Claude Universite

Bernard -Lyon

M Le Professeur

Frédéric Fleury

Hamda Ben

Hadid M Le Professeur Didier

Revel M Le Professeur

Philippe Chevalier

M Fabrice

Vallée M Alain Helleu

Composantes Sante

J Etienne

La Mme

C Professeure

Burillon

D Bourgeois

Vinciguerra

Y Matillon

A-M Professeure

Schott

Judith Bovee

Frederic Blanchard

Valérie Decouvelaere

Jean-Philipe Michot

Isabelle Treilleux

Delphine Jury

Mélanie Léone

Marie-Jeanne Roux

Vincent Commelin

David Monderer

Morgane Monchanin

Iseulys Richert

Margaux Vienne

Emeline Brandinelli

Sandrine Wittman

Tala Shakarian

Ann-Catherine Jallas

Keywords: Chondrosarcoma, lymphocyte, PD-1, PD-L1, macrophages, immunotherapy, PI3K/Akt/mTOR, immunosurveillance

À l'issue de la rédaction de cette recherche, je suis convaincue que la thèse est loin d'être un travail solitaire. En effet, je n'aurais jamais pu réaliser ce travail doctoral sans le soutien d'un grand nombre de personnes dont la générosité , la bonne humeur et l'intérêt manifestés à l'égard de ma recherche m'ont permis de progresser dans cette phase délicate de « l'apprenti-chercheur ».

En premier lieu, je tiens à remercier mon directeur de thèse, Jean-Yves Blay, et ma codirectrice, Aurélie Dutour, pour la confiance qu'ils m'ont accordée en acceptant d'encadrer ce travail doctoral, pour les multiples conseils et pour toutes les heures qu'ils ont consacré à diriger cette recherche. J'aimerais égalementleurs dire à quel point j'ai apprécié leurs grandes disponibilités et leurs respect sans faille des délais serrés de relecture des documents que je leurs ai adressé. Enfin, j'ai été extrêmement sensible à leurs qualités humaines d'écoute et de compréhension tout au long de ce travail doctoral.

Je souhaiterais exprimer ma gratitude à Christophe Caux pour m'avoir conseillé tout au long de cette thèse sur la partie immunologie. Je le remercie également pour son accueil chaleureux à chaque fois que j'ai sollicité son aide.

COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE

Résumé Introduction

Les chondrosarcomes représentent 20% de tous les sarcomes osseux. Ces tumeurs dont l'incidence est estimée à 1/200 000 par an, sont caractérisées par la production d'une matrice cartilagineuse par les cellules tumorales, une faible vascularisation et un faible pourcentage de cellules en division. Du fait notamment de l'abondance de matrice extracellulaire et de leur faible densité vasculaire, les chondrosarcomes sont particulièrement chimio-et radiorésistants. En conséquence, la prise en charge thérapeutique de ces tumeurs n'a que peu évolué au cours des 30 dernières années et le seul traitement efficace à l'heure actuelle reste une résection chirurgicale large de la tumeur. Des approches d'immunothérapies ont récemment généré un grand enthousiasme dans la recherche anticancéreuse ; ces thérapies exploitent la capacité de l'immunité innée et adaptive pour contenir la croissance de cellules cancéreuses et les détruire. Les points de contrôle « checkpoints » immunitaires sont des protéines de signalisation permettant le maintien de la tolérance du soi et de limiter les réponses immunitaires physiologiques dans les tissus périphériques ; détournés de leur fonction, ils peuvent également permettre l'immunotolérance vis-à-vis des tumeurs. Des inhibiteurs de ces checkpoints immunitaires (Cytotoxic T lymphocyte antigen 4 (CTLA-4) ou de programmed cell death protein 1 (PD-1) et son ligand (PD-L1) induisent des réponses immunitaires contre les cellules cancéreuses, in vivo chez l'homme. Ils restaurent l'activité antitumorale des cellulles immunocompétentes et pourraient augmenter l'immunogénicité tumorale induite par les thérapies conventionnelles (chimiothérapie, radiothérapie) ou les thérapies ciblées. L'impact de ces immunothérapies est mal connu dans les sarcomes, et notamment le chondrosarcome. Il apparait donc intéressant d'étudier l'environnement immunitaire du chondrosarcome et d'évaluer les possibilités de combiner des inhibiteurs de checkpoints immunitaires à des thérapies de première ligne, comme les inhibiteurs de la voie PI3K/Akt/mTOR. Jusqu'à présent, très peu d'études se sont intéressées à l'environnement immunitaire du chondrosarcome et à son implication dans la progression et la réponse tumorales. Notre groupe a construit un projet de recherche s'intéressant à ces questions et explorant le rôle joué par le checkpoint immunitaire PD1/PDL1 dans la progression de cette tumeur afin de déterminer si PD1 et PDL1 peuvent être de nouvelle cibles thérapeutiques pour les patients atteints de chondrosarcome et s'ils peuvent être joint à des thérapies de première ligne. Cidessous, sont présentés les résultats obtenus jusqu'à présent. Les analyses immunohistochimiques réalisées à l'aide de marqueurs des lymphocytes (CD3, CD4, CD8) et des macrophages (CD163) ont permis de mettre en évidence la présence d'infiltrats immunitaires (lymphocytes et macrophages) en périphérie des nodules cartilagineux dans la zone fibreuse de la tumeur. Nous avons pu montrer qu'une forte présence des lymphocytes T (CD3+, CD4+, CD8+) est corrélée à une meilleure survie et un moindre taux de rechutes des patients. A l'inverse, une corrélation inverse entre la présence de macrophages (CD163+) et le taux de survie des patients est observée. Le ratio cellules CD8+/cellules CD163+ est inversement corrélé à l'agressivité de la tumeur : dans les tumeurs de faible grade et non invasives une plus forte proportion de cellules CD8+ par rapport aux cellules CD163+ est retrouvée alors que les chondrosarcomes invasifs de haut grade présentent une faible proportion de cellules T CD8+ et une plus forte proportion de cellules CD163+.

