found to be safe and tolerable among GBM patients [START_REF] Idbaih | Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed Ultrasound in Patients with Recurrent Glioblastoma[END_REF]. Two other phase 1 and phase 2 clinical trials are currently in progress to evaluate UMBO's efficacy in combination with carboplatin in patients with recurrent GBM (NCT03744026).

Conclusion

GBM is an aggressive primary brain tumour with dismal prognosis. Over the last 15 years, no new drugs were found to be superior to TMZ. The long-term limited efficacy of TMZ is explained, at least partly, by the effect of MDR proteins (ABCB, ABCC1 and ABCG2).

Accumulating evidence are rising to connect the effect of chemotherapies and ABC proteins. The clinical role of ABC proteins is still under investigation and the failure of previous clinical trials raised several questions regarding the strategies to overcome MDR in GBM. A few clinical recommendations are currently being reported in the literature regarding future clinical trials. Firstly, all drugs that are going to be used in the trials should be tested against the major ABC proteins (ABCB, ABCC1 and ABCG2). A wide range of in vitro and in vivo models could allow a precise testing of the novel drugs [START_REF] Tamaki | The controversial role of ABC transporters in clinical oncology[END_REF]. Secondly, newly available non-invasive diagnostic imaging approaches i.e. PET scanning have the potential to determine whether ABCB1 or other transporters are functioning to reduce drug accumulation and whether inhibition can change drug uptake in solid tumours (Fomichov et al., Bauer et al., 2016). Furthermore, dual ABC inhibitors with a high specificity could be developed. Indeed, for example, the ABCB1 specific inhibitor zosuquidar enhanced sunitinib brain concentration in mice, but not to the same level as the dual inhibitions of ABCB1 and ABCG2 [START_REF] Oberoi | Pharmacokinetic assessment of efflux transport in sunitinib distribution to the brain[END_REF]. Following these recommendations could lead to the design of clinical trials that might successfully demonstrate the therapeutic potential of ABC protein inhibition in GBM treatment.

Table 9: Analysis of published studies that show the effects of ABC transporters on chemotherapeutic agents used in GBM.

Effect ABC Transporter involved

Drug involved Reference

High dose of cyclosporine 1 doubles the plasma concentration of etoposide among glioma patients ABCB1 Cyclosporine [START_REF] Lum | Alteration of etoposide pharmacokinetics and pharmacodynamics by cyclosporine in a phase I trial to modulate multidrug resistance[END_REF] Nimodipine 2 enhances sensitivity to procarbazine in viability tests in vitro using PDCLs obtained from glioblastoma patients ABCB1 and ABCG2 Nimodipine [START_REF] Durmaz | The effects of anticancer drugs in combination with nimodipine and verapamil on cultured cells of glioblastoma multiforme[END_REF] Paclitaxel in combination with valspodar 3 significantly decreases the tumour volume of U-118 MG tumors compared to the control and paclitaxel groups in mice ABCB1 Valspodar [START_REF] Fellner | Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo[END_REF] Vincristine exposure induces an elevated expression of ABCG1 in rats' brain. This effect could lead to the assumption that ABCB1 is partially responsible for the observed resistance of a relapsing tumours. ABCB1 Vincristine [START_REF] Balayssac | Patterns of P-glycoprotein activity in the nervous system during vincristine-induced neuropathy in rats[END_REF] Overexpression of ABCG2 in human GBM cell lines is associated with mitoxantrone resistance.

ABCG2

Mitoxantrone [START_REF] Rao | Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells[END_REF] GBM cell lines overexpressing ABCB1 exhibit high resistance to carmustine, carboplatin and etoposide ABCB1

Carmustine, carboplatin and Etoposide [START_REF] Nakai | Enhanced MDR1 expression and chemoresistance of cancer stem cells derived from glioblastoma[END_REF] Elacridar 4 sensitizes GBM cell lines to dasatinib. Homozygous knockout of ABCG2 in mice results in a better overall survival compared to the wild type when treated with dasatininb ABCB1 and ABCG2 Elacridar [START_REF] Agarwal | Active efflux of Dasatinib from the brain limits efficacy against murine glioblastoma: broad implications for the clinical use of molecularly targeted agents[END_REF] Similar brain-to-plasma concentration was observed for sunitinib in both ABCB1 and ABCG2 knockout mice model and with elacridar treatment in mice. However, mild effect was observed with the zosuquidar 5 and no effect with KO143 6 ABCB1 and ABCG2

Elacridar, KO143 and Zosuquindar [START_REF] Oberoi | Pharmacokinetic assessment of efflux transport in sunitinib distribution to the brain[END_REF] Inhibition of ABCB1 and ABCG2 with ABT-888 improves the efficacy of TMZ therapy in GBM patients. ABCB1 and ABCG2 ABT-888 [START_REF] Lin | ABCB1, ABCG2, and PTEN determine the response of glioblastoma to temozolomide and ABT-888 therapy[END_REF] Mdr1-/-mice show a higher concentration of irinotecan compared to mdr1a+/+ mice when both exposed to the same dose of irinotecan. ABCB1 Irinotecan [START_REF] Goldwirt | Irinotecan and temozolomide brain distribution: a focus on ABCB1[END_REF] MRP1 inhibition enhanced Vincristine and Etoposide but not TMZ chemotherapeutic effect however the combined inhibition of MRP1 and P glycoprotein (P-gp) using Reversan 7 increased TMZ response in GBM PDCLs

ABCC1

MK571 and Etoposide [START_REF] Tivnan | Inhibition of multidrug resistance protein 1 (MRP1) improves chemotherapy drug response in primary and recurrent glioblastoma multiforme[END_REF] Inhibition of ABCB1 and ABCG2 with verapamil and KO143 increases the efficacy of TMZ when combined with MGMT inhibitors.

ABCB1 and ABCG2

Verapamil and KO143 [START_REF] Tomaszowski | Multidrug Efflux Pumps Attenuate the Effect of MGMT Inhibitors[END_REF] Melatonin enhances ABCG2 promoter methylation hence sensitizes GBM cell lines to mitoxantrone, doxorubicin, TMZ

ABCG2

Melatonin [START_REF] Martin | Melatonin-induced methylation of the ABCG2/BCRP promoter as a novel mechanism to overcome multidrug resistance in brain tumour stem cells[END_REF] Limited drug delivery into brain tumors may significantly limit the efficacy of rucaparib 8 combined with TMZ in GBM ABCB1 and ABCG2 Rucaparib [START_REF] Parrish | Efficacy of PARP Inhibitor Rucaparib in Orthotopic Glioblastoma Xenografts Is Limited by Ineffective Drug Penetration into the Central Nervous System[END_REF] AZD2461 9 has a limited brain permeability in vivo due to its efflux by ABCB1 protein.

ABCB1

AZD2461 (de Gooijer et al., 2018a) Downregulation of ABCB1 and ABCG2 by Bone morphogenetic protein 7 sanitize the GBM stem cells to the clinically relevant dose of TMZ.

ABCB1 and ABCG2 BMP7 [START_REF] Tso | Bone morphogenetic protein 7 sensitizes O6-methylguanine methyltransferase expressing-glioblastoma stem cells to clinically relevant dose of temozolomide[END_REF] ABCE1 downregulation enhance the efficacy of TMZ in GBM cells (U87 and A172). ABCE1 TMZ (Zhang and Chen, 2018) ABCG2 knockdown in several GBM cell lines resulted in upregulation of other drug transporters ABCB1 and ABCC3 when treated with TMZ. ABCG2 TMZ. [START_REF] Chua | Characterization of a side population of astrocytoma cells in response to temozolomide[END_REF] KIAP -anti apoptotic protein-sensitizes U251 cells to TMZ through reduction of the ABCC1 expression ABCC1 TMZ (Liu et al., 2015b) The study was not conclusive. Only 7% of the 125 cases studied showed detectable MDR1 expression, suggesting that ABCB1 was not a major contributor to drug resistance in the selected cohort ABCB1 TMZ [START_REF] Fruehauf | In vitro drug response and molecular markers associated with drug resistance in malignant gliomas[END_REF] Histone-lysine N-methyltransferase (EZH2) enzyme silencing decreases the ABCB1, ABCC1 and ABCG2 mRNA and protein levels, which would lead to reduce efflux pump activity ABCC1, ABCB1 and ABCG2 EZH2 [START_REF] Fan | Inhibition of EZH2 reverses chemotherapeutic drug TMZ chemosensitivity in glioblastoma[END_REF] TMZ treatment upregulate the expression of ABCC3 compared to control mice. ABCC3 protect natural killers from TMZ. A GL261 syngeneic mouse model was used in this study. ABCC3 TMZ [START_REF] Pessina | The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma[END_REF] LRIG1, human EGFR inhibitor, reversed MDR in GBM cell lines (U87 and U251) by inhibiting EGFR and secondary ABCB1 and ABCG2 ABCB1 and ABCG2 TMZ (Liu et al., 2015a) CDK6 knockdown in GBM cell line (U251) resulted in significant downregulation of MDR1, MRP which enhanced the TMZ response ABCB1, ABCC1 TMZ [START_REF] Li | Knockdown of CDK6 enhances glioma sensitivity to chemotherapy[END_REF] The antitumor efficacy of TMZ against three different intracranial tumor models was significantly enhanced by a homozygous knockout of Abcb1a/b and Abcg2 genes. ABCB1 and ABCG2 TMZ (de Gooijer et al., 2018b) The single nucleotide polymorphism (SNPs) in the MDR1 gene exon12 C1236T is an independent predictive factor for prediction of the TMZ treatment in GBM patients.

ABCB1 TMZ [START_REF] Schaich | A MDR1 (ABCB1) gene single nucleotide polymorphism predicts outcome of temozolomide treatment in glioblastoma patients[END_REF] Overexpression of MDR and MRP in GBM cells (U87, U251, U373) is associated with a high resistance to TMZ.

ABCB1 and ABCC1

TMZ [START_REF] Liang | Overexpression of ILK promotes temozolomide resistance in glioma cells[END_REF] Activated EGFR kinase enhanced the ability of GBM cells (U87 and T98G) to resist TMZ through the upregulation of MDR1 ABCB1 TMZ [START_REF] Munoz | Temozolomide induces the production of epidermal growth factor to regulate MDR1 expression in glioblastoma cells[END_REF] Loss and gain of function for MDR1 showed an enhanced and reduced efficacy of TMZ in GBM cell lines (U87 and T98G) ABCB1 TMZ (Munoz et al., 2015b) Inhibition of ABCG2 enhanced the efficacy of TMZ and is considered a promising therapeutic target in GBMs ABCG2 TMZ [START_REF] Emery | Expression and function of ABCG2 and XIAP in glioblastomas[END_REF] TMZ downregulate the expression of ABCB1 in GBM stem cells ABCB1 TMZ [START_REF] Riganti | Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/beta-catenin pathway[END_REF] MDR1 and ABCG2 is responsible for the resistant of recurrent GBM to TMZ. Following a TMZ exposure in U87 and T89G cells 8 folds expression MDR1 and 4 folds for ABCG2 in the cells compared to naive cells was recorded using real time PCR.

ABCB1 and ABCG2 TMZ (Munoz et al., 2015a) Multiple inhibition of the MDR1 protein showed no enhanced associated with an enhanced sensitivity of TMZ in GBM cell line (T98G) ABCB1, ABCB1 TMZ [START_REF] Peignan | Combined use of anticancer drugs and an inhibitor of multiple drug resistance-associated protein-1 increases sensitivity and decreases survival of glioblastoma multiforme cells in vitro[END_REF] Downregulation of p-glycoprotein enhance the efficacy of TMZ in GBM U87 cell line ABCB1 TMZ [START_REF] Zhang | EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of Pglycoprotein inhibition[END_REF] ABCA13 overexpression is associated with a decreased progression free survival in univariate and multivariate analyses in GBM patients. ABCA13 TMZ (Drean et al., 2018a) Carbonic anhydrase XII (CAXII) sensitizes primary GBM cells to TMZ by reduction of ABCB1 protein activity. ABCB1 TMZ [START_REF] Salaroglio | Carbonic Anhydrase XII Inhibitors Overcome P-Glycoprotein-Mediated Resistance to Temozolomide in Glioblastoma[END_REF]) [START_REF] Johnstone | Multiple physiological functions for multidrug transporter P-glycoprotein?[END_REF][START_REF] Vasiliou | Human ATP-binding cassette (ABC) transporter family[END_REF] ABCC 13

• Anion efflux.

• Drug resistance • Nucleoside transport [START_REF] Vasiliou | Human ATP-binding cassette (ABC) transporter family[END_REF])

ABCD 4
• Mainly expressed in peroxisomes.

• ABCD2 fatty acid transport and a major modifier locus for clinical diversity in X-linked ALD [START_REF] Vasiliou | Human ATP-binding cassette (ABC) transporter family[END_REF])

ABCE/ABCF 1 ABCE 3 ABCF

• Along with ABCE1, ABCF members have ATP-binding domains, but no transmembrane domains, making transporter function unlikely • Mainly regulate protein synthesis or expression [START_REF] Vasiliou | Human ATP-binding cassette (ABC) transporter family[END_REF] ABCG 5

• Transport of diverse drug substrates, sterols, and lipids • ABCG4 is expressed in macrophages • Drug resistance [START_REF] Vasiliou | Human ATP-binding cassette (ABC) transporter family[END_REF]) (Linton andHiggins, 2007, Zolnerciks et al., 2011).

Figure 18 Represents the methodology of this literature review. The key words (ABC transporters, glioblastoma, TMZ, and chemotherapy) were used in PubMed and Google scholar search engines. 151 articles appeared in the results, then a few steps were carried out to exclude the duplicated and review articles. 91 abstracts were then reviewed and from the abstract, only articles that studied ABC transporters or GBM were selected. Full texts were obtained for all 36 selected articles.

Figure 19: Summary of methods that are being developed to overcome ABC transporters. Panel A: represent the development of nanocarriers that allow the drug to enter via endocytosis. Panel B shows another method by using a partial or complete antagonist that can be administered in combination with the efflux pumps substrates and as a result enhance the activity of the substrates.