Ceci indique un rôle potentiel pour les macrophages et les lymphocytes T CD8+ dans l'invasion du chondrosarcome ; les cellules CD163+ favoriseraient la progression tumorale alors que les cellules T CD8+ la contiendraient.

Des analyses similaires ont été réalisées dans le modèle de chondrosarcome de Swarm. Ces études ont montré un profil d'infiltration immunitaire de cette tumeur similaire à celui observé pour le chondrosarcome humain, avec une présence de lymphocytes (CD3, CD8) et de macrophages (CD163) à la périphérie de la tumeur et dans les interstices fibreux. Comme chez l'humain, le ratio CD8+/CD163+ a été calculé, il est inversement corrélé à la progression tumorale et confirme le rôle potentiel des macrophages CD163+ et des lymphocytes T CD8+ dans la progression de chondrosarcome. Par ailleurs, nous avons confirmé le rôle de cellules immunitaires dans le contrôle de la progression tumorale. Des études de cytotoxicité réalisées à l'aide de splénocytes de rats sains ou porteurs de tumeurs ont montré que la présence de la tumeur induit une réponse immunitaire anti cancer chez le rat. En effet, une action cytotoxique spécifique est observée pour les splénocytes de rats porteurs de chondrosarcome (cytotoxicité de 27% contre des chondrocytes à un ratio effecteur cible de 100/1), réponse qui n'est pas obtenue avec des splénocytes de rats sains.

2-Rôle des différentes populations immunitaires dans la progression du chondrosarcome

La présence de macrophages semble contribuer à la croissance du chondrosarcome, tandis que celle des lymphocytes semble associée à un ralentissement de la croissance tumorale. Afin de vérifier cette hypothèse, nous avons inhibé l'une après l'autre ces populations immunitaires dans le modèle de chondrosarcome de rat. Ces traitements ont été administrés pendant 5 semaines sur des rats porteurs de tumeur progressive, et montrent qu'en absence de lymphocytes T les tumeurs progressent significativement plus vite. A l'inverse, en l'absence de macrophages M2, un ralentissement significatif de la progression tumorale est observé.

Ces données indiquent que les macrophages (principalement les macrophages M2) exercent une action protumorale et pourraient être une cible d'intérêt pour les futures approches thérapeutiques. Ces résultats confirment également le rôle des lymphocytes T dans le contrôle de la croissance du chondrosarcome et cette action antitumorale pourrait être améliorée par des approches d'immunothérapie ciblant les checkpoints immunitaires.

3-Effet des inhibiteurs de la voie PI3K/Akt/mTOR sur la réponse immunitaire

Les inhibiteurs de la voie PI3K/Akt/mTOR réduisent significativement la progression tumorale dans le modèle de chondrosarcome de rat. De plus, ils n'affectent pas l'infiltration des populations immunes (macrophages et lymphocytes) dans le microenvironnement de la tumeur et une diminution de l'expression de PD-L1 dans les tumeurs de rats et dans des lignées cellulaires de sarcomes humains.

Par contre, les inhibiteurs testés réduisent la cytotoxicité des splénocytes de rat face à des cellules de chondrosarcomes de rat. Ils ont aussi comme effet de réduire la sécrétion de cytokine pro-inflammatoire au niveau systémique.

Cela suggèrent que les inhibiteurs de la voie PI3K/Akt/mTOR ont un effet immunosuppresseur et qu'ils seraient inopportun des les associer à une immunothérapie.

4-Checkpoint immunitaire PD-1/PD-L1 et le chondrosarcome

Nous avons étudié l'expression de PD-1/PD-L1 dans le chondrosarcome humain et de rats. Ce checkpoint immunitaire joue un rôle dans l'échappement tumoral.

En effet, au niveau tumoral, la liaison de PD-1 et PD-L1 inhibe la prolifération de lymphocytes T cytotoxique (CTL), leur production de cytokines et leur cytotoxicité, favorisant ainsi la prolifération tumorale.

Nous avons mis en évidence l'expression de PDL1 par les cellules tumorales à la fois dans les échantillons de chondrosarcome humain et dans le modèle de chondrosarcome de Swarm. Des cellules immunitaires PD1+ ont été trouvées à la fois dans le chondrosarcome humain et de rat.

Ces résultats montrent qu'une immunothérapie visant les « immunocheckpoints » PD-1/PD-L1 est possible pour le chondrosarcome. Nous avons aussi démontré que l'étude des populations qui infiltrent le chondrosarcome pourrait servir de biomarqueur potentiel pour ces patients. Nous étudions actuellement le rôle de PD-1/PD-L1 dans le chondrosarcome à l'aide du modèle de chondrosarcome de Swarm en utilisant un anticorps bloquant PD-L1. Nous allons également confirmer les observations conduites dans les chondrosarcomes humains en augmentant la cohorte de patients analysés et étendre l'étude aux sous types plus rares de chondrosarcomes. ------------------1.1 Epidemiology ---------------------------------------------------------------------------1.2 Risks factors ---------------------------------------------------------------------------

1.3

Classification ---------------------------------------------------------------------------

1.4

Survival and Prognosis factors -----------------------------------------------------1.5 Current treatment ----------------------------------------------------------------------

Surgery ------------------------------------------------------------------------1.5.2 Conventional therapies applied to chondrosarcoma ---------------

A) Radiation therapy ----------------------------------------------------------------B) Chemotherapy --------------------------------------------------------------------

1.6

Chondrosarcoma therapeutic resistance ----------------------------------------1.7 New therapeutic approaches for chondrosarcoma --------------------------- 1.7.3 Targeting the chondrogenic matrix of CHS to deliver drugs -----

Targeting chondrosarcoma microenvironment ----------------------1.7.2 Bone resorption osteoclast activity -------------------------------------

1.8

Targeting therapies for chondrosarcoma ---------------------------------------- ------------------------------------------------------------1.8.2 Apoptosis and chondrosarcoma:

HDAC inhibitors -

BH3 mimetics for chondrosarcoma treatment -----------------------

Hedgehog pathway and chondrosarcoma ----------------------------1.8.4 Inhibitors of IDH ½ Mutant Proteins ------------------------------------1.8.5 mTOR pathway inhibitors -------------------------------------------------6.2.6 Effectors T cells/cytotoxic activity of SRC splenocytes ---------6.2.7 Inflammation and chondrosarcoma progression -----------------

6.2.8 Take home message / main results -------------------------------------

6.3

Modulation of immune response in chondrosarcoma by PI3K pathway inhibitor ------------------------------------------------------------

PI3K pathway inhibitors and tumor progression -----------------6.3.2 PI3K pathway inhibitors and cytotoxic immune populations ----------------------------------------------------------------6.3.3 PI3K pathway inhibitors and inflammation -------------------------6.3.4 Take home message / results ----------------------------------------

6.4

Immune checkpoint targeting ----------------------------------------------------

Expression of PD-1 / PD-L1 in chondrosarcoma ----------------

A) Conventional human chondrosarcoma ------------------------------B) SRC --------------------------------------------------------------------------

Modulation of PD1/PDL1 expression in SRC ---------------------

A) By targeted therapies ----------------------------------------------------B) By blocking antibody ----------------------------------------------------- --------------------------------------------------------------------------------7.1 Prognosis value of T lymphocytes infiltrates in chondrosarcoma ------7.1.1 T lymphocytes in human chondrosarcoma ------------------------

Take home message / main results ---------------------------------

Discussion

T lymphocytes in SRC model : immunological

microenvironment in SRC is similar to human immunological environment -------------------------------------------

Impact of CD3+ and CD8+ T lymphocytes on

chondrosarcoma progression in SRC model ---------------------

7.2

Prognosis value of macrophages in chondrosarcoma --------------------

Macrophages infiltrates in human chondrosarcoma ------------

List of the figures and tables

Figure 1.1:Histological aspect of conventional chondrosarcoma ------------------------------------------Figure 1.2:Histological grading of chondrosarcomas ---------------------------------------------------------Figure 2.1:Evolution of the hallmarks of cancer over the past decade -----------------------------------Figure 2.2:Innate and adaptive immunity -------------------------------------------------------------------------Figure 2.3:The two type of adaptive immune response -------------------------------------------------------Figure 2.4:Direct cancer recognition by the innate immune system --------------------------------------Figure 2.5:"Signal-strength model" of T cell differentiation --------------------------------------------------Figure 2.6:Granzyme/Perforin mediated cytotoxic action of CTL ------------------------------------------Figure 2.7:The prognostic impact of T cells in tumor ---------------------------------------------------------Figure 2.8:The three phases of cancer immunoediting. ------------------------------------------------------Figure 2.9:A proposed model for the elimination phase of the cancer immunoediting process ----Figure 2.10: Co-stimulatory and inhibitory signal of TCR ----------------------------------------------------Figure 2.11: Immune checkpoint in cancer ----------------------------------------------------------------------Figure 3.1:Immunotherapy against tumor cells -----------------------------------------------------------------Figure 5.1:Transplantable orthotopic Swarm rat chondrosarcoma ----------------------------------------Figure 5.2:The targets and effects of each immunomodulator treatment --------------------------------Figure 6.1:Immune infiltrate in human chondrosarcoma -----------------------------------------------------Figure 6.2:Immune infiltrates correlation with chondrosarcoma's survival ------------------------------Figure 6.3:Immune infiltrates correlation with chondrosarcoma's progression -------------------------Figure 6.4:Correlation between CD8+, CD163+ cells counts and tumor size --------------------------Figure 6.5:Correlation between CD8+, CD163+ cells counts and tumor agressivity -----------------Figure 6.6:Correlation between CD8+ lymphocytes and the CD163+ macrophages in tumor's microenvironment -- ----------------------------------------------------------------------Figure 6.9:Correlation between CD8+, CD163+ cells counts and tumor size -------------------------Figure 6.10:CD8/CD163 ratio and tumor agressivity ---------------------------------------------------------Figure 6.11:Effects of selective depletion on SRC immune infiltrates -----------------------------------Figure 6.12:Effects of selective depletion on SRC progression -------------------------------------------Figure 6.13:Cytotoxic response in chondrosarcoma ---------------------------------------------------------Figure 6.14:Inflammatory response in chondrosarcoma ----------------------------------------------------Figure 6.15:PI3K, Akt and mTOR inhibitors affect SRC progression ------------------------------------Figure 6.16:PI3K, Akt and mTOR inhibitors increase SRC survival -------------------------------------Figure 6.17:Effect of PI3K pathway inhibitors on SRC immune environment -------------------------Figure 6.18:Effect of PI3K pathway inhibitors on cytotoxic response in chondrosarcoma ---------Figure 6.19:Effect of PI3K pathway inhibitors on inflammation in chondrosarcoma -----------------Figure 6.20:Expression of PD-L1 and PD-1 in human conventional chondrosarcoma -------------Figure 6.21:Expression of PD-L1 and PD-1 in SRC - ------------------------------------------------------- 6.24:Blocking of rat PD-1 immune checkpoint using a murine PD-L1 blocking antibody -Table 1.1:10 years survival of chondrosarcoma patients in function of the histological grade -----Table 2.1:The Principal immune cells population in cancer -------------------------------------------------Table 2.2:Classification of tumor-associated antigens that are recognized by T cells ---------------Table 3.1:Interferons studies in bone sarcoma. ----------------------------------------------------------------Table 3.2:TAAs expression in sarcoma tumor samples and cell lines ------------------------------------Table 3 In the following paragraphs we will present the different types of chondrosarcoma, their conventional treatments as well as the therapeutic options being currently explored. Given the heterogeneity of this group of tumor we will focus on the most encountered chondrosarcoma: the conventional central chondrosarcoma.