Panel C shows the antibody drug conjugates approach that relies on an antibody to carry the substrates when binding to its ligand. Panel D represents the combined effect of using microbubbles and low intensity ultrasound to open the BBB. These four methods have been used in vitro and in vivo to develop strategies to overcome ABC efflux pumps [START_REF] Li | Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies[END_REF].

Introduction

Glioblastoma (GBM) is the most common and aggressive glioma in adults. The latest World Health Organization (WHO) guideline classifies GBM as grade IV glioma [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF]. Over the last years, massive efforts have led to a better understanding of the pathology and the genetic of GBM (deSouza et al., 2016). To date, the most effective and approved standard therapeutic regimen is maximum surgical resection of the tumor followed by concurrent chemoradiation and adjuvant chemotherapy with temozolomide [START_REF] Louis | The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary[END_REF]. Despite this very intensive therapeutic regimen, newly diagnosed GBM patients have a dismal outcome with a median overall survival (OS) below 18 months [START_REF] Marenco-Hillembrand | Trends in glioblastoma: outcomes over time and type of intervention: a systematic evidence based analysis[END_REF]. The main known prognostic factors are: (i) age, (ii)

Karnofsky performance status -KPS-, (iii) MGMT promoter methylation status, and (iv) IDH mutational status [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF].

Immunotherapies have dramatically improved the prognosis of melanoma [START_REF] Leven | Immune Checkpoint Inhibitors in Melanoma: A Review of Pharmacokinetics and Exposure-Response Relationships[END_REF] and other non-neurological solid tumors [START_REF] Leven | Immune Checkpoint Inhibitors in Melanoma: A Review of Pharmacokinetics and Exposure-Response Relationships[END_REF]. In the setting of primary brain cancer, results from clinical trials are still disappointing [START_REF] Muftuoglu | Results From the CheckMate 143 Clinical Trial: Stalemate or New Game Strategy for Glioblastoma Immunotherapy[END_REF]. Nonetheless, specific GBM patients responded, supporting the identification of biomarkers to stratify patients in the prescription of immunotherapies. Immune checkpoint proteins such as Cluster of differentiation 80 (CD80; known as B7-1) and CD86

(known as B7-2) are expressed on the surface of tumor cells [START_REF] Ville | Co-Stimulatory Blockade of the CD28/CD80-86/CTLA-4 Balance in Transplantation: Impact on Memory T Cells[END_REF].

Furthermore, CD80 protein expression was observed in infiltrative tumor lymphocytes in melanoma [START_REF] Hersey | Expression of the co-stimulatory molecule B7 on melanoma cells[END_REF]. Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and CD28 are located on T-lymphocytes. Both CD28 and CTLA-4 proteins bind to their ligands on the antigen presenting cells and major histocompatibility complex (MHC) [START_REF] Wei | Fundamental Mechanisms of Immune Checkpoint Blockade Therapy[END_REF]. CTLA-4 has a higher affinity to CD80 and CD86, and when bound to its ligands, T-lymphocytes remain inactive and exhausted [START_REF] Rowshanravan | CTLA-4: a moving target in immunotherapy[END_REF].

` 102
Antibodies targeting CTLA-4 were used in both preclinical studies in multiple solid tumors, resulting in many ongoing clinical trials [START_REF] Letendre | Ipilimumab: from preclinical development to future clinical perspectives in melanoma[END_REF]. Ipilimumab (anti-CTLA-4) has also shown responses in patients with brain metastases, highlighting efficacy within the central nervous system [START_REF] Savoia | Ipilimumab (Anti-Ctla-4 Mab) in the treatment of metastatic melanoma: Effectiveness and toxicity management[END_REF]. Expression of the most extensively studied immune checkpoint protein, programmed death-ligand (PD-L1), was inversely correlated with OS in GBM patients [START_REF] Nduom | PD-L1 expression and prognostic impact in glioblastoma[END_REF]. However, the expression of CD80 and CD86 in GBM tissues and their prognostic significance have not been reported yet.

This study investigated the mRNA and protein expression of CD80 and CD86 in newly diagnosed GBM patients, aged below 70 and with KPS above 70 treated with the standard of care. In addition, we have investigated possible correlations with prognosis.

Materials and methods

Patient samples

OncoNeuroTek

IHC staining

Paraffin-embedded tissue blocks (5-7 μm) from biopsies of newly diagnosed GBM patients were received from the ONT biobank. Tissue sections were deparaffinized using xylene and rehydrated. For antigen retrieval, each slide was embedded in citrate buffer at pH 4.0 and heated for 15 min in the microwave at 800w. 10% goat serum with 5% fetal bovine serum in 0.2% triton phosphate buffer saline was used as a blocking buffer. 3%

hydrogen peroxide was used to block tissue peroxidation. Anti-human CD80 antibody (A16039; Abclonal) and anti-human CD86 antibody (A2353; Abclonal) were used at 1:500 dilution in blocking solution and incubated on the tissue slides overnight at room temperature. Avidin-Biotin Complex (ABC) kit was used as a signal enhancer before the incubation in DAB (3,3′-Diaminobenzidine). Slides were embedded in hematoxylin dye and rinsed with tap water for nuclear staining; gradual alcohol and xylene baths were used for `survminer::surv_cutpoint` function, which determines the cut point based on the highest/lowest value of the log-rank statistics (low or high expression values). Then these categories were used for Kaplan-Meier analysis or Cox proportional hazard regression modeling testing for each variable independently or to adjust for multiple variables including CD80/CD86 expressions and MGMT promoter methylation status. P-values lower than 0.05 were considered significant (Greenbaum et al., 2003, van Nieuwenhuijze and[START_REF] Van Nieuwenhuijze | The Molecular Control of Regulatory T Cell Induction[END_REF])

Results

Patients and tumors characteristics

Forty-seven patients with a confirmed GBM diagnosis fulfilled the inclusion criteria:

14 men and 33 women (sex ratio m/w = 0.42). The patients' median age at diagnosis was 55.9 years (range: 24.3-69.5 years). KPS was 70 and above in all patients. The median OS is 559 days (range 31 -2539) and the median PFS is 266 days (range 26-1355). The IDH status was evaluated as mutant for two patients (4.3%) while wildtype for 45 (95.7%).

Furthermore, MGMT promoter was methylated in 16 patients (34%) and unmethylated in 31 (66%). All patients received the standard of care first-line treatment regimen.

CD80 and CD86 expression at mRNA and protein level

At the mRNA level, CD86 expression was quantitatively higher than CD80 expression (Figure 20-A). In agreement with mRNA expression, IHC analysis showed that the expression of CD86 is higher than CD80 in our discovery cohort (Figure 20-B). Based on the IHC staining, CD80 and CD86 are observed in the cell membrane and/or cytoplasm (Figure 21). Following protein quantification, we observed a positive correlation between mRNA and protein expression of CD86 (spearman coefficient of correlation Rho=0.28; p=0.08; Figure 22-A). However, we observed a weaker correlation between mRNA and protein expression for CD80 (p=0.108; Rho= 0.25; Figure 22-B).

Prognostic value of CD80 and CD86 expression

Our patient's cohort was used as a discovery cohort (ONT cohort), while the TCGA dataset was used as a validation cohort. In a univariate analysis, mRNA expression of CD80 and CD86 was not correlated with OS in both ONT cohort and TCGA dataset (Table 11).

On the other hand, CD80 and CD86 mRNA high expression was significantly associated with shorter PFS (p=0.04 and p=0.005, respectively; Figure 23,A,B). Next, these findings were validated using TCGA cohort; higher CD80 and CD86 expressions were correlated with shorter PFS (p-value=0.04 and p=0.002 respectively; Figure 23, C andD).

Interestingly, higher CD86 protein expression was associated with shorter PFS in the ONT cohort (p<0.005; Table 12). CD80 and CD86 protein expressions were not available in the TCGA dataset for validation purpose.

As expected, MGMT promoter methylation was associated with longer PFS and longer OS in ONT cohort (p<0.05 and p<0.05 respectively) and TCGA dataset (p<0.05 and p<0.05 respectively) (Table 11 andTable 12). Furthermore, IDH mutations were also associated with better OS and PFS in the TCGA database (p<0.05 and p<0.05 respectively); however, in ONT cohort, the limited number of IDH-mutant GBM did not allow a robust analysis (n=2). In multivariate analysis, CD80 mRNA expression did not provide additional prognostic information to MGMT promoter methylation in ONT cohort. On the other hand, multivariate analysis of CD86 mRNA expression was an independent prognostic factor for PFS in the TCGA dataset only (p<0.05; Figure 24). We have observed a similar trend (p=0.27; Figure 24) in the ONT cohort, yet the trend was not significant, which could be related to the lower patient numbers (n=47) in the ONT cohort compared to (n=121) in TCGA dataset.

Discussion

CD80 and CD86 molecules play an essential role in influencing the immune recognition of GBM cells. They bind to the CD28 molecule with a costimulatory signal for T-lymphocytes activation. On the other hand, they bind to CTLA-4, resulting in an immunosuppressive effect. CTLA-4 has a higher affinity to CD80 and CD86, making these molecules' role in immunosuppressive effect higher than their stimulatory effect (van [START_REF] Van Nieuwenhuijze | The Molecular Control of Regulatory T Cell Induction[END_REF]. The current study has linked CD80 and CD86 expression on GBM tumor microenvironment to PFS.

We observed a low correlation between mRNA and protein expression of CD80.

However, a better correlation was observed between CD86 protein and mRNA expression.

Low correlation between the mRNA and protein expression might be due to posttranscriptional mechanisms involved in turning mRNA into protein. Another reason could be related to the stability of both mRNA and protein in our patient's samples. Finally, there is a possible error and noise in protein quantification and mRNA extraction that could influence mRNA stability and protein expression [START_REF] Greenbaum | Comparing protein abundance and mRNA expression levels on a genomic scale[END_REF].

The number of patients (n=47) in ONT cohort is lower than the number of patients in the TCGA dataset (n=121). The higher number of TCGA GBM samples could be one reason that affected the statistical analysis and provided a better prognostic value in the TCGA dataset compared to the ONT cohort. Indeed, the availability of GBM samples with comprehensive clinical and biological annotations and fulfilling the inclusion criteria is a limitation for ONT cohort. Larger patient cohort is needed to evaluate the prognostic value of CD80 and CD86 expression in GBM samples. In our protein analysis, co-staining of CD80 and CD86 is needed to determine these proteins' expression in different immune cell populations. Furthermore, other immune checkpoint proteins could be evaluated in future studies.

The expression of 50 immune checkpoint molecules was investigated in breast cancer. The study showed that high expression of co-stimulatory immune checkpoint molecules was associated with better PFS. However, no significant effect on prognosis was associated with CD80 and CD86 expression in the selected cohort [START_REF] Fang | Prognostic value of immune checkpoint molecules in breast cancer[END_REF]. Feng et al. reported that low expression of CD80 is a predictive biomarker for poor prognosis in adenocarcinoma [START_REF] Feng | Low expression of CD80 predicts for poor prognosis in patients with gastric adenocarcinoma[END_REF]. Furthermore, CD80 and CD86 were found to be potential biomarkers for better prognosis survival in nasopharyngeal carcinoma. [START_REF] Chang | Expression of CD80 and CD86 costimulatory molecules are potential markers for better survival in nasopharyngeal carcinoma[END_REF]. Additionally, the molecular characterization of PD-L1 expression was correlated with other checkpoint proteins, i.e., CD80, highlighting that higher level of immunosuppression are associated with GBM compared to lower grade gliomas (LGG) [START_REF] Wang | Molecular and clinical characterization of PD-L1 expression at transcriptional level via 976 samples of brain glioma[END_REF]. In myeloma cell lines, silencing the CD28-CD86 pathway resulted in significant cell death of myeloma cells [START_REF] Gavile | CD86 regulates myeloma cell survival[END_REF]. A recent study constructed a more robust model, using GBM and LGG data from the TCGA and CGGA (Chinese glioma genomic atlas), and identified that low expression of CD86 molecules is a good prognostic indicator for OS. PFS analysis were not applied in this study [START_REF] Qiu | A Prognostic Microenvironment-Related Immune Signature via ESTIMATE (PROMISE Model) Predicts Overall Survival of Patients With Glioma[END_REF].

In 2017, Berghoff et al. described a specific signature to predict the success of TMZ in MGMT-methylated patients. They showed that the TME signature could be used to indicate an individual's TMZ sensitivity. The TME was identified to be different between IDH-mutant and IDH-wildtype tumors. A richer tumor infiltrative lymphocyte (TIL) and a higher expression of PD-L1 were observed in IDH-wildtype tumors [START_REF] Berghoff | Correlation of immune phenotype with IDH mutation in diffuse glioma[END_REF].

However, to date, no studies have linked MGMT promoter methylation status with the TME. A recent research article has studied the expression of immune checkpoint inhibitor Tim3 and MGMT promoter methylation status. They identified that a high expression of Tim3 in MGMT-unmethylated patients is linked to poor prognosis (Zhang et al., 2020b).

Pratt et al. have reported that the expression of PD-L1 is a negative prognostic biomarker in recurrent IDH-wildtype GBM [START_REF] Pratt | Programmed Death Ligand 1 Is a Negative Prognostic Marker in Recurrent Isocitrate Dehydrogenase-Wildtype Glioblastoma[END_REF]. In line with these findings, our study supports that the expression of immune checkpoint inhibitors may inhibit T-lymphocytes and anti-tumor reaction.

CD86 expression could be used as potential biomarkers predicting the efficacy of ipilimumab in GBM patients. Furthermore, it could be used as a biomarker for patients' stratification for future clinical trials. Our study suffers from limitation of retrospective studies with limited number of patients. Nonetheless, our results were validated in an independent dataset and support investigations of immune checkpoint molecules as potential prognostic biomarkers and potential predictive biomarkers of response to immunotherapies in GBM. Email : ahmed.idbaih@aphp.fr

Tables, figures, and legends to figures

Introduction

Glioblastoma (GBM) is the most malignant primary brain tumor in adults, with a median overall survival below 18 months after initial diagnosis [START_REF] Ostrom | The epidemiology of glioma in adults: a "state of the science[END_REF].