--------------------------------------------------------------------------- Figure 6.7:CD8/CD163 ratio and tumor agressivity ----------------------------------------------------------- Figure 6.8:Immune infiltrate in SRC model
------------------------------------------------------------------------------------------------ Figure
.3:Vaccines immunotherapy in sarcomas -------------------------------------------------------------- Table 6.1:Clinical information of chondrosarcoma patients ------------------------------------------------- Table 6.2:Inflammatory cytokines in chondrosarcoma -------------------------------------------------------

List of abbreviations

1.Epidemiology

The estimated overall incidence of CHSs is 1 in 200,000 per year (2), and it is the third most frequent malignant bone tumor after multiple myeloma and osteosarcoma. It is estimated that CHSs account for approximately 20~30% of primary malignant bone tumors (3).

Chondrosarcoma most commonly arise in adults aged 30 to 70 years, with a peak around 40 to 60. Chondrosarcoma has a male to female ratio of 1.5 to 1. It is most common in the femur, humerus, ribs and on the surface of the pelvis.

Risks factors

Most often, chondrosarcoma happens from normal cartilage cells. It may also arise from a pre-existing benign bone or cartilage tumor. Patients with enchondroma, with Ollier's disease (multiple enchondromatosis) or Maffucci's syndrome (multiple enchondromas and hemangiomas) are at much higher risk of chondrosarcoma than the normal population and often present in the third and fourth decade (4).

1.3 Classification

Except for their origin in preexisting benign cartilaginous diseases conditions, secondary chondrosarcomas are similar to conventional chondrosarcomas in many aspects.

Chondrosarcoma is a heterogeneous group of tumor; as a consequence many chondrosarcoma subtypes are described. More than 90% of chondrosarcoma are conventional chondrosarcoma. Among these, 90% are low grade to The higher the grade, the more likely the tumor is to spread and metastasize.

Grade 1 lesions rarely metastasize; whereas 10-15% of grade 2 lesions and more than 50% grade 3 lesions metastasize. Chondrosarcoma are often slow evolving tumors. Thus the reported 5 years survival rates are higher than most of tumors depend on the grade of chondrosarcoma. The reported 5-year survival for grade 1 chondrosarcoma ranged from 82% to 99%. The 5-year survival for grade 2 chondrosarcoma ranged from 63% to 92%. The 5-year survival for grade 3 chondrosarcoma ranged from 0% to 77%. The lowest (0%) survival was displayed in a study looking at a very small subgroup of patients treated with an intralesional resection (5).

As no therapeutic treatment besides surgery is available for chondrosarcoma, Identification of prognostic factors is important to select the optimal treatment strategy. To direct efforts in the development of new therapeutic strategies the identification of proven prognostic factors of central chondrosarcoma is important, even though the treatment options are still currently limited.

Various studies have suggested that histologic grade, size, stage, and anatomic location of the lesion are important prognostic features (8,9). In an analysis of 2,890 patients with chondrosarcoma from the Surveillance, Epidemiology, and End Results (SEER) database, only grade and stage were identified as independent prognostic factors for survival (10). For Fiorenza and al.,on 153 non-metastatic chondrosarcoma patients based on a multivariate analysis only extracompartimental spread and high histologic grade were a risk factor (11).

1.5 Current treatment

The clinical management of chondrosarcoma remains a challenging problem as this is highly resistant to currently available chemotherapy and radiation treatment regimens (2,12,13). Surgery remains the cornerstone in CHS treatment. Several studies have demonstrated the benefit of resection of tumor with wide surgical margins (14)(15)(16).

The goal of chondrosarcoma resection is complete tumor removal with microscopically negative margins. Intralesional surgery (microscopically positive margins) should be avoided because it is associated with a high risk of local recurrence regardless of whether the patient receives perioperative radiation therapy or chemotherapy; if intralesional surgery has been performed, re-excision of the tumor bed is recommended (17). For low-grade chondrosarcomas of the extremities, intralesional surgery is acceptable, as it has been reported to have a low rate of recurrence (18). However, there are a significant number of chondrosarcomas, especially those arising in the pelvis or the spine, that are difficult to remove with a wide surgical margin (19,20). In these instances, local recurrence is almost inevitable with major function and life threatening consequences.

Conventional radiation therapy (RT) has limited to arguably no effect on conventional chondrosarcoma (13,21). Currently, there is not enough data to support its routine use in patients with this disease. However, RT may be considered if there is incomplete resection, for palliation of symptoms, in patients with advanced or unresectable tumors, or for mesenchymal chondrosarcoma (4,13). Proton beam therapy, carbon ion therapy and neutron therapy have however been reported to induce tumor control in inoperable patients (22) (23).

Different studies have reported the lack of efficacy or limited efficacy of conventional chemotherapy in patients with advanced chondrosarcoma (24). The chondrosarcoma subtypes for which the highest benefits of chemotherapy can be observed are mesenchymal and dedifferentiated chondrosarcoma (24,25).

Indeed a recent report from the Italiano show an improvement in dedifferentiated chondrosarcoma and mesenchymal chondrosarcoma patients survival that are treated respectively as if they had osteosarcoma and Ewing sarcoma (25).

Similar results were reported by Anderson (24).

1.6 Chondrosarcoma Therapeutic resistance

The biological mechanisms of chondrosarcoma resistance to cytotoxic are multifactorial. The low percentage of dividing cells within a poorly vascular matrix, are thought to be involved in chondrosarcoma therapeutic resistance (4).

Indeed, chemotherapies are most effective on rapidly dividing cells while chondrosarcoma are slow progressing tumors and the low vascularity of the tumor limits access of chemotherapeutic drugs to tumor cells (4,26). Moreover, chondrosarcomas express high levels of the multidrug resistance-associated protein P-glycoprotein, a product of the ABCB1 gene (27) one of the most effective pump efflux for chemotherapies.

The mechanism of radiation resistance in chondrosarcomas is better understood.