Despite remarkable efforts in the neuro-oncology field to develop new therapeutic alternatives, temozolomide (TMZ) remains today the standard first-line chemotherapy in GBM treatment [START_REF] Ostrom | The epidemiology of glioma in adults: a "state of the science[END_REF][START_REF] Pace | European Association for Neuro-Oncology (EANO) guidelines for palliative care in adults with glioma[END_REF]. For over five decades, significant efforts have been put into the development of new anti-cancer therapies for GBM including anti-neoplastic agents [START_REF] Atiq | Anti-neoplastic Potential of Flavonoids and Polysaccharide Phytochemicals in Glioblastoma[END_REF], molecular targeted drugs [START_REF] Touat | Glioblastoma targeted therapy: updated approaches from recent biological insights[END_REF], immunotherapeutic approaches [START_REF] Weenink | Immunotherapy in Glioblastoma: Current Shortcomings and Future Perspectives[END_REF], and angiogenesis inhibiting compounds [START_REF] Wang | New Directions in Anti-Angiogenic Therapy for Glioblastoma[END_REF]; however, the prognosis of patients has hardly improved [START_REF] Al-Kharboosh | Advances in Brain Tumor Surgery for Glioblastoma in Adults[END_REF].

The existence of the blood-brain barrier (BBB) as a specificity of the central nervous system (CNS) blood vessels prevents most systemic therapeutic compounds from reaching the brain parenchyma and GBM cells [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF]. Structural and functional changes of the BBB in GBM are frequent. They lead to changes of the BBB, called the blood-tumor barrier (BTB), allowing some chemotherapies to reach the tumor. Although the BTB enhances the delivery of some chemotherapeutic agents, large therapeutic antibodies have no chance to reach the brain at their therapeutic levels [START_REF] Ait-Belkacem | Monitoring therapeutic monoclonal antibodies in brain tumor[END_REF].

Several innovative strategies are known to enhance the delivery of chemotherapeutic agents and antibodies [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF]. Ultrasound-mediated blood-brain barrier opening (UMBO) using low-intensity pulsed ultrasound (LIPU) is one of the safe and effective methods to enhance the delivery of chemotherapeutic agents in preclinical (Zhang et al., 2020a) and clinical settings [START_REF] Idbaih | Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed Ultrasound in Patients with Recurrent Glioblastoma[END_REF]. Indeed, UMBO showed adequate safety and efficacy in recurrent GBM patients [START_REF] Idbaih | Safety and Feasibility of Repeated and Transient Blood-Brain Barrier Disruption by Pulsed Ultrasound in Patients with Recurrent Glioblastoma[END_REF]. LIPU is delivered to the brain simultaneously with an intravenous injection of micron-sized bubbles for a few minutes, allowing the microbubbles to oscillate. Microbubbles oscillation produces a mechanical stretching on vessel walls that allows a transient opening of tight junctions [START_REF] Sheikov | Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles[END_REF].

The choice of therapeutic agents to deliver after UMBO is crucial and remains a point of discussion among researchers and regulators. Direct stimulation of the immune system with ICBs (e.g., PD-1/PD-L1) showed promising effects alone or with other chemotherapies in multiple cancers. Ipilimumab was the first humanized anti-CTLA-4 approved by the American Federal Drug Administration (FDA) to treat inoperable melanoma [START_REF] Tarhini | Tremelimumab: a review of development to date in solid tumors[END_REF].

Five years later, Atezolizumab was the first humanized anti-PD-L1 approved by the FDA to treat advanced or metastatic urothelial carcinoma [START_REF] Hsu | A Comprehensive Review of US FDA-Approved Immune Checkpoint Inhibitors in Urothelial Carcinoma[END_REF]. PD-L1 proteins are expressed as surface molecules by cancerous cells such as GBM cells [START_REF] Hao | PD-L1 Expression in Glioblastoma, the Clinical and Prognostic Significance: A Systematic Literature Review and Meta-Analysis[END_REF] and provide a tumor escape mechanism when bound to PD-1 proteins at the surface of activated T-lymphocytes leading to their exhaustion [START_REF] Azoury | Immune Checkpoint Inhibitors for Cancer Therapy: Clinical Efficacy and Safety[END_REF]. Nivolumab and ipilimumab have limited or no efficacy in GBM patients and Avelumab monotherapy (anti-PD-L1) showed a minor impact on progression-free survival (NCT03047473).

In the present study, we evaluated the effect of anti-PD-L1 and anti-CTLA-4 alone and in combination with UMBO in GBM mouse models. GL261 was transduced with a luciferase/mKate2 vector described before [START_REF] Plessier | New in vivo avatars of diffuse intrinsic pontine gliomas (DIPG) from stereotactic biopsies performed at diagnosis[END_REF]. GL261-luciferase and Nfpp10-luciferase cells (1.4 x 10 5 cells /2 µL) were inoculated into the right caudate nucleus-putamen (AP +10 mm, DV +0.25 mm, ML +0.15 mm) of 7-8 weeks old C57BL/6 females using a stereotactic injection frame (David Kopf Instruments Tujunga, CA). Mice were imaged using the IVIS Spectrum (PerkinElmer) 10 minutes following a 2 mg subcutaneous injection of luciferin (Sigma, L9504). The growth of GL261-luciferase and Nfpp10-luciferase cells was confirmed by two IVIS imaging one week apart following intracranial cell injection. During the characterization of tumor growth in our mouse models, we observed that mice with bioluminescence values lower than 5.00+E05 would not develop GBM tumors. Therefore, we have chosen to include only mice with bioluminescence values over 5.00+E05 photon/second. Mice were randomly placed into treatment arms once they passed the bioluminescence cutoff value.

Materials and Methods

Cell culture and in vivo studies

Animals were treated with 200µL of anti-CTLA-4 (Bristol-Myers Squibb, G1-XAS-Ab), anti-PD-L1 (Genentech, 6E11) and IgG1 (BXCELL, BE0083) for four doses. Unless specified otherwise, animals were sacrificed when they showed signs of tumor-associated illness (20% body weight loss or changes in behavior or posture).

Calibration of low-intensity pulsed ultrasound device

The ultrasound transducer (CarThera ® ) used in this study was calibrated before each experiment. The aim was to map the ultrasound field and to determine the electrical set point that the generator uses during the experiments to obtain the targeted acoustic pressure in situ. The calibration was performed using degassed water at room temperature with a 200 µm needle hydrophone (HNC0200, ONDA). A 2D acoustic field was scanned at 5 mm from the transducer surface with a 3-axis computer-controlled motorized positioning system (UMS, Precision Acoustics, U.K.). A transducer velocity pattern of 1.05

MHz was then obtained by acoustic holography. The 3D acoustic field was computed from this pattern with the Rayleigh integral. The hydrophone was positioned at the spatial peak pressure determined from the 3D acoustic field. The ratio between the active electrical power drawn by the transducer measured with an oscilloscope and the square of the spatial peak acoustic pressure was measured. This ratio is used as a calibration coefficient by the generator during subsequent experiments: the active electrical power needed to obtain the targeted pressure is calculated by the generator using this coefficient at the beginning of manipulation, and the generator adjusts its set point to obtain the specified active electrical power measured internally.

Ultrasound-mediated blood-brain barrier opening

UMBO was delivered to both UMBO and UMBO plus anti-PD-L1 groups. Anti-PD-L1 (6E11 Genentech) was administered intraperitoneally in all experiments at a dose of 200 µg 1 hour before UMBO application. UMBO device for preclinical studies was manufactured by CarThera ® . Mice were maintained under anesthesia with isoflurane (2%, 2L/min O2). For each UMBO application, 200 µL of SonoVue ® per mouse was injected by the retro-orbital route less than 10 seconds before the start of ultrasound application.

1MHz LIPU was delivered to the brain through a transducer at an 0.3 MPa acoustic pressure with a pulse length of 23.8 milliseconds (25,000 cycles) and a frequency of 1Hz for two minutes. For each sonication, UMBO was validated using a control mouse. Each control mouse was injected intravenously (IV) with a solution of 2.7% Evans blue (Sigma, E2129) in phosphate buffer saline (PBS) at a dose of 4 mL/kg 10 minutes post UMBO application. UMBO test mice were sacrificed 15 minutes following Evans' blue injection, and their brain was harvested. The passage of Evans blue was assessed both visually and by ZEISS Axio-Scan fluorescence imaging of cryosectioned brains.

Pharmacokinetic analysis of therapeutic antibodies with and without UMBO

Thirty-six mice were used in the pharmacokinetic experiment. Mice were separated in control and UMBO groups. Six-time points were selected as follows: 0.15, 0.3, 3, 6, 24, 48 and 96 hours. Each mouse received a 200 µg of nivolumab (Bristol-Meyers Squibb, New

York, NY, USA) intravenous injection 10 minutes following the BBB opening. 100 μL of blood was collected through cardiac puncture using a pre-heparinized syringe. The serum was collected by centrifugation of the blood at 3500 rpm for 10 minutes.

All samples (plasma and brain) were then analyzed using ultra-performance liquid chromatography (UPLC) system coupled to mass spectrometry (LC-MS/ MS; MS-8060, Shimadzu, Japan). Quantifications were achieved in multiple reaction monitoring (MRM) mode, and electrospray ionization was operated in a positive mode. Peak integration and quantification were performed using LabSolutions Insight LC-MS software. Nivolumab was quantified with signature peptide ASGGITFSNSGMHWVR.

MRI data acquisition

MRI acquisitions were performed using a preclinical 11.

mRNA sequencing

Twelve mice with a confirmed tumor of comparable sizes (as measured by bioluminescence imaging) were included in this experiment. Mice were divided into four groups. UMBO group, anti-PD-L1 group, UMBO plus anti-PD-L1, and InVivoPure pH 6.5

Dilution Buffer (BXCELL, IP0065) for vehicle groups. Two treatment sessions (day 21 and 24) were administered in this experiment. Mice were sacrificed 24 hours after the last treatment by cervical dislocation, and the right hemisphere was snap-frozen in 2 ml RNALater (Thermofisher AM7020). Lysing Matrix D (MBio, 6913050) was used to homogenize the brain tissues. mRNA was extracted using Maxwell RSC simply RNA automated RNA purification kit (Promega, AS1340). RNA quality was analyzed using high sensitivity RNA chips (TapeStation). For RNA sequencing, NovaSeq 6000 sequencer ( 200cycles, 800 million reads) and reagent kit were used. Following data alignment and normalization, we applied the publicly available RSTUDIO package called mMCP [START_REF] Petitprez | The murine Microenvironment Cell Population counter method to estimate abundance of tissue-infiltrating immune and stromal cell populations in murine samples using gene expression[END_REF] to characterize the tumor microenvironment changes before and after UMBO.

Immunohistochemistry (IHC)

Detection of anti-PD-L1 6E11 mouse antibody by IHC is irrelevant due to the crossreactivity with mouse antibodies. Instead, A 150 KDa rat IgG2 antibody targeting PD-L1 was used in our IHC staining (BXCELL, #BE0101). Goat anti-rat IgG antibody (BA-9400) was used to detect the anti-PD-L1. CD8+ T-lymphocytes were detected using CD8 alpha antibody (1:1000, BioRad, #MCA48R) while Iba1 protein was detected using 1:1000, Abcam, #ab178846. Mouse brains were fixed overnight in 4% paraformaldehyde (PFA) then immersed in 10% sucrose for cryoprotection, then were cryosectioned. 10 µm cryosections were harvested using Leica CM1950 cryostat. Slides were stored at -80°C until analysis.

Quantitative digital droplet polymerase chain reaction (ddPCR)

GL261 tumor-bearing mice four weeks following cell inoculation were used in the ddPCR experiment. A single UMBO treatment was completed, and 30 minutes later, blood (100 µl) was collected in heparinized tubes through cardiac puncture. Whole blood DNA was extracted automatically using Maxwell ® Blood DNA Purification Kit (AS1010). QX200

ddPCR EvaGeen ® was utilized to detect mKate2 and Luciferase genes in the extracted DNA.

Primer3Plus web interface was used to design mKate2 and Luciferase primers and was purchased from Life Technologies. The following forward (FR) and reverse (RV) primers were used: luciferase-FR, TCCACGATGAAGAAGTGCTC; luciferase-RV, AGGCTACAAACGCTCTCATC; mKate2-FR, GGTGAGCGAGCTGATTAAGG; and mKate2-RV, GGGTGTGGTTGATGAAGGTT.

Flow cytometry

Twenty mice with a confirmed tumor of comparable sizes were included in this experiment. Mice were separated into four groups: UMBO group, anti-PD-L1 (Genentech, 6E11) group, UMBO plus anti-PD-L1, and vehicle group as described above. One treatment session was delivered in this experiment. Mice were perfused using cold distilled phosphate buffer saline (DPBS) ~16 hours after treatment. Brains were isolated immediately and stored in 2 mL ice-cold Hanks' balanced salt solution (HBSS). According to the manufacturer's protocol, the right hemisphere was isolated and mixed in the enzyme mix solution from the adult brain dissociation kit (Miltenyi Biotec, #130-107-677).

Cells gentleMACS ® Octo Dissociator with Heaters (#130-096-427) and gentleMACS C Tubes (#130-093-237) were used to perform mice brain dissociation. The number of dissociated cells was calculated using Scepter ® 3.0 Handheld Cell Counter.

Samples were acquired on a spectral flow cytometer (Aurora, Cytek) and analyzed by FlowJo software (FlowJo, LLC). Briefly, cells were selected based on their morphology, doublets, and dead cells were excluded using (Biolegend, #423107) while tumor cells were excluded based on their mKate expression. Monocytes (Ly6C + Ly6G -), neutrophils (Ly6C + Ly6G + ) were excluded from non-tumoral live cells using Ly-6C (Biolegend, #128036) and Ly-6G (Biolegend, #127617). Microglia were identified based on their expression of CD11b + and CD45 low using CD45 (Biolegend, #103131) and CD11b (Biolegend, #101255).

Activated microglia were identified as CD68 + using (Biolegend, #137003). F4/80 marker (Biolegend, #123117) was used to determine macrophages in the CD45 high CD11b + cell population. CD206 marker (Biolegend, #141729) was used to distinguish between subpopulations of macrophages. Lymphocytes CD4 + (Biolegend, #100541) and CD8 + (Biolegend, #100737) were identified on the CD45 + CD11b -fraction of non-tumoral live cells. The percentage of each subpopulation was calculated and using in our flow cytometry analyses.