Radiation resistance may be related to local tissue hypoxia as has been described in other tumors, the relative low levels of oxygen preventing the formation and propagation of radiation-induced reactive oxygen species that cause single and double strand DNA damage (28). Expression of HIF-1α by chondrosarcomas in association with increased levels of Bcl-2 can contribute to both radio resistance and chemoresistance (29). p16 ink4a , a tumor suppressor protein, also appears to have a role in radiation resistance of chondrosarcomas (13).

Because no systemic therapy has proven effective, chondrosarcoma are like the gastrointestinal stromal tumors of the 1990s, before the rise of targeted therapies and Gleevec. A variety of strategies are being developed to help overcome the limited effectiveness of current radiotherapy and chemotherapy on chondrosarcomas (30). Some of which will be presented in the following chapter.

1 1.7 New therapeutic approaches for chondrosarcoma CHS remains a major challenge in both diagnosis and therapy since conventional chemotherapy has proven to be largely ineffective, with no significant progress made for approximately three decades (31).

Improving chondrosarcoma clinical management is therefore a challenging problem and novel therapeutic approaches are needed.

The increasing understanding of chondrosarcoma biology, mechanisms of chondrosarcoma pathogenesis have prompted the development of new therapeutic approaches over the last decade. Some of these approaches are taking advantages of targeting chondrosarcoma environment. Others are targeting molecular pathways involved in tumorigenesis or that have been described as over-activated or mutated in chondrosarcoma or bone sarcoma.

As numerous strategies are being tested, the following paragraphs will present, on exhaustive way, some of these new therapeutic approaches for chondrosarcoma.

One of the approaches being investigated is to take advantages of phenotypic microenvironmental features of CHS (that have been considered for many years as limitations), instead of fighting them.

Recent scientific datas in the microenvironment and molecular pathways involved in CHS have highlighted the role of matrix metalloproteinases, bone niche, hypoxia-inducible factor (HIF), and mTOR (32)(33)(34). Treatments targeting tumoral microenvironment have therefore been suggested as complements to surgical excision (30,31,35).

Chondrosarcoma induce a local osteolytic reaction with bone remodeling at the site of tumor on gross and histopathological specimens, a phenomenon mediated by the action of osteoclasts, monocyte-derived bone resorbing cells (36).

Teams postulated that osteoclasts in the tumor microenvironment could be exploited to prevent tumor-mediated bone destruction and control tumor growth (37).

A few years ago, Heymann's group published the therapeutic relevance of using zoledronic acid (ZOL; an inhibitor of osteoclasts) in the SRC model. They showed that zoledronic acid, directly inhibits SRC cell cycle progression, induces apoptosis, and reduces peri-skeletal tumor growth in the SRC model (38). Zoledronic acid slows down rat primary chondrosarcoma development, recurrent tumor progression after intralesional curettage and increases overall survival (38). Since then, others have reported the promising effects of bisphosphonates not only on chondrosarcoma proliferation and invasion in vitro, in animal models but also in clinical practice (39)(40)(41)(42).

One strategy is to exploit the chondrogenic nature of the tumor in order to selectively deliver drugs or radioisotopes to tumoral tissue. This strategy is being explored by team of JM Chezal and E Miot Noirault. They have developed cartilage-targeting compounds for therapeutic or imaging purposes by linking a quaternary ammonium function to anti-inflammatory drugs, chondroprotective agents, cytotoxic agents and 99m Tc chelate. They have shown the capacity of these compounds to localize to the tumor site after in vivo administration (43)(44)(45).

Regarding the therapeutic potential of these agents, this team generated a quaternary ammonium derivative of melphalan a conventional DNA alkylating agent (Mel-AQ) in order to enhance both the delivery and the specificity of cytotoxic agents in chondrosarcoma tissue (43). They showed in vitro that Mel-AQ had a cytotoxic activity against bone tumor lines (HEMC-SS and Saos-2 cell lines), whereas no effect was observed with melanoma cells or chondrocytes.

Moreover, Mel-AQ showed its ability to inhibit tumor cell growth in vivo using the syngeneic Swarm rat chondrosarcoma (SRC) model. The antitumor efficacy of this agent and its good tolerance in animals make it a promising candidate for chondrosarcoma treatment [START_REF] Peyrode | Quaternary ammonium-melphalan conjugate for anticancer therapy of chondrosarcoma: in vitro and in vivo preclinical studies[END_REF].

1.8 Targeted therapies for chondrosarcoma

Recent advances in the understanding of molecular pathways involved in chondrosarcoma development have suggested several molecular targets for novel adjuvant therapies for the tumor, such as the inhibition of parathyroid hormone-related protein signaling, matrix metalloproteinases [START_REF] Bovee | Emerging pathways in the development of chondrosarcoma of bone and implications for targeted treatment[END_REF], p38 mitogen-activated protein kinase pathways [START_REF] Halawani | p38 MAP kinase signaling is necessary for rat chondrosarcoma cell proliferation[END_REF], the treatment with peroxisome proliferator-activated receptor-γ ligands [START_REF] Nishida | Inhibition of human chondrosarcoma cell growth via apoptosis by peroxisome proliferator-activated receptor-gamma[END_REF], hypoxia-inducible factor (HIF), and mTOR (32,34).

The transcriptional regulating effects of histone acetylation or deacetylation play important roles in specific gene expressions of chondrocytes [START_REF] Tsuda | Transcriptional coactivators CREB-binding protein and p300 regulate chondrocyte-specific gene expression via association with Sox9[END_REF][START_REF] Imamura | Transcriptional Coactivators CREB-binding protein/p300 increase chondrocyte Cd-rap gene expression by multiple mechanisms including sequestration of the repressor CCAAT/enhancer-binding protein[END_REF][START_REF] Furumatsu | Sox9 and p300 cooperatively regulate chromatin-mediated transcription[END_REF]. Moreover it has also be shown that treatment with the histone deacetylase (HDAC) inhibitor trichostatin A enhances the expression of the α1 chain of type II collagen (COL2A1) and aggrecan in human primary chondrocytes [START_REF] Furumatsu | Sox9 and p300 cooperatively regulate chromatin-mediated transcription[END_REF].