Statistical tests

Statistical analysis was performed using Prism software (GraphPad). Data are shown as mean values plus and minus standard error of the mean (SEM). Statistical significance of differences between groups was verified using appropriate statistical tests. Significance level were denoted with asterisks: * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001, and **** for p ≤ 0,0001.

Results

Anti-PD-L1 increases survival of GL261-bearing mice and Nfpp10 -bearing mice

Pilot studies using both GL261-luciferase and Nfpp10-luciferase mouse models were performed. These two experiments aimed to determine the effect of anti-PD-L1 and anti-CTLA-4 in our GBM mouse model and select the best candidates to combine with UMBO.

Anti-PD-L1 antibody alone has shown a limited effect on survival of GL261-bearing (Figure 26-B), even though a slight regression in tumor growth was seen (Figure 26-A).

Anti-CTLA-4 treatment did not affect tumor growth (Figure 26-A) or animal survival (Figure 26-B). No treatments had an impact on mice's body weight (Figure 26-C).

Interestingly, anti-PD-L1 antibody showed better efficacy in Nfpp10 GBM mouse model compared to GL261-bearing mice (Figure 26-E). Anti-PD-L1 treatment reduced tumor growth in some mice (Figure 26-D) and increased the number of long-term survivors (3/6) (Figure 26-E).

Blood-brain barrier integrity in GBM bearing mice

We evaluated the BBB integrity in both mouse models. Assessment of BBB disruption was performed using 1.2 mg of Hoechst 33342 (Sigma) diluted in PBS was injected intravenously 20 min prior to sacrifice. Hoechst staining was not detected in normal brain tissue (Figure 27-A), yet higher staining intensity was observed in brain tissue harvested from Nfpp10-bearing mice compared to GL261-bearing mice. Those as mentioned earlier could indicate a higher BBB permeability in the Nfpp10 GBM mouse model. Overall, this makes the anti-PD-L1 antibody the best candidate to be combined with UMBO in the GL261 GBM mouse model.

UMBO is safe and effective in immunocompetent mice

UMBO parameters were previously optimized in our setting using athymic nude mice [START_REF] Dréan | Temporary blood-brain barrier disruption by low intensity pulsed ultrasound increases carboplatin delivery and efficacy in preclinical models of glioblastoma[END_REF]. The safety and the efficacy of UMBO were then evaluated in C57BL/6 mice. We have selected 0.3 MPa for a safe and effective UMBO. UMBO was optimized to target the right hemisphere, and BBB opening was confirmed macroscopically (Figure 27 We observed tolerability of GL261-bearing mice to repeated biweekly UMBO and UMBO did not affect mice's weight (Figure 27-A). Furthermore, the Kaplan-Meier estimate shows no significant difference in the OS between UMBO and non-treated groups in the GL261 GBM mouse model (Figure 27-A). Overall, the UMBO parameters that are used for repeated UMBO opening are safe and tolerable in GL261-bearing mice.

UMBO dramatically increased the efficacy of anti-PD-L1 in GL261-bearing mice

We attempted to investigate any possible positive outcome from UMBO in combination with anti-PD-L1 in the GL261 GBM mouse model. We initially hypothesized that anti-PD-L1 efficacy was limited because the BBB protects GBM cells from exposure to a therapeutic level of anti-PD-L1.

Mice with comparable bioluminescence values were divided into five groups: (i) UMBO group, (ii) anti-PD-L1 group, (iii) UMBO plus anti-PD-L1 group, (iv) IgG1 group, and (v) IgG1 plus UMBO group. In each treatment protocol, all mice received 200 µl of warm saline injection before anesthesia to prevent any possible hypothermic effect.

Intraperitoneal injection of anti-PD-L1 injection was given 60 min before sonication to ensure anti-PD-L1 absorption. We have not observed any toxic effect in UMBO plus anti-PD-L1 treated mice versus control mice (Figure 28-C). UMBO and anti-PD-L1 did not show an early impact against tumor growth (Figure 28-D); however, a delayed effect on tumor size was observed two weeks after the last dose of treatment (Figure 28-F).

Interestingly, mice received an anti-PD-L1 antibody with UMBO showed a (13/17) 76 % long-term survivors (over 100 days) compared to (4/15) 26 % in anti-PD-L1 alone and (0/16) 0% in control groups. Kaplan-Meier estimate shows a significant difference (p<0.05) in UMBO's overall survival plus anti-PD-L1 treated mice versus anti-PD-L1 alone treated mice. Furthermore, a higher significance difference (p=0.0001) was observed in UMBO plus anti-PD-L1 treated mice compared to the IgG1 plus UMBO treated mice in the GL261 GBM mouse model.

UMBO increased the penetration of ICBs into brain parenchyma

The BBB can easily block large therapeutic agents as antibodies. With UMBO, we attempted to deliver antibodies to the brain parenchyma. IHC staining of anti-PD-L1

(BXCELL, BE0101) confirms UMBO's ability to deliver anti-PD-L1 to the right hemisphere brain parenchyma (Figure 29-A). Furthermore, an already clinically optimized nSMOL LC-MS [START_REF] Iwamoto | Application of nSMOL coupled with LC-MS bioanalysis for monitoring the Fc-fusion biopharmaceuticals Etanercept and Abatacept in human serum[END_REF]

UMBO increased circulating GL261-DNA to the peripheral blood circulation

Additionally, we aimed to evaluate molecules circulation between tumor brain and blood with and without UMBO. Therefore, we evaluated whether UMBO could enhance the leakage of circulating tumor DNA to the bloodstream. GL261-bearing mice four weeks following GL261 cell grafting were used in the experiment. We have observed a significant elevation (p<0.01) in the number of copies for both m-Kate and luciferase in the UMBO treated group compared to the control.

UMBO increased CD8 + T-lymphocytes in the brain and modulate microglia's phonotype.

Microglia staining in anti-PD-L1 plus UMBO treated group shows a phenotype of activated microglia. Double nuclear staining in the UMBO plus anti-PD-L1 treated GL261bearing mice show a possible induction of microglia's cell division (Figure 30). Therefore, our foreseen flow cytometry analysis of activated microglia would provide additional evidence of UMBO's effect on microglia's activations.

Additionally, we observed an enhanced passage of CD8 + T-lymphocytes in UMBO plus anti-PD-L1 compared to anti-PD-L1 treated GL261-bearing mice. This effect is currently under investigation by flow cytometry analysis of microglia, macrophages, CD3 + , CD8 + , and CD4 + T-lymphocytes.

Discussion

Patients with GBM have a dismal outcome with a median overall survival below 18 months with the current standard of care, including concurrent administration of TMZ and radiotherapy followed by adjuvant TMZ. Since 2005,TMZ has remained the first-line chemotherapy in treating GBM patients. TMZ is an alkylating agent with a small molecular weight (194.15 g/mol) that readily passes BBB. As suggested from TMZ clinical pharmacokinetics studies between 20-30% of TMZ reaches the brain following oral administration [START_REF] Ostermann | Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients[END_REF] however, large therapeutic agents, i.e., antibodies, do not cross the BBB in physiological conditions. UMBO and several innovative strategies continuously evolve to overcome the BBB by increasing drug delivery [START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF].

Immunotherapies, including ICIs and cell therapies, have revolutionized multiple solid tumors' treatments through activating the general antitumor immune response.

CheckMate-143 phase 3 clinical trial was then initiated to evaluate the effect of nivolumab versus bevacizumab. Unfortunately, nivolumab did not demonstrate higher efficacy compared to bevacizumab. Several reasons might explain the low efficacy of ICIs in GBM: (i) low tumor mutation load, (ii) lack of predictor of response and lack of selection of patients, (iii) low penetration of ICIs within the brain parenchyma, (iv) low peripheral priming, (v) local immunosuppression and (vi) low penetration of T-lymphocytes [START_REF] Beccaria | Ultrasound-induced blood-brain barrier disruption for the treatment of gliomas and other primary CNS tumors[END_REF].

We explored the BBB as the limitation for antibody and lymphocytes penetration and priming and attempted to evaluate UMBO's effect on the penetrating large therapeutics to the brain and modulating the immune microenvironment in GBM mouse model. Our data confirmed the limited efficacy of ICIs efficacy in the Gl261-bearing and Nfpp10-bearing mouse models. Consistent with our data, preclinical evaluation of anti-PD-L1 and anti-CTLA-4 in GL261-bearing mice model showed comparable limited efficacy of both antibodies in a GL261 GBM mouse model. In this study, they have used a different treatment regimen, yet the efficacy was comparable [START_REF] Reardon | Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model[END_REF].

To the best of our knowledge, this is the first research article that report a dramatic increase in the overall survival of GL261-bearing mice when treated with UMBO plus anti-PD-L1. Indeed, 76% of GL261-bearing mice treated with anti-PD-L1 plus UMBO survive longer than 100 days compared to 26% for GL261 mice treated with anti-PD-L1 alone.

Next, we tried to understand the mechanisms involved in the anti-tumor effect. We initially hypothesized that the BBB was responsible for the limited efficacy by blocking anti-CTLA-4 and anti-PD-L1 from reaching the GBM tumor. This hypothesis is consistent with a recent study that reported an enhanced efficacy of ICIs following their delivery to brain tumors [START_REF] Guo | Deliver anti-PD-L1 into brain by p-hydroxybenzoic acid to enhance immunotherapeutic effect for glioblastoma[END_REF]. Additionally, a recent study, illustrated that focused ultrasound enhanced the delivery of an intrasnal delivery of anti-PD-L1 but not overall survival of GL261-bearning mice. UMBO plus 200 µg of the anti-PD-L1 biweekly treatment regimen was used to maintain the higher concentration of anti-PD-L1 within the brain parenchyma.

Immune checkpoint blockade with anti-PD-L1 was performed on day 14 post-inoculation to allow for T-lymphocytes depletion [START_REF] Aslan | Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas[END_REF].

Using LC-MS/MS and IHC we reported an enhancement of nivolumab and anti-PD-L1 concentrations in the brain parenchyma. In our setting, we reported that UMBO enhanced antibody's concentration up to 28 folds compared to control. UMBO was optimized to disturb one hemisphere; however, in our PK analysis, we have used a wholebrain homogenization method; therefore, local concentrations of nivolumab could have been even higher. Consistent with our data, a study has shown that UMBO enhanced the delivery of bevacizumab ~149 KDa to the brain parenchyma by 5.7 to 56.7 folds compared to non-sonicated brain in a glioma mouse model [START_REF] Liu | Focused Ultrasound Enhances Central Nervous System Delivery of Bevacizumab for Malignant Glioma Treatment[END_REF].

GBM tumors have low chances of extracranial metastases with negligible risk for GBM spreading after surgical brain biopsies [START_REF] Lun | The natural history of extracranial metastasis from glioblastoma multiforme[END_REF]. UMBO stimulates a detectable peripheral circulation of GL261 DNA. We have observed a significant elevation of mKate2 and luciferase DNA 30-mintues following UMBO. Consistent with our data, a recently published article investigated the possibility of using UMBO for liquid biopsies in GBM models. They have observed a detectable level of green fluorescent protein mRNA 20-mins following UMBO in the GL261 mouse model [START_REF] Zhu | Focused Ultrasound-enabled Brain Tumor Liquid Biopsy[END_REF].

Here, we have investigated the effect of peripheral circulation of DNA to extrapolate the possibility of priming effect. The priming effect could activate naïve T-lymphocytes through their exposure to new antigens. As mentioned previously, the BBB is protecting the tumor from T-lymphocytes infiltration and immune activation. Thus, we have shown that the possibility of detecting GL261 tumors in the peripheral circulation might activate the global antitumor effect. Further functional demonstration of lymphocyte activation should be performed to evaluate any priming effect of UMBO.

Our results showing microglia activation in the UMBO plus anti-PD-L1 treated GL261-bearing mice suggest a possible mechanism for the observed enhanced therapeutic efficacy of anti-PD-L1. Our flow cytometry analysis is consistent with a newly published article that observed a high ratio of Iba1 staining in sonicated brain regions compared to the non-sonicated one. However, this observation was not significant [START_REF] Sinharay | In vivo imaging of sterile microglial activation in rat brain after disrupting the blood-brain barrier with pulsed focused ultrasound: [18F]DPA-714 PET study[END_REF]. PD-L1 is expressed on the cell surface of both GL261 and microglia (Chen et al., 2019b). A possible effect on microglia phenotype might be related to the combined effect of UMBO and anti-PD-L1 delivery to the brain parenchyma.

Activated microglia might have an impact on the cytotoxic effect against GL261 tumor cells [START_REF] Li | Microglia activation triggers oligodendrocyte precursor cells apoptosis via HSP60[END_REF].

To date, there is no clear evidence on the effect of UMBO on T-lymphocytes passage to the brain. We have not observed any significant elevation in the percentage of CD8 + and CD4 + T-lymphocytes at one timepoint (~16 hours). This effect might be related to the timing of sample collection. We have not evaluated the effect of our treatment regimen at later time points. We have seen a delayed antitumor effect in UMBO and anti-PD-L1 group which could be related to a delayed effect on T-lymphocytes. Furthermore, we have not analyzed any subpopulations of CD8 + T lymphocytes i.e., PD-1 + CD8 + T-lymphocytes.

Syngeneic mice models and especially the GL261 mouse model is one limitations of this study. GL261 mouse model has a high mutation load which is not consistent with GBM patients. Not to mention, a heterogeneity of responses in the GL261 mouse model was reported when treated with ICIs in vivo [START_REF] Aslan | Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas[END_REF]. Another limitation of our findings is the inability to demonstrate functional analysis of the role of UMBO in priming naïve T-lymphocytes through their exposure to new antigens. Additional functional analysis on the effect of UMBO plus anti-PD-L1 would explain the dramatic effect on OS that was observed in our study.