HDAC inhibitors represent a novel class of antineoplastic agents and several HDAC inhibitors are currently under clinical trial [START_REF] Garcia-Manero | Histone deacetylase inhibitors: a review of their clinical status as antineoplastic agents[END_REF]. The antitumor activity of a HDAC inhibitor, depsipeptide (FK228, FR901228), for chondrosarcomas has been tested both in vitro and in vivo given possible implication in cellular differentiation of chondrosarcomas. Depsipeptide induced, cell cycle arrest, and apoptosis in chondrosarcoma cells in vitro [START_REF] Zhu | Histone deacetylase inhibitors repress chondrosarcoma cell proliferation[END_REF]. In addition, HDAC inhibitor modulates the expressions of extracellular matrix genes in chondrosarcoma cells [START_REF] Sakimura | The effects of histone deacetylase inhibitors on the induction of differentiation in chondrosarcoma cells. Clinical cancer research : an official journal of the American Association for[END_REF]. In vivo, the effect of these inhibitors on chondrosarcoma progression was evaluated in a murine xenograft model. In these settings it led to a significant inhibition of tumor growth in a dose-dependent manner without toxicities [START_REF] Sakimura | The effects of histone deacetylase inhibitors on the induction of differentiation in chondrosarcoma cells. Clinical cancer research : an official journal of the American Association for[END_REF].

Another HDAC inhibitor (SAHA) showed similar effect on chondrosarcoma cells proliferation and induced cell autophagy in vitro, inhibited tumor progression in in vivo model [START_REF] Yamamoto | Suberoylanilide hydroxamic acid (SAHA) induces apoptosis or autophagyassociated cell death in chondrosarcoma cell lines[END_REF]. Thus the novel HDAC inhibitors might be a promising tool to use against chondrosarcomas.

Bcl-2 mediates an anti-apoptotic function principally by blocking Apaf1-mediated caspase-9 activation. Bcl-2 is expressed at a high level in osteosarcoma and in 63%-71% of high-grade conventional chondrosarcoma. For the rare chondrosarcoma subtypes, the team of J Bovee demonstrated Bcl-2 expression in 26% of dedifferentiated chondrosarcomas, while 86% of clear cell chondrosarcomas and 96% of the mesenchymal chondrosarcomas expressed Bcl-2. They additionally evaluated the expression of the Bcl-2 family member Bclxl and demonstrated that in dedifferentiated chondrosarcoma, displaying low expression of Bcl-2, high expression of Bcl-xl was found [START_REF] Van Oosterwijk | Screening for potential targets for therapy in mesenchymal, clear cell, and dedifferentiated chondrosarcoma reveals Bcl-2 family members and TGFbeta as potential targets[END_REF].

Moreover, using a large panel of chondrosarcoma cell lines, they were able to demonstrate that chemoresistance in chondrosarcoma is not due to the large amount of extracellular matrix or due to multidrug resistance pump activity, but is caused by the expression of Bcl-2 family members [START_REF] Van Oosterwijk | Restoration of chemosensitivity for doxorubicin and cisplatin in chondrosarcoma in vitro: BCL-2 family members cause chemoresistance[END_REF]. Therefore, Bcl-2 may be a promising target for therapy for chondrosarcoma. BH-3 mimetics like compounds developed by Abbot (ABT-737 and ABT-263) are synthetic small-molecule inhibitors of anti-apoptotic proteins B-cell lymphoma-2 (Bcl2) and Bcl-xL, were recently reported to have an anti proliferative effect (decrease cell viability) in vitro in 2 chondrosarcoma cell lines and improve animal's survival in an xenograft murine model of chondrosarcoma [START_REF] Morii | BH3 mimetics inhibit growth of chondrosarcoma--a novel targeted-therapy for candidate models[END_REF].

In recent years, the Hedgehog (HH) signaling pathway has emerged as a critical determinant of cancer initiation, progression and metastasis of many types of cancers [START_REF] Nagao | Role of GLI2 in the growth of human osteosarcoma[END_REF][START_REF] Ng | The Hedgehog's tale: developing strategies for targeting cancer[END_REF]. HH signals have been shown to be active in chondrosarcoma [START_REF] Tiet | Constitutive hedgehog signaling in chondrosarcoma up-regulates tumor cell proliferation[END_REF]. In chondrosarcoma, HH signaling is not responsible for malignant transformation but likely plays a role in cartilage tumorigenesis by maintaining tumor cells in a less differentiated but proliferative state [START_REF] Tiet | Constitutive hedgehog signaling in chondrosarcoma up-regulates tumor cell proliferation[END_REF].

HH pathway can be targeted at different levels. A study focusing on Gli1 showed that knockdown of Gli1 expression by siRNA downregulated the expression of major HH signal pathway members, including IHh, PTHrP, PTCH1, and SMO. siGli1 decreased the proliferation rate, induced cell cycle G2/M phase arrest by downregulating cyclinB and Rb phosphorylation, thereby rendering chondrosarcoma cells quiescent and preventing tumor progression. These data indicated that for chondrosarcoma, therapeutic effects might be achieved by Gli1 inhibitors [START_REF] Sun | Gli1 inhibition suppressed cell growth and cell cycle progression and induced apoptosis as well as autophagy depending on ERK1/2 activity in human chondrosarcoma cells[END_REF].

Another effector of HH pathway that was targeted by a synthetic inhibitor is SMO.

IPI926 is small-molecule that directly targets SMO and had shown activity in xenograft models of basal cell carcinoma and medulloblastoma before being tested in chondrosarcoma. It was shown to be a very effective antitumor agent in primary chondrosarcoma xenografts [START_REF] Campbell | Hedgehog pathway inhibition in chondrosarcoma using the smoothened inhibitor IPI-926 directly inhibits sarcoma cell growth[END_REF].

A Phase 2 multicenter trial evaluating the safety and efficacy of IPI-926 in patients with metastatic or locally advanced (unresectable) chondrosarcoma was initiated (clinical trial NCT01310816) and did not showed promising results or proved disappointing (NCT01310816) even if long term non progressive patients are still under treatment (unpublished data).

Isocitrate dehydrogenase (IDH) mutations were recently identified in approximately 50% of chondrosarcomas and drugs are currently under development to inhibit mutant IDH enzymatic activity [START_REF] Amary | IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours[END_REF]. Isocitrate dehydrogenase proteins consist of three regulatory enzymes (IDH1, IDH2, and IDH3) that catalyze the NADP + -dependent oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (alpha-KG) in the Krebs cycle Mutations in the IDH family of genes recently have been described in patients with a variety of cancers, among which chondrosarcomas [START_REF] Kosmider | Mutations of IDH1 and IDH2 genes in early and accelerated phases of myelodysplastic syndromes and MDS/myeloproliferative neoplasms. Leukemia : official journal of the Leukemia Society of America[END_REF][START_REF] Marcucci | IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study[END_REF]. Somatic mutations also have been identified in up to 70% of primary conventional central chondrosarcomas, and 54% of dedifferentiated chondrosarcomas [START_REF] Amary | IDH1 and IDH2 mutations are frequent events in central chondrosarcoma and central and periosteal chondromas but not in other mesenchymal tumours[END_REF][START_REF] Meijer | Genetic characterization of mesenchymal, clear cell, and dedifferentiated chondrosarcoma[END_REF][START_REF] Schaap | Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 in tumors[END_REF]. Mutated IDH1 or IDH2 are active catalytic enzymes. in vitro treatment of IDH1 mutant chondrosarcoma cell lines with an IDH1 inhibitor, induced apoptosis, and inhibited tumorigenic activity [START_REF] Tinoco | The biology and management of cartilaginous tumors: a role for targeting isocitrate dehydrogenase[END_REF][START_REF] Li | Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells[END_REF]. Clinical trials on IDH inhibitors in chondrosarcoma are underway.

One of the signaling pathways that has been extensively studied over the last years is the PI3K/Akt/mTOR signaling cascade. Mutations and aberrant activations of this pathway effectors are frequently detected in malignancies including sarcoma and have been linked to cancer development, chemoresistance and radiation resistance mechanisms [START_REF] Bovee | Emerging pathways in the development of chondrosarcoma of bone and implications for targeted treatment[END_REF]. In cancers, PI3K/mTOR promotes cell survival and proliferation, apoptotic resistance and increasing data also link this pathway to tumor invasion and metastasis (25).

The emergence of pharmacological compounds directed against mTOR represents a major breakthrough for cancer-targeted therapies. First-generation mTOR inhibitors, i.e. rapalogs, have shown anticancer activities in routine clinical setting as well as in clinical trials [START_REF] Ito | In vivo antitumor effect of the mTOR inhibitor CCI-779 and gemcitabine in xenograft models of human pancreatic cancer[END_REF][START_REF] Mabuchi | RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer[END_REF][START_REF] Nathan | Mammalian target of rapamycin inhibitors as possible adjuvant therapy for microscopic residual disease in head and neck squamous cell cancer[END_REF]. Two sets of recent data suggest that the inhibition of mTOR pathway could be an effective systemic treatment for chondrosarcoma.

The first one was generated by the group of Merimsky who showed in a case report then in a clinical trial that the association of a rapalog with cyclophosphamide is efficient in controlling chondrosarcoma progression [START_REF] Merimsky | Targeting the mammalian target of rapamycin in myxoid chondrosarcoma[END_REF][START_REF] Bernstein-Molho | Clinical activity of mTOR inhibition in combination with cyclophosphamide in the treatment of recurrent unresectable chondrosarcomas[END_REF].

The second one concerns preclinical data generated by our group. Using an orthotopic rat chondrosarcoma model, we have shown that RAD001 inhibits chondrosarcoma proliferation, significantly reduced tumor growth and delayed tumor recurrence [START_REF] Perez | Inhibition of chondrosarcoma growth by mTOR inhibitor in an in vivo syngeneic rat model[END_REF].

In vivo this agent was able to significantly slow down chondrosarcoma progression in settings where the conventional agent doxorubicin didn't have a therapeutic effect.

The discrepancy between the in vivo activity of rapalogs and lack of direct activity on tumors cells in vitro seems to indicate that tumor microenvironment plays a role in chondrosarcoma response to rapalog, as it has been reported for other therapeutic agents [START_REF] David | Direct anti-cancer effect of oncostatin M on chondrosarcoma[END_REF].

The results obtained in this study, prompted us to explore the role played by tumor microenvironment, more precisely by the immune tumor environment, in chondrosarcoma progression and response to conventional and/or targeted therapy.

In order to better understand immune systems populations involvement in chondrosarcoma, the next chapter will present in a summarized way the role of immune system in cancer.

Cancer and the immune system in carcinomas and sarcomas

Cancer is a disease of unrestricted proliferation of cells. The uncontrolled growth of cells that characterize cancer can be caused by many factors, such as mutations in oncogenes and tumor suppressor genes as well as virally encoded proteins; elements causing deregulation of the cell cycle.

The accumulation of mutations in oncogenes or tumor suppressor genes, lead to tumorigenesis which endows the mutation-rich cell with the hallmarks typically present in cancer (Figure 2.1). These include, sustained proliferation, resistance to cell death, angiogenesis, evasion of growth suppressors, replicative immortality, genomic instability, escape from immune destruction, tumorpromoting inflammation, deregulated cellular energetics as well as invasive characteristics (79) (figure 2.1).

The involvement of immune system in the cancer has only been in considered for decades. It was confirmed in human tumors since the advent of therapeutic immunosuppression developed in transplant settings and experience in HIV patients, but confirmed as a potential therapeutic strategy only since the beginning of the 80' with a limited efficacy of recombinant cytokines IFNa and IL-2. The introduction of immune checkpoint inhibitors demonstrated the crucial therapeutic potential of immune system manipulation since 5 years.