Conclusions

Our study showed statistically significant increased brain penetration and efficacy of anti-PD-L1 in GL261-bearing mice when delivered by UMBO. We have also provided clear evidence of the possible safe and effective delivery of large therapeutic agents using UMBO. Further investigations are needed to confirm the impact of UMBO on brain penetration and efficacy of chemotherapeutic agents and anti-PD-L1 to overcome the resistance of GBM to the current treatments. TME in GBM is peculiar compared to TME in non-neurological cancers. Indeed, the brain is an immune-privileged organ, glymphatic recently discovered in animal models and is primarily active during sleep and neuropathological disorders. Besides, the BBB protects the brain, limiting the exchange between the normal and tumor brain and the rest of the body. By controlling these exchanges, the BBB reduces endogenous (i.e., host immune system) and exogenous (i.e., medicines) anti-tumor molecular and cellular interventions against brain cancer. This thesis main goal is to investigate how TME modulation such as BBB disruption may overcome the resistance of GBM to anti-tumor treatments.

A. ABC transporters as a GBM TME therapeutic target

A few ABC transporters are predominantly expressed in ECs of the BBB [START_REF] Mahringer | ABC transporters at the blood-brain barrier[END_REF]. Indeed, ABCB1 and ABCG2 are expressed in ECs of the BBB, while others (ABCC, ABCG2) can be found in other cells such as astrocytes and neurons [START_REF] Linton | Structure and function of ABC transporters: the ATP switch provides flexible control[END_REF][START_REF] Shen | ABC transporters and drug efflux at the blood-brain barrier[END_REF][START_REF] Mahringer | ABC transporters at the blood-brain barrier[END_REF].

We reviewed the effect of ABC transporters in GBM chemoresistance. Furthermore, we summarized all the original research articles that have been published to discuss the role of ABC transporters in GBM chemoresistance, and the strategies that have been developed to overcome their negative effects for therapy purposes.

In the literature review article, we showed that ABCB1, ABCG2, and ABCC1 are the most studied ABC transporters in GBM. Furthermore, we discussed the failure of ABC protein blocking strategy to show significant clinical benefit. Although a limited number of clinical trials were initiated to modulate ABC proteins, the clinical benefit from these studies was hardly met and could be related to a few reasons. Firstly, the study design was not optimal, i.e., in the early clinical trials, the patients were not stratified based on high expression of ABC proteins, and a precise evaluation of the role of ABCB1 and ABCG2 transporters in patients could not be conducted. Secondly, modulators of ABC proteins could change the pharmacokinetics of other drugs reducing their anti-tumor properties.

Finally, the dose of inhibitors selected to inhibit the ABC protein was perhaps not sufficient, or a higher dose could not be applied safely in patients.

The selection of cancer cell lines is a crucial step in in vitro studies dedicated to GBM.

Many commercial human cell lines are available for GBM. Our group showed previously that patient-derived cell lines (PDCL) recapitulated better ABC gene expression patterns of human GBM compared to commercial cell lines and can thus be considered a better model to test the biology of ABC proteins in GBM. Besides, we found that fetal bovine serum that is usually added to the cell culture medium for commercial GBM cell lines modulates resistance to TMZ. Moreover, the high passaging number of commercial GBM cell lines could change the expression level of ABC protein and could lead to conclusions irrelevant to newly diagnosed human tumors.

Finally, we summarized the strategies that are developed to overcome the ABC transporters-induced chemoresistance in GBM. Strategies vary from partial to complete chemical or physical inhibition of ABC transporter to approaches that overcome ABC efflux pumps i.e., (i) nanocarriers technologies, (ii) antibody-drug conjugates and (iii) UMBO.

Inhibition of ABC transporters limits the efflux of therapeutic agents from ECs to blood and increase their penetration into the normal and the tumor brain. In addition to chemical or pharmaceutical inhibition of ABC proteins, physical disruption approaches may also bypass the BBB. Indeed, recently, ultrasound application was shown to inhibit the expression of ABC transporters. UMBO could decrease the expression of ABCB1 protein in cerebral vessels without affecting the integrity of other proteins [START_REF] Choi | Diminished Expression of P-glycoprotein Using Focused Ultrasound Is Associated With JNK-Dependent Signaling Pathway in Cerebral Blood Vessels[END_REF]. More studies should be performed to evaluate the role of UMBO to bypass the BBB and their role in enhancing the efficacy of chemotherapies in GBM.

B. Clinical significance of GBM TME protein

The expression of CD80 and CD86 in GBM tissues and their prognostic significance was not reported before. We have investigated the mRNA and protein expression of CD80 and CD86 in newly diagnosed GBM patients aged below 70 and with KPS above 70 treated with the standard of care. We have observed a link between CD80 and CD86 expression to prognosis and, more specifically, PFS in our local discovery cohort and the TCGA dataset.

CD80 and CD86 are expressed in the GBM tumor bulk. We assumed that CD80 and within the normal and the tumor brain [START_REF] Hodges | Mutational burden, immune checkpoint expression, and mismatch repair in glioma: implications for immune checkpoint immunotherapy[END_REF][START_REF] Galstyan | Blood-brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy[END_REF][START_REF] Beccaria | Ultrasound-induced blood-brain barrier disruption for the treatment of gliomas and other primary CNS tumors[END_REF].

Our first data confirmed the limitation of ICBs efficacy in GL261 and Nfpp10 GBM mouse model. Consistent with our data, preclinical evaluation of anti-PD-L1 and anti-CTLA-4 in GL261 showed comparable limited efficacy of both antibodies in GL261 GBM mouse model. Their study has used a different treatment regimen, yet the efficacy was comparable [START_REF] Reardon | Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model[END_REF]. Therefore, we have investigated ICBs combined with UMBO in murine preclinical models of GBM to overcome some of these limitations.

UMBO parameters, i.e., acoustic pressure, time, microbubbles dose, and ultrasound waves frequency, influence UMBO's safety and efficacy. The safety and the efficacy of UMBO were evaluated in C57BL/6 mice bearing GL261 tumor cells. UMBO was optimized to target the right hemisphere where the GBM cells are grafted. Additionally, each mouse model should be subjected to an in-depth evaluation of the UMBO parameters. Here, I evaluated UMBO parameters and treatment frequency in GL261 GBM mouse models. T1weighted MRI results described previously showed a marked gadolinium contrast enhancement within an hour following UMBO. The large area of UMBO opening in animal models suggest a larger delivery of the therapeutic antibodies to the normal and tumor brain, a larger penetration of T-lymphocytes within the normal and tumor brain, and a larger circulation of tumor antigen from the brain to blood for better priming.

Antibody sequence is crucial in the nano-surface and molecular-orientation limited (nSMOL) proteolysis method to measure the antibody through mass spectroscopy (LC-MS). nSMOL method identify a signature peptide i.e., nivolumab signature peptide was identified as ASGGITFSNSGMHWVR. To identity the signature peptide antibody sequencing should be performed. We have obtained our murine anti-PD-L1 antibody from Genentech, USA through material transfer agreement. This agreement did not allow any sequencing of the antibody. Therefore, we have used nivolumab for this purpose. On the other hand, we have optimized our in-house ELISA analysis to study the pharmacokinetics of anti-PD-L1. This experiment is currently under analysis.

Nivolumab concentration in the brain parenchyma was enhanced up to 28 folds following UMBO. In our setting, UMBO was optimized to distributing one hemisphere. In our pharmacokinetics analysis, we have used a whole-brain homogenization method instead of one hemisphere homogenization. Consequently, local concentrations of nivolumab within the sonicated hemisphere are probably higher. Consistent with our data, a study has shown that UMBO enhanced the delivery of bevacizumab ~149 KDa to the brain parenchyma by 5.7-to 56.7-fold compared to non-sonicated brain in a glioma mouse model [START_REF] Liu | Focused Ultrasound Enhances Central Nervous System Delivery of Bevacizumab for Malignant Glioma Treatment[END_REF]. An already planned experiment with optimized ELISA analysis is going to be done in the foreseen weeks to identify the pharmacokinetics of anti-PD-L1 (Genentech, 6E11) with and without UMBO.

In the other direction, we have investigated the passage of tumor molecules from the tumor bulk to blood flow stream. Significant number of GL261 copies were identified in the blood stream of mice treated with UMBO 30 min prior to blood collection. We can demonstrate that tumor DNA in whole blood is higher in GL261-bearing mice treated with UMBO compared to their counterparts without UMBO. We hypothesized that this increase of tumor DNA passage may increase T-lymphocytes priming. However, this hypothesis remains to be tested. Furthermore, additional experiments to study the effect of UMBO at different time point to follow the fate of GL261 DNA in the blood stream overtime.

We initially hypothesized that the BBB was responsible for the limited efficacy by mainly blocking anti-CTLA-4 and anti-PD-L1 from reaching the GBM tumor. This hypothesis is consistent with two recent studies that reported an enhanced efficacy of ICBs following their brain tumor . Interestingly we have observed an enhanced efficacy of anti-PD-L1 when delivered after UMBO. 76 % long-term survival (over 100 days) compared to (4/15) 26 % in anti-PD-L1 alone in GL261-bearing mice.

A possible effect on microglia's phenotype might be observed as an effect and anti-PD-L1 delivery plus UMBO to the brain parenchyma. Therefore, our foreseen flow cytometry analysis of immune cell populations is inevitable to support this observation.

General Conclusion

The clinical role of ABC proteins is still under investigation, and the failure of previous clinical trials raised several questions regarding the strategies to overcome MDR in GBM A wide range of in vitro and in vivo models could allow precise testing of the novel drugs.

One of the most important models is the utilization of low passaging of PDCLs which carry the molecular features of ABC efflux pumps. Furthermore, dual ABC inhibitors with a high specificity could be developed for more effective treatment strategies.

There are multiple immune pathways involved in antitumor immunity. Because of the complexity and evolution of tumor immune responses, it is impossible to rely on blockade of one or two inhibitory pathways to elicit long-lasting or curative antitumor effects for many patients. Combination therapies are being developed i.e., between (i) chemotherapies and ICBs, (ii) ICBs and vaccines, (iii) block multiple immune inhibitory pathways or provide agonists to activate the immune stimulatory pathways. It is reasonable to develop markers to guide future combination therapy strategies and for patient stratifications to hopefully increase response rates in clinical trials.

Finally, my thesis opens new avenues for the efficacy of UMBO to deliver antibodies and in the treatment of GBM. Indeed, further studies should be warranted before the translation of this work to clinical trials. ICBs plus chemotherapies are currently being evaluated in clinical trials. Combinational therapies of UMBO plus TMZ and ICBs could be evaluated as a next step. Furthermore, UMBO was able to deliver both chemotherapies and antibodies to the brain. Additional treatments as cellular therapy or vaccinations could be delivered using UMBO in vivo studies.

  presentation and diagnosis of GBM 1.2.3 Standard treatment of GBM 1.2.4 Prognostic factors in GBM patients 2. GBM CELL BIOLOGY AND THEIR MICROENVIRONMENT 2.1 GBM cells and heterogeneity 2.1.1 Tumor cells origin, and intratumor cell heterogeneity 2.1.2 Four signaling pathways are disrupted in GBM 2.2 Tumor cell microenvironment 2.2.1 Niches and stem cells of GBM 2.2.2 Cellular components of TME 2.2.3 Glioma-associated neovascularization 2.2.4 The blood-brain barrier 3. THERAPEUTIC STRATEGIES TO MODULATE THE TUMOR MICROENVIRONMENT 3.2 Modulation of the immune system 3.2.1 History and concept of immune system's modulation 3.2.2 Antitumor immune response 3.2.3 Tumor escape mechanisms 3.2.4 Immunotherapy 3.2.5 Monoclonal antibodies 3.2.6 Monoclonal antibodies against checkpoint proteins 3.3 Overcoming, disrupting, or bypassing the BBB 3.3.1 The BBB limits drug penetration into normal and tumor tissue 3.3.2 Role of ultrasound-mediated BBB opening 3.3.3 UMBO and antibodies delivery to the brain 3.3.4 Ultrasound mediated BBB opening in clinical settings THESIS OBJECTIVES RESULTS Role of Multi-Drug Resistance in Glioblastoma Chemoresistance: Focus on ABC Transporters CD80 and CD86: expression and prognostic value in newly diagnosed glioblastoma Temporary blood-brain barrier disruption by low-intensity pulsed ultrasound increases anti-PD-L1 delivery and efficacy in glioblastoma mouse

  Simplified classification of diffuse gliomas according to the WHO 2016 publication ............................................................................................................................................. Table 2: Therapies for recurrent GBM patients ................................................................................. Table 3: Examples of molecular and innovative therapeutic strategies used in clinical trials for GBM treatment ................................................................................................................... Table 4: Prognostic factors in GBM patients ..................................................................................... Table 5: Molecular characteristics of identified subtypes ............................................................. Table 6: Summary of GBM escape mechanisms ............................................................................... Table 7: Available commercial microbubbles used in combination of ultrasound mediated BBB opening ..................................................................................................................... Table 8: Original research articles that combined UMBO with antibodies and macromolecules delivery. ................................................................................................................. Table 9: Analysis of published studies that show the effects of ABC transporters on chemotherapeutic agents used in GBM...................................................................................... Table 10: Identified subfamilies of ABC transporters and their physiological functions. ..

Figure 1 :

 1 Figure 1: Incidence of primary brain tumor's subtypes illustrated as percentage ...............

Figure 2 :

 2 Figure 2: MRI imaging of GBM patient ................................................................................................

Figure 3 :

 3 Figure 3: MGMT promoter methylation as a prognostic factor in GBM..................................

Figure 4 :

 4 Figure 4: Main signaling pathways disrupted in GBM. RTK, TP53 and RB pathways and their implications on apoptosis, senescence, and cell cycle progression. ......................

Figure 5 :

 5 Figure 5: Niches organizations in GBM. ..............................................................................................

Figure 6 :

 6 Figure 6: GBM microenvironment components ...............................................................................

Figure 7 :

 7 Figure 7: Glioma-associated neovascularization ..............................................................................

Figure 8 :

 8 Figure 8: BBB is formed of different types of cells tightly knit together .................................

Figure 9 :

 9 Figure 9: Anticancer immunity can be described as a cycle leading to an accumulation of immune-stimulating factors that enhance T-lymphocytes responses .............................

Figure 10 :

 10 Figure 10: The concept of immunoediting. ........................................................................................

Figure 11 :

 11 Figure 11: A list of the therapeutic antibodies currently available and associated with their potential targets of immune checkpoint pathways on T-lymphocytes and antigen presenting cells. ...................................................................................................................

Figure 12 :

 12 Figure 12: Mechanism of ultrasound mediated BBB opening ...................................................

Figure 13 :

 13 Figure 13: Two photon microscopy to visually follow the leakage of a Dextranconjugated Texas Red through a micro-vessel wall after UMBO ......................................

Figure 14

 14 Figure 14 Efficacy/safety: impact of acoustic pressure on treated rats. ..................................

Figure 15 :

 15 Figure 15: Cellular structure of the blood-brain barrier.. .............................................................

Figure 16 :

 16 Figure 16: Molecular structure of ABC transporters. ......................................................................

Figure 17 :

 17 Figure 17: Functional analysis of ABC transporters ......................................................................... Figure 18 Represents the methodology of this literature review.. ............................................

Figure 19 :

 19 Figure 19: Methods that are being developed to overcome ABC transporters....................

Figure 20 :

 20 Figure 20: Data distribution of CD80 and CD86 mRNA and protein expression in ONT cohort ....................................................................................................................................................

Figure 21 :

 21 Figure 21: Represent the expression of CD86 and CD80 proteins in paraffin sectioned GBM samples. .....................................................................................................................................

Figure 22 :

 22 Figure 22: Spearman correlations between the mRNA expression and protein expression of CD80 and CD86 in newly diagnosed glioblastoma patients ........................................

Figure 23

 23 Figure 23 CD80 and CD86 mRNA expression and outcome in GBM in both ONT cohort and TCGA dataset. ............................................................................................................................

Figure 24 :

 24 Figure 24: Proportional hazards: multivariate analysis of CD86 protein expression and mRNA expression. .............................................................................................................................

Figure 25 :

 25 Figure 25: Sequences of the forward and reverse primers for CD80, CD86 and PPIA .....

Figure 26 :

 26 Figure 26: Anti-PD-L1 increased survival of GL261 and Nfpp10 -bearing mice. ................

Figure 27 :

 27 Figure 27: UMBO is safe and effective in immunocompetent mice. .......................................

Figure 28 :

 28 Figure 28: UMBO dramatically increases the efficacy of anti-PD-L1 in GL261-bearing mice. .......................................................................................................................................................

Figure 29 :

 29 Figure 29: UMBO increased the penetration of ICBs into brain parenchyma.. ...................

Figure 30 :

 30 Figure 30: UMBO modulates microglia phenotype and enhance the presence of CD8 + Tlymphocytes in GL261 tumors.. ....................................................................................................

Figure 1 :

 1 Figure 1: Incidence of primary brain tumor's subtypes illustrated as percentage. Adapted from (Ostrom et al., 2017).

Figure 2 :

 2 Figure 2: MRI imaging of GBM patient. Panel a1: T1-weighted MRI imaging. Panel a2: the central necrotic area is stained in yellow. Panel b1: T1-weighted MRI imaging with gadolinium contrast enhancement. Panel b2: The contrast uptake zone colored in red corresponds to the proliferative zone of the tumor. Panel c1: FLAIR T2-weighted MRI imaging. Panel c2: The area colored in green corresponds to an area of vasogenic edema in which tumor cells are present[START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF].

Figure 4 :

 4 Figure 4: Main signaling pathways disrupted in GBM. RTK, TP53 and RB pathways and their implications on apoptosis, senescence, and cell cycle progression. In addition, TERT promoter mutations, not shown in this figure, are found in ~80% of GBM[START_REF] Chen | Malignant glioma: lessons from genomics, mouse models, and stem cells[END_REF].

Figure 5 :

 5 Figure 5: Niches in GBM. Panel A: Perivascular niche. Panel B: Hypoxic niche. Panel C: Invasive niche[START_REF] Hambardzumyan | Glioblastoma: Defining Tumor Niches[END_REF] Bergers, 2015, Broekman et al., 2018).

  Regulatory T-lymphocytes (Tregs) are considered a small population of CD4 + Tlymphocytes expressing FoxP3 transcription factor. Tregs are a subpopulation of circulating lymphocytes with immune suppressive effects. Tregs also express CD25 and CTLA-4, and their role in GBM is still under investigation. Studies have shown that glioma associated Tregs are most likely originating from the thymus. They migrate within the GBM bulk via chemotactic attraction from the TME (González-Tablas[START_REF] Pimenta | Tumor cell and immune cell profiles in primary human glioblastoma: Impact on patient outcome[END_REF]

Figure 7 :

 7 Figure 7: Several steps of glioma associated neovascularization: The first step starts with vascular cooption (A) followed by angiogenesis (B), vasculogenesis (C), vascular mimicry (D), and GBM-endothelial cell trans differentiation (E). Adapted from (Hardee and Zagzag, 2012).

  immunity cells are alerted to the presence of a growing tumor. The following two steps occur when the captured antigens on MHC class I and MHC class II molecules are presented to T-lymphocytes by DCs, triggering the activation and the priming of effector T-lymphocytes against tumor-specific antigens. At this stage, the immune response is initiated, with the ratio of T effector lymphocytes to T-regulatory lymphocytes presenting a critical determinant in this response.

Figure 9 :

 9 Figure 9: Anticancer immunity can be described as a cycle leading to an accumulation of immunestimulating factors that enhance T-lymphocytes response (Chen and Mellman, 2013).

Figure 10 :

 10 Figure 10: The concept of immunoediting. Adapted from (Vesely et al., 2011).

Figure 11 :

 11 Figure 11: A list of the therapeutic antibodies currently available and associated with their potential targets of immune checkpoint pathways on T-lymphocytes and antigen presenting cells. PD-1, CTLA-4, PD-L1 and CD80 are targets with available antibodies to block their activity.

Figure 12 :

 12 Figure 12: Mechanisms of UMBO. Panel A: represent the values of SH acoustic emission in blue detected in real-time (Sonocloud device for small animal). On the other hand, BB in red, is maintained with low values to reduce adverse effects. Panel B: Although the mechanisms potentially involved in UMBO in combination with microbubbles are poorly understood, accumulative evidence suggest that the mechanism of stable cavitation is responsible for the transient opening of the BBB. Adapted from[START_REF] Beccaria | Ultrasound-induced blood-brain barrier disruption for the treatment of gliomas and other primary CNS tumors[END_REF]

Figure 13 :

 13 Figure 13: Two photon microscopy to visually follow the leakage of a Dextran-conjugated Texas Red (10 KDa) through a micro-vessel wall after UMBO. Numbers correspond to the time in second following UMBO[START_REF] Cho | Two-photon fluorescence microscopy study of cerebrovascular dynamics in ultrasound-induced blood-brain barrier opening[END_REF].

Figure 14

 14 Figure 14Example of efficacy/safety: impact of acoustic pressure on treated rats. High acoustic pressure induces large UMBO with a significant tissue damage and associated hemorrhage. Consistent with such effect, duration of sonication (long sonication's time is associated with tissue damage) and microbubbles doses. The opening is visible by the entry of Evans blue into the brain parenchyma on the right hemisphere of a rat brain. Hematoxylin and eosin stains of brain sections allow identification of tissue damage[START_REF] Shin | Focused ultrasound-mediated noninvasive blood-brain barrier modulation: preclinical examination of efficacy and safety in various sonication parameters[END_REF]

  , reviewing the literature to understand the role of ABC transporters in GBM resistance to chemotherapy. B. Secondly, exploring the expression of immune checkpoint proteins, mainly CD80 and CD86, in the GBM TME and their role as prognostic markers in newly diagnosed GBM patients.C. Finally, evaluating the role of the UMBO to improve and to understand response to ICB GBM mouse model.A. Review of the literature to understand the role of ABC transporters in GBM chemotherapy resistanceGBM is the most frequent and the most aggressive primary cancer of the brain in adults. Without doubt, therapeutic strategies to overcome GBM resistance are continuously failing. Not to mention, after initial efficacy, GBM cells acquire resistance to TMZ and other chemotherapeutic agents via multiple mechanisms, including the expression of ATP-binding cassette (ABC) efflux proteins. These transporters are involved in normal physiological functions, i.e., physiological cholesterol transport and elimination of toxins, but also it plays a role in pathological conditions, i.e., chemotherapies drug resistance. In humans, each ABC protein has specific tissue locations and specific functions.

  Manuscript 1 (Submitted): Mohammed AHMED, Maite VERREAULT, Xavier DECLAEVE, Ahmed IDBAIH 2021. Role of Multi-Drug Resistance in Glioblastoma Chemoresistance: Focus on ABC Transporters, Elsevier Publishing Company • Manuscript 2 (Submitted): Mohammed AHMED, Isaias. HERNANDEZ., Franck BIELLE, Maite VERREAULT, Julie LEROND, Agusti ALENTORN, Marc SANSON, Ahmed IDBAIH 2021. CD80 and CD86: expression and prognostic value in newly diagnosed glioblastoma. Pathology and Oncology Research Journal • Manuscript 3 (in Preparation): Mohammed AHMED, Mohammed Ahmed1,2*, Nolwenn LEMAIRE, Emie QUISSAC, Rana SALAM, Isaias HERNANDEZ, Coralie L GUERIN, Lea GUYONNET, Noël ZAHR, Laura MOUTON, Mathieu SANTIN, Alexandra PETIET, Charlotte SCHMITT, Guillaume BOUCHOUX, Marc SANSON, Maite Verreault, Alexandre Carpentier, Ahmed IDBAIH, 2021. Increased brain delivery of anti-programmed deathligand 1 using low-intensity pulsed ultrasound-mediated blood-brain barrier opening is associated with increased anti-tumor efficacy and microglia activation in glioblastoma mouse models.

  they contain the Walker A motif -a phosphate-binding structure-, Walker B motif, and Walker C motif. C motif is specific for ABC proteins while Walker A and B are present in all ATP dependent proteins. The TMDs include six or ten transmembrane helices depending on ABC superfamily members. There are two types of transmembrane helices: the inward part (open to the cytoplasm) and the outward part (open to the extracellular environment).

  ABC proteins hydrolyze ATP to efflux chemical agents against their concentration gradient. The active transport cycle starts with binding a substrate, e.g., xenobiotic to a high-affinity structure formed by the TMDs and two ATP molecules binding NBDs. A conformational contribution forms the ATP binding sites at the NBDs residues from each NBD monomer. As a result, a conformational change in TMDs occurs from either outward to inward (importer) or vice versa (exporter) allowing the NBD units to form a dimer. The NBD dimer induces a major conformational change on the TMDs, allowing the xenobiotic to be translocated across the plasma membrane. The hydrolysis of ATP allows the NBD dimers to be dissociated and again inducing the TMDs conformational change, resulting in the xenobiotic to be released. A final step of restoration of the open NBD-dimer conformation then takes place (Figure

Figure 15 :

 15 Figure 15: The blood-brain barrier (BBB) is formed of different types of cells tightly knit together.Highly specialized endothelial cells (ECs) surround blood vessels and form part of the BBB. In addition to brain ECs various cells contribute to the structure of BBB. Pericytes (represented in green) are attached to endothelium cells via gap junctions whilst astrocytes end feet (represented in purple) surround endothelial cells of the BBB, providing structural and functional support to these cells. Five mechanisms are known to regulate the entry of molecules to the brain. The efflux pumps pathway is considered a mechanism of active transport through the BBB[START_REF] Drean | Blood-brain barrier, cytotoxic chemotherapies and glioblastoma[END_REF].

Figure 16 :

 16 Figure16: A full ABC transporter consists of four building units. The first two building units called TMDs are formed by six transmembrane segments. TMD1 and TMD2 are colored in green and pale red, respectively. The two other building units are called NBDs, NBD1 (pale blue) and NBD2 (red). ABC half transporters have only one TMD and one NBD and need to dimerize to become a functional protein. Additionally, some other ABC transporters have an additional TMD unit that is conjugated to the N-terminus of the protein and called "Long" ABC transporter[START_REF] Deeley | Transmembrane transport of endo-and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins[END_REF].

Figure 17 :

 17 Figure 17: ABC transporters are transmembrane proteins capable of actively transporting a xenobiotic from the intracellular to the extracellular compartments. This active transport requires the hydrolysis of ATP to provide the energy necessary for the transport. The active transport cycle starts with the binding of the xenobiotic to a high-affinity structure formed by the TMDs (step 1). As a result, a conformational change makes the NBD units more exposed for ATP binding. The NBD dimer induces a major conformational change on the TMDs allowing the xenobiotic to be translocated (step 2). The hydrolysis of ATP allows the NBD dimers to be dissociated and again induces a TMDs conformational change (step 3). A final step of restoration of the open NBD-dimer conformation then takes place (step 4)(Linton and Higgins, 2007, Zolnerciks et al., 2011).

  (ONT) is a local brain tumor tissue bank collecting samples from patients operated at the University Hospital La Pitié-Salpêtrière. All samples were collected with informed consent from patients. The inclusion criteria of the discovery local cohort (47 patients) were as follow: (i) newly diagnosed and histologically verified GBM, (ii) age at diagnosis is below 70 years, (iii) KPS above 70%, (v) known MGMT promoter methylation status, (vi) known IDH status, (vii) treated with the standard first-line therapeutic regimen including chemoradiation and adjuvant temozolomide and, (viii) a documented clinical follow-up. The validation cohort (121 patients, the cancer genome atlas, TCGA cohort) clinical information and RNA-seq data (read counts) were downloaded from the National Cancer Institute's Genomic Data commons (GDC) Data portal and from the NCBI GEO GSE62944, respectively.

Figure 20 :

 20 Figure 20: Panel A Violin plot to visualize the data distribution of CD80 and CD86 mRNA expression in ONT cohort Panel B shows CD80 and CD86 protein expression in ONT cohort.

Figure 21 :

 21 Figure 21: Represent the expression of CD86 and CD80 proteins in paraffin sectioned GBM samples. Panel A: high expression of CD86. Panel B: low expression of CD86. Panel C: High expression of CD80. Panel D: low expression of CD80.

Figure 22 :

 22 Figure 22: Panel A-B Spearman correlations between the mRNA expression and protein expression in CD86 (Panel A) and CD80 (Panel B). X-axis indicated mRNA expression values while Y-axis indicates protein expression value as percentage of positive IHC signals (CD86 in Panel A and CD80 in Panel B).

Figure 23 Figure 24 :

 2324 Figure 23 CD80 and CD86 mRNA expression and outcome in GBM in both ONT cohort and TCGA dataset. Panel A: Kaplan-Meier PFS estimates in GBM patients in relation to CD86 (ONT database) Panel B: Kaplan-Meier PFS estimates in GBM patients in relation to CD80 (ONT cohort). C: Kaplan-Meier PFS estimates in GBM patients in relation to CD86 (TCGA dataset). D: Kaplan-Meier PFS estimates in GBM patients in relation to CD80 (TCGA dataset)

Figure 25 :

 25 Figure 25: Sequences of the forward and reverse primers for CD80, CD86 and PPIA. Universal Probe Library numbers that were used in our RT-PCR.

4F

  Pharmacokinetics and Therapeutic Drug Monitoring unit, INSERM, CIC-1901, UMR ICAN 1166, Pitié-Salpêtrière Hospital, Sorbonne Université, AP-HP, F-75013 Paris, France. 5 Centre de Neuroimagerie de Recherche, Institut du Cerveau -Paris Brain Institute -ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière, Paris, France 6 CarThera, Institut du Cerveau et de la Moelle épinière (ICM), F-75013, Paris, France 7 Sorbonne Université, Institut du Cerveau -Paris Brain Institute -ICM, Inserm, CNRS, AP-HP, Hôpital de la Pitié Salpêtrière, DMU Neurosciences, Service de Neurologie 2-Mazarin, .ahmed.icm@gmail.com Ahmed Idbaih, M.D, Ph.D. Sorbonne Université, Institut du Cerveau-Paris Brain Institute -ICM, Inserm, CNRS, AP-HP, Hôpital de la Pitié Salpêtrière, DMU Neurosciences,

GL261

  cells were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum and 1% Penicillin/Streptomycin. Cells were passaged twice weekly according to their confluence. Nfpp10-luciferase (NF1, PTEN, and TP53 deficient) as described in[START_REF] Friedmann-Morvinski | Dedifferentiation of Neurons and Astrocytes by Oncogenes Can Induce Gliomas in Mice[END_REF]. GBM cells were obtained from Dr Gabriele Bergers' laboratory. Nfpp10-luciferase cells were maintained in culture using DMEM/F12 (Gibco; Life Technologies) culture medium supplemented with 1% penicillin-streptomycin, EGF (20 ng/mL), and FGF (20 ng/mL; Preprotech), Heparin 2 µg/ml (Sigma H33930) and N-2-supplement 1/100 (Gibco 17502-048). The animal ethics committee at the Ministry of Higher Education and Research in Paris, France, approved all protocols involving live mice (protocol #17503 and #26137). C57BL/6 mice were purchased from Charles River and were given a week of acclimation before starting any experiment.

  7 T MRI scanner (Biospec, Bruker BioSpin, Germany) equipped with a CryoProbe dedicated to mouse brain imaging(Biospec, Bruker BioSpin, Germany). The total MRI experiment time was approximately 80 min per mouse (including MRI settings, acquisitions, and gadolinium injection), during which the animals were anesthetized with 1% isoflurane in O2 (2 L/min). The respiration rate was monitored via a respiration pillow sensor, and the body temperature was maintained using a heated water circuit incorporated into the cradle. The head was placed in a prone position and restrained stereotaxically by a bite bar and ear pins. For each animal, the protocol consisted in : (i) acquiring pre-gadolinium enhancement anatomical T1-weighted (T1w) images using a Multi-Slice Multi Echo (MSME) sequence with the following parameters: T.R. = 400 ms, T.E. = 5 ms (one single echo), four averages, 14 slices, and resolution = 60x60x500 μm 3 , (ii) following injection of a total volume of 100 µL of gadolinium (DOTAREM ® , Guerbet, Aulnay-sous-Bois, France) at 0.5 mM and at physiological temperature in the tail vein of the mouse outside the MRI scanner, (iii) acquiring post-gadolinium T1w images using the same sequence as used for (i) and, (iv) acquiring post-gadolinium injection T2*-weighted (T*2w) images using a Multi Gradient Echo (MGE) sequence. MGE sequence was acquired with the following parameters: T.R. = 80 ms, ten echoes ranging from T.E. = 2.7 ms to 35.1 ms (echo spacing = 3.6 ms), and isotropic resolution of 60x60x60 μm 3 .

  -B) and by fluorescence microscopy with a BBB opening on the right hemisphere (Figure27-C). Next, we evaluated UMBO parameters and treatment frequency in GL261 GBM mouse models. T1w MRI (Figure27-E) showed a marked gadolinium contrast enhancement within an hour following emission of ultrasound.

Figure 26 :

 26 Figure 26: Anti-PD-L1 increased survival of GL261 and Nfpp10 -bearing mice. Animals were treated with anti-PDL-1, IgG1 antibody, anti-CTL-4, and vehicle for four doses. Panel A: bioluminescence measures normalized to the first measured value performed on day 7 after cell inoculation in GL261-bearing mice model. Each dot represents values for one animal and the line represents the mean value for the group. Bioluminescence signal was measured weekly. Dotted lines represent the days of treatments. Panel B: Kaplan Meier curves in GL261-bearing mice. Panel C: mean of animal body weight in each group over time. Panel D: bioluminescence measures normalized to the first measured value performed on day 7 after cell inoculation in Nfpp10 mouse model. Dotted lines represent the days of treatments. Panel E, Kaplan Meier curves in Nfpp10-bearing mice. Panel F: mean of animal body weight in each group over time.

Figure 27 :

 27 Figure 27: UMBO is safe and effective in C57BL/6 mice. Panel A: BBB integrity in GL261-bearing and Nfpp10-bearing mice. Hoechst staining (in green) was not detected in normal (N) brain tissue. However, higher staining intensity was observed in the tumor (T) area in Nfpp10 model compared to GL261 model. Panel B-C: Evans blue staining was enhanced in sonicated brain hemisphere compared to the control hemisphere visually (Panel B) and by fluorescence (Panel C; Evans blue in red, DAPI in blue) in a cryo-sectioned mouse brain. Panel D: schematic representation of Sonocloud® device used in our setting and timeline used for UMBO plus anti-PD-L1 treatments. Panel E: T1W MRI showed a marked gadolinium contrast enhancement within an hour following the ultrasound emission. The lower two T1-MRI images were obtained after UMBO (pre-gadolinium left MRIs; and post gadolinium right MRIs).

Figure 28 :

 28 Figure 28: UMBO dramatically increases the efficacy of anti-PD-L1 in GL261-bearing mice. Panel A: Repeated UMBO application alone does not affect OS and mouse body weight in GL261-bearing mice compared to non-treated mice. Panel B: Schematic representation of the timeline used for UMBO plus anti-PD-L1 treatments. Panel C: All treatments have no impact on mice body weight. Panel D: bioluminescence measures normalized to the first measured value performed on day 7 after cell inoculation in GL261-bearing mice model. Each dot represents values for one animal and the line represents the mean value for the group. Bioluminescence signal was measured weekly. Dotted lines represent the days of treatments. Panel E: UMBO plus anti-PD-L1 increased overall survival: *p<0.05 compared to anti-PD-L1 alone and ***p<0.0001 compared to vehicle-treated group Panel F: Comparison of tumor size in UMBO plus anti-PD-L1 at day 45 compared to anti-PD-L1 treated mouse in GL261-bearing mice.

Figure 29 :

 29 Figure 29: UMBO increased the penetration of ICBs into brain parenchyma. Panel A: IHC staining of anti-PD-L1 (BXCELL, in the brain parenchyma showing greater staining in the UMBO-targeted right hemisphere. Panel B: Pharmacokinetic analysis of Nivolumab concentration in the C57BL/6 mice blood and brain. Panel C: Brain/plasma ratio of nivolumab concentration over time. UMBO enhanced the brain/plasma ratio of nivolumab compared to control mice. Panel D: ddPCR analysis of Luciferase DNA in the blood 30 minutes following UMBO. Panel E: ddPCR analysis of mKate2 DNA in the blood 30 minutes following UMBO.

Figure 30 :

 30 Figure30: UMBO plus anti-PD-L1 activates microglia and modulates microglial phenotype. Panel A: Flow cytometry analysis of the percentage of total microglia in all different groups (n=4-5). Panel B: UMBO plus anti-PD-L1 significantly enhanced (*p<0.05) the percentage of CD68 + cells than anti-PD-L1 alone. Panel C-D: UMBO plus anti-PD-L1 did not influence CD4 + and CD8 + T-lymphocytes percentages compared to other groups. Panel E-F: No significant difference in CD206 + and CD206 - macrophages in all groups. UMBO plus anti-PD-L1 did not modulate macrophages' expression. Panel G: Green: Iba1 microglia/macrophages Blue: DAPI nuclear staining; microglia staining in anti-PD-L1 plus UMBO (right photos) treated group confirm a phenotype of activated microglia. Double nuclear staining in the UMBO plus anti-PD-L1 staining shows a possible induction of microglia cell division.

  CD86 are mainly expressed by GBM cells. Nonetheless, additional studies are required to identify cells expressing these proteins considering all tumor bulk cell populations (i.e., tumor cells and TME components). Low expression of CD80 and CD86 are associated with better prognosis in terms of PFS in newly diagnosed GBM. CD80 and CD86 act as Tlymphocytes inhibitors; we hypothesized that CD80-low/CD86-low GBM is more permissive for cytotoxic T-lymphocytes. In the same line, CD80-high/CD86-high GBM should respond better to anti-CTLA-4 immunotherapeutic antibodies.Although we failed to demonstrate that CD80 and CD86 are independent prognostic factors in newly diagnosed GBM, probably due to the limited statistical power of our local discovery cohort, a trend was observed. This observation supports investigations of GBM TME features as prognostic or predictive factors in GBM. Indeed, composite biomarkers based on patient characteristics, tumor characteristics, and TME characteristics might be more powerful to guide prognostic evaluation and drug prescription[START_REF] Russell | Characterization of biomarkers to immune checkpoint blockade therapy across solid tumors[END_REF] C. UMBO enhances delivery and efficacy of ICBs through modulation of the GBM immune TMEImmunotherapies, including ICBs and cell therapies, have revolutionized the treatment of multiple solid tumors through activating the general antitumor immune response. In GBM, the earliest clinical trials evaluating the effect of nivolumab monotherapy or in combination with ipilimumab showed a limited efficacy with multiples toxicities.CheckMate-143 phase 3 clinical trial was then initiated to evaluate the effect of nivolumab versus bevacizumab. Unfortunately, nivolumab did not demonstrate higher efficacy over bevacizumab. A few reasons might explain the low efficacy of ICBs in GBM: (i) low tumor mutation load within GBM cells, (ii) lack of predictor of response to guide prescription, (iii) low penetration of ICBs within the brain parenchyma, (iv) low peripheral immune priming in lymph nodes, (v) local TME immunosuppression, (vi) low penetration of lymphocytes

Table 11 :

 11 Univariate analysis for OS in both ONT cohort and TCGA dataset. .....................

Table 12 :

 12 Univariate analysis for PFS in both ONT cohort and TCGA dataset .....................

Classification of primary brain tumors in adults 1.1 Primary brain tumors in adults

		O6-Methylguanine-DNA Methyltransferase Major histocompatibility complex Introduction	MGMT MHC
		An inhibitor of ABCC1 and ABCC4	MK571
		Magnetic resonance imaging	MRI
	1.	Monoclonal antibodies	MAbs
		arms 1p and 19q Multidrug resistance-associated protein	1p/19q MRP
		Depatuxizumab Mafodotin Molecular weight	ABT-414 MW
		Antibody-drug conjugates Nucleotide-binding domains	ADC NBD
		X-linked adrenoleukodystrophy Neurofilament Light	ALD NEFL
		Alternative lengthening of telomeres Not otherwise specified	ALT NOS
		Adenosine triphosphate Overall survival	ATP OS
		Alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler OncoNeuroTek database	ATRX ONT
		Blood-brain barrier Programmed death-1 receptor	BBB PD-1
		Carmustine Patient-derived cell line	BCNU PDCL
		Breast cancer resistance protein Platelet-derived growth factor receptor A	BCRP PDGFRA
		Bone morphogenetic protein 7 Programmed death ligand-1	BMP7 PD-L1
		Blood-tumor barrier Positron emission tomography	BTB PET
		Carbonic Anhydrase XII Progression-free surival	CA XII PFS
		Cyclin dependent kinase inhibitor 2A P-glycoprotein	CDKN2A P-gly
		Convection enhanced delivery Phosphatase and tensin homolog	CED PTEN
		Central nervous system Tumor suppressor retinoblastoma	CNS pRB
		Computed tomography Proto-oncogene, NF-KB subunit	CT RELB
		Cytotoxic T-lymphocyte-associated antigen-4 Role of 5	CTLA-4 RO5
		Dendritic cells Receptor tyrosine kinase	DC RTK
		Endothelial cells Src homology 2	EC SHR2
		Extracellular matrix Solute carrier family 12-member 5	ECM SLC1245
		Epidermal growth factor receptor Single nucleotide polymorphism	EGFR SNP
		Fluid-attenuated inversion recovery Synaptotagmin 1	FLAIR SYT1
		Food and Drug administration Tumor associated macrophages	FDA TAM
		Gamma-aminobutyric acid receptor subunit alpha-1 The Cancer Genome Atlas	GABRA1 TCGA
		Glioma-associated neovascularization T cells receptor	GAN TCR
		Glioblastoma Transforming growth factor-beta	GBM TGF-B
		Glioblastoma stem cells Trans membranous domain	GSC TMD
		Gray-radiation Tumor microenvironment	GY TME
		Histone H3 mutation Temozolomide	H3K27m TMZ
		hypoxia-inducible factor-1 alpha Tumor necrosis factor receptor 1 superfamily Member 1A	HIF-1α TNFRSF1A
		Immune checkpoint blocking antibodies Tumor necrosis factor receptor type 1-associated death domain protein	ICB TRADD
		Isocitrate dehydrogenase T regulatory lymphocytes	IDH Tregs
		Interferon Tumor treating fields	IFN TTF
		Immunohistochemistry Ultrasound-mediated blood-brain barrier opening	IHC UMBO
		Interleukin World health organization	IL WHO
		Kilo Dalton	Kda
		Karnofsky Performance Scale	KPS
		MRP1 and MRP4 I inhibitor	MDM2
		Multidrug resistance	MDR
		Proto-oncogene, receptor tyrosine kinase	MERTK

Table 1 :

 1 Simplified classification of diffuse gliomas according to the WHO 2016 publication

	Tumor type and grade	IDH	IDH	IDH mutant
		mutant	wildtype	1p/19q co-
				deletion

Table 2 :

 2 Therapies for recurrent GBM patients

	Therapies for recurrent GBM patients
	Carmustine (BCNU) wafers	A biodegradable polymer containing 3.85% carmustine
		applied in the surgical lesion when patients are candidate for
		second surgery (Xiao et al., 2020).
	Bevacizumab	10 mg/kg once every two weeks (Wick et al., 2017)
	BCNU	150-200 mg/m² (single dose or divided over two days) over
		six weeks OR
		75-100 mg/m²/day for two days over six weeks
	Lomustine (CCNU)

Table 3 :

 3 Examples of molecular and innovative therapeutic strategies used in clinical trials for GBM treatment are summarized from the clinicaltrial.gov official website (21/03/2021)

	Intervention	Experimental	Status	Phase Clinical trial reference
	Molecular targeted	Marizomib	Active, not	III	NCT03345095
	therapies	(proteasome	recruiting
		inhibitor)
		Regorafenib	Recruiting	II/III	NCT03970447
		(pan-Tyrosine
		kinases inhibitor)
	Passive and active	Autologous	Recruiting	II	NCT04115761
	immunotherapy	dendritic cells
		vaccination
		Nivolumab (anti-	Active, not	II	NCT03367715
		PD-1) and	recruiting
		Ipilimumab (anti-
		CTLA-4)
		EGFRvIII and	Completed	I/II	NCT01454596
		chimeric antigen
		receptor T cell
	Medical devices	Ultrasound	Recruiting	I/II	NCT04528680
		mediated BBB
		opening
		(Sonocloud 9) in
		combination
		with albumin-
		bound Paclitaxel

Abbreviations: EGFRvIII: Epidermal growth factor receptor variant III; PD-1: programmed death-1; CTLA-4; cytotoxic T-lymphocyte-associated antigen-4

Table 5 :

 5 Molecular characteristics of identified subtypes[START_REF] Verhaak | Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1[END_REF]

	Abbreviations: PDGFRA: platelet-derived growth factor receptor A; EGFR: epidermal growth factor
	receptor; PTEN: phosphatase and tensin homolog; NEFL: neurofilament Light; GABRA1: gamma-
	aminobutyric acid receptor subunit alpha-1; SYT1: synaptotagmin-1; SLC12A5: solute carrier family
	12 member 5; CDKN2A: cyclin dependent kinase inhibitor 2A); 17q11.2: chromosome 17q11.2
	deletion; NF-kB; TRAAD: tumor necrosis factor receptor type 1-associated death domain protein;
	RELB: RELB proto-oncogene, NF-KB subunit; TNFRSF1A: tumor necrosis factor receptor 1
	superfamily Member 1A; MET: proto-oncogene, receptor tyrosine kinase.

Table 6 :

 6 Summary of GBM escape mechanisms[START_REF] Razavi | Immune Evasion Strategies of Glioblastoma[END_REF]

	Category	Biomarkers or	Major source	Effect
		Mechanism
	CNS	BBB	Anatomical	Decrease the chance of immune cells
			barrier	infiltration to the brain
	TME	IL-6, IL-10, TGF-	Microglia/TAMs,	Blocks T-cell activation and proliferation,
		β, IL-1	GBM cells and	suppresses NK cell activity, promotes
			endothelia cells.	Treg activity, promotes tumor growth
				and invasion
	Immune	PD-L1, CTLA-4	GBM cells,	Significantly reduce T-cell proliferation
	checkpoints		microglia/TAMs,	and increase T cell exhaustion
			T-Cells
	Regulatory T-	CCL22, CCL2	GBM cells	Modulate T cell activity and attracts
	lymphocytes			Tregs to the tumor site
	Tumor-associated	VEGF, IL-6, EGF,	Microglia/TAMs Promote tumor growth,
	macrophages	TGF-β1, MIC-1		immunosuppression, and tumor
				vascularity
	Abbreviations: IL-6: interlueikin-6; TGF-β : Transforming growth factor beta; CCL22: C-C motif
	chemokine 22; EGF: epidermal growth factor: MIC-1: macrophage inhibitory cytokine 1

Table 7 :

 7 Available commercial microbubbles. Each type of microbubbles differs in its composition (capsule and gas), size, half-life, and concentration.

		SonoVue ®	Definity ®	Optison ®
	Company	Bracco Diagnostics	Lantheus	E-Cardio
	Encapsulated gas	Hexafluoride	Octafluoropropane	Octafluoropropane
		sulphide
	Capsule	Phospholipids	Phospholipids	Albumin
	Diameter of	2.5	1.1 -3.3	2-4.5
	microbubbles (μm)
	Concentration	1 -5 x 10 8	1.2 x 10 10	5 -8 x 10 8
	(bubbles/ml)

Table 8 :

 8 Original research articles that combined UMBO with antibodies and macromolecules delivery.

	Antibody	Model	Results	Reference
	Herceptin	Healthy mice	Proof of concept of safety	(Kinoshita et
			and efficacy of Herceptin	al., 2006a)
			delivery
	Anti-D4 receptor	Healthy mice	Enhanced the delivery of	(Kinoshita et
	antibody		anti-D4 receptor antibody	al., 2006b)
			in the brain
	VEGF-targeting, drug-	C-6 Glioma rat	Reduction in tumor	(Fan et al.,
	loaded MBs	model	volume with enhanced	2013)
			overall survival
	Trastuzumab and	Brain metastasis	Combination of focused	(Kobus et al.,
	Pertuzumab	in rat model	ultrasound and	2016)
			antibodies enhanced the
			overall survival
	Bevacizumab	Glioma mouse	Significant enhancement	(Liu et al.,
		model	of bevacizumab	2016)
			concentration (5-57 folds)
			and increased survival
	Anti-β-amyloid protein	Alzheimer's	Focused ultrasound	(Alecou et al.,
	antibody	model in rabbits	decreased B amyloid	2017)
		(high-cholesterol	plaque
		diet)
	Tau-specific antibody	Alzheimer's	Focused ultrasound	(Janowicz et
		Mouse model	increased the local	al., 2019)
			delivery of tau antibody
			in the brain parenchyma
	Macromolecule	Healthy mice	Focused ultrasound	(Valdez et al.,
	(dextrans MW 3, 70 and		enhanced the delivery of	2020)
	500KDa)		different dextran sizes in
			the brain.

4 Ultrasound mediated BBB opening in clinical settings

  During the last two decades, Sunnybrook Research Institute in Canada established and developed transcranial ultrasound systems to open the BBB under MRI guidance. A commercial transcranial ultrasound system implementing this approach was developed by the company Insightec ® and is currently evaluated in clinical trials for several neurological disorders, including GBM (NCT03712293). The noninvasive system's main disadvantage compared to the implantable SonoCloud-9 ® device is its cost (including a 3-hour MRI immobilization per treatment) and the limited BBB disruption volume. The company NaviFUS

® developed another noninvasive transcranial system. They recently launched a neuronavigational-guided clinical trial to open the BBB to enhance bevacizumab delivery in GBM patients (NCT04446416). The device's main strength is that it uses the neuronavigational system to target a specific brain region. It provides a cheaper alternative than the Insightec device; however, this device is still under testing and validation.

Table 10 :

 10 Identified subfamilies of ABC transporters and their physiological functions.

	1	Immunosuppressant medication with ABCB1 blocking activity	5	A potent ABCB1 inhibitor, has reached clinical trials.
	2	Calcium channel blockers with ABCB1 blocking activity	6	Experimental drugs with ABCG2 inhibition activity.
	3	An experimental cancer drug with ABCB1 inhibition properties. It is a derivative of cyclosporine.	7	It is an experimental drug with ABCC1 and ABCB1 inhibition.
	4	An experimental small molecule that has a dual ABCB1 and ABCG2 inhibition	8,9	Poly (ADP ribose) polymerase (PARP) inhibitors

Table 11 : Univariate analysis for OS in both ONT cohort and TCGA dataset.

 11

	Characteristics	ONT

Table 12 : Univariate analysis for PFS in both ONT cohort and TCGA dataset

 12

	Characteristics	ONT

Increased brain delivery of anti-programmed death-ligand 1 using low-intensity pulsed ultrasound-mediated blood-brain barrier opening is associated with increased anti-tumor efficacy and microglia activation in glioblastoma mouse models

	Mohammed Ahmed 1,2* , Nolwenn Lemaire 1 , Emie Quissac 1 , Rana Salam 1 , Isaias Hernández-
	Verdin 1 , Coralie L Guerin 3 , Lea Guyonnet 3 , Noël Zahr 4 , Laura Mouton 5 , Mathieu Santin 5 ,
	Alexandra Petiet 5 , Charlotte, Schmidt 6 , Guillaume Bouchoux 6 , Marc Sanson 7 , Maite
	Verreault 1 , Alexandre Carpentier 6,7 , Ahmed Idbaih 7*
	1 Sorbonne Université, Institut du Cerveau -Paris Brain Institute -ICM, Inserm, CNRS, APHP,
	Hôpital de la Pitié Salpêtrière, Paris, France
	2 Faculté de Médecine Paris-Sud -Université Paris-Saclay, 91190, Saint-Aubin, France.

3

Curie Institute, Cytometry Department, F-75006, Paris, France.

Acknowledgments

This work was supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement #766069 (GLIO-TRAIN). We would like to acknowledge the online BioRender platform that provided all the tools and icons to draw the manuscript's graphs. The research leading to these results has received funding from the program Investissements d'avenir" ANR-10-IAIHU-06. Institut Universitaire de Cancérologie. INCA-DGOS-Inserm_12560 (SiRIC CURAMUS) benefits from support from Institut National du Cancer, ministère des Solidarités et de la Santé and Inserm. Acknowledgments This work was supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement #766069 (GLIO-TRAIN). The research leading to these results has received funding from the program Investissements d'avenir" ANR-10-IAIHU-06. Institut Universitaire de Cancérologie. INCA-DGOS-Inserm_12560 (SiRIC CURAMUS) benefits from support from Institut National du Cancer, ministère des Solidarités et de la Santé and Inserm. OncoNeuroTek biobank Acknowledgments This work was supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement #766069 (GLIO-TRAIN), INCA-DGOS-Inserm-12560 (SiRIC CURAMUS), Institut National du Cancer, ministère des solidarités et de la Santé and Inserm.

Authors' contributions

MA performed the literature analysis, drawing of the figures, writing of the manuscript and final approval of the final version. AI participated in the original concept of the article, reviewing, and editing of the figures, reviewing the manuscript and final approval of the manuscript. MV and XD participated in reviewing the figures, reviewing the manuscript and final approval of the manuscript.

Conflicts of interest

No potential conflicts of interest were disclosed AI reports grants and travel funding from Carthera (September 2019), research grants from Transgene, research grants from Sanofi, research grants from Air Liquide, travel funding from Leo Pharma

Quantification of IHC staining

Following all slides' imaging, three regions of interest with known dimensions (528*528 µm) were randomly selected for each tissue section and quantified using an inhouse quantification Fiji code. Shortly, each image was imported to the Fiji program [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. Using the color deconvolution tool, the area positive for DAB staining was isolated and quantified using a semi-automated in-house generated code.

The percentage of DAB positive areaswas calculated, and the mean value from the three images was calculated and used in the survival analysis.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT PCR)

RNA samples were obtained from ONT bank and used to synthesize cDNA. Reverse transcription of RNA samples was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, K1442) according to the manufacturer's recommendations with 100-250 ng of RNA. qPCR was used to quantify the expression levels of CD80 and CD86 in patients. PPIA gene was used as a house keeping reference gene for normalization. Primer's sequences are listed in (Figure 25). cDNA samples were analyzed using the Light Cycler Probe Master mix 2× (Roche, 04887301001) and the UPL detection system (Roche, 04483433001) in a Light Cycler 96 (Roche). For each qPCR, two independent experiments were completed with duplicate samples in each experiment. The mean of 2^-(CT gene of interest -CT PPIA ) from the two different experiments was used in all analyses.

Statistical analysis

A Violin plot was used to visualize our data's full distribution (GraphPad Prism).

Spearman correlation between the expression values (RNA or protein) and age was evaluated to discard age bias. Survival analysis was performed by finding a supervised cutoff value for the CD80 expression or the CD86 expression independently using the

Conflicts of interest

No potential conflicts of interest were disclosed.

Author's contribution

MA, MV designed the primers, obtained the tissues. MA performed the experiments.

MA, AI designed the experiments, wrote the manuscript, and approved the final version of the manuscript. IHV performed the statistical analysis and revised the manuscript. All authors reviewed the manuscript.

Abstract

Therapeutic antibodies targeting immune checkpoints have limited efficacy in the overall population of glioblastoma (GBM) patients. Limited penetration of these large molecules within the normal and the tumor brain may explain at least partly these disappointing results. We hypothesized that increasing brain penetration of immune checkpoint inhibitors using low intensity pulsed ultrasound-mediated blood-brain barrier opening (UMBO) may increase their tumor bioavailability and their efficacy against GBM.

In syngeneic GBM-bearing immunocompetent mice, we show that UMBO is able to open safely and repeatedly the blood-brain barrier using Evans's blue dye imaging, immunofluorescence, and MRI. UMBO is associated with (i) increased penetration of immune checkpoint inhibitor within the brain when delivered intravenously and (ii) increased circulation of tumor DNA within the bloodstream. Finally, we report here that the combination of UMBO and anti-PD-L1 therapeutic antibody increases dramatically the survival of GBM-bearing mice compared to their counterparts treated with anti-PD-L1 alone. Our study highlights the blood-brain barrier as a limitation to overcome to increase efficacy of immune checkpoint inhibitors in GBM and supports clinical trial combining UMBO and anti-PD-L1 in GBM patients.

Keywords: Sonocloud, GL261 mouse model, BBB opening, antibody delivery

Conflicts of interest

No potential conflicts of interest were disclosed.

Author's contribution