There has been remarkable progress in both the understanding of the immune system and of ways to turn it on and off to treat cancer. Given these changes in perspective, cancer immunotherapy was Science magazine's 2013 advance of the year [START_REF] Couzin-Frankel | Breakthrough of the year 2013[END_REF]. As a result of this enthusiasm, many immunotherapeutic approaches have been developed and some are now in clinical trials for all type of cancers including sarcomas. In this section of the introduction, we will briefly present the immune system, its involvement in cancer surveillance. The mechanisms that cancer cell has developed to evade this surveillance will then be introduced and some immunotherapeutic strategies discussed.

Since the knowledge and studies of immune system implication in sarcoma are comparatively way behind what was done in carcinomas, we will only present the immune system in cancer in general with a focus on sarcomas when the data are available and/or different from what is generally accepted in cancer immunity.

Because it is impossible to cover all aspects of the immune response in a single manuscript, we will focus on what is useful for the readers (even non immunologists) to understand the results of this work.

2.1 The immune system in a nutshell

The natural task of the immune system is to protect the human body from infections by bacteria, viruses, and parasites but also against it's own cells dysfunction.

The immune system is composed of two parallel and closely related components: the innate immunity and the adaptive immunity.

The innate immunity, often called the 'first line of defense', defends the host within minutes after exposure to a noxious stimulus. Innate immune response may initiate acute inflammation, which involves the accumulation and activation of phagocytes. The innate immune system uses non-clonal "pattern recognition" receptors (PRRs), which do not recognize human structures but do recognize patterns found on microbes. These receptors recognize pathogen-associated molecular patterns (PAMP), such as single-stranded viral DNA, and as a consequence induce activation and effector functions in innate immune cells. The intruding pathogen will be captured, internalized, and killed by effectors of the innate immunity (macrophages, NK). At the same time, elements of the pathogen, so-called antigens, will be transported from the site of infection to secondary lymphoid organs, such as lymph nodes and the spleen, to be presented to the adaptive arm of the immune system. This presentation is performed by professional antigen-presenting cells (APC), of which dendritic cells (DC) are the most potent [START_REF] Abbas | Cellular and molecular immunology[END_REF]. Each of these cell populations express receptor specific for a particular antigen.

A T cell will therefore sample antigens presented by many different DC in the lymph node. The encounter with its cognate antigen will activate this T cell, which will proliferate and produce cytokines. Activated T cells can then migrate to the site of infection in response to chemokines produced by the innate immune cells that first encountered the pathogen [START_REF] Abbas | Cellular and molecular immunology[END_REF].

T cells populations are divided into helper T (Th) cells, which are CD4+, and mainly produce cytokines, and 'killer T cells, which express CD8 and can recognize and kill pathogen-infected cells that present viral antigens on their surface. Some of the activated, antigen-specific lymphocytes will persist as memory T cells even after the infection is cleared and can be rapidly re-activated if they encountered the same antigen/pathogen.

Immune system function to protect body from aggressions and cellular dysfunction; whether its role was also to contain non virally induced tumors was a matter of debate. The concept of immune surveillance, which was proposed in the 1950s states that a physiological function of the immune system is to recognize and destroy clones of transformed cells. The following paragraphs will present the actors of tumor immune response and mechanisms of immunosurveillance and immunoediting.

2.Tumoral immune response

Tumor microenvironment is composed of stromal cells, blood vessels, and innate and adaptive immune cells populations. The lasts are inflammatory infiltrate that cause a local chronic inflammation. Infiltrating leukocytes in the tumor microenvironment may exert a dual role on tumor development and progression. Tumor cells can be directly eliminated by the immune cells, which had developed an antitumoral immune response. Tumor cells can also recruit and instruct immune cells to favor tumor growth and progression. The recruited immune cell consists of many players, including innate and adaptive immune populations. Cells that infiltrate cancer and their role are recapitulated in table below.

Table 2.1: The Principal immune cells population in cancer

Cells from the innate immunity can directly recognize cancer cells via specific receptors (resume on figure 2.4) and participate to lymphocytes recruitment to induce an antitumoral action (Fig 2 .4). Here we have done a short review on the role of the three principal innate immunity effectors within cancer microenvironment: macrophages, natural killer cells and dendritic cells.

A)

Plasticity is a well-known characteristic of macrophages: they are able to adapt their phenotypes in function of signals they receive from the microenvironment [START_REF] Sica | Macrophage plasticity and polarization: in vivo veritas[END_REF]. Tumor associated Macrophages (TAM) are divided into two antagonistic populations: M1 and M2 macrophages.

M1 macrophages are induced by the exposure to Th1 cytokines (e.g., IFN-γ) alone or in combination to microbial components (e.g., LPS). M1 macrophages participate in Th1 response and are mediators of resistance against tumor cells. Typically M1 polarized macrophages are producers of effector molecules (e.g., reactive oxygen and nitrogen intermediates) and inflammatory cytokines (e.g., IL-1β, TNF, IL-6). They produce high levels of IL-12, and display a low expression of . In general, cytokines produced by M1 in cancer induce an anti-tumoral response.

The cytokines IL-4 and IL-13 inhibit this classical activation and induce the alternative M2 form of macrophage polarization [START_REF] Gordon | Monocyte and macrophage heterogeneity[END_REF]. Unlike M1, M2 macrophages produce high amounts of IL-10 and low amounts of IL-12, express CD14 and CD163 surface proteins and display poor antigen presenting capacity [START_REF] Noel | Alternatively activated macrophages during parasite infections[END_REF]. Moreover, they suppress Th1 adaptive immunity, participate in the resolution of inflammation, and promote wound healing, angiogenesis, and tissue remodeling [START_REF] Biswas | Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm[END_REF]. Immunological stimuli, such as IFN-γ or IFNα, can reprogram immunosuppressive M2 macrophages into immunostimulatory M1 cells [START_REF] Duluc | Interferon-gamma reverses the immunosuppressive and protumoral properties and prevents the generation of human tumor-associated macrophages[END_REF][START_REF] Palma | Tumortargeted interferon-alpha delivery by Tie2-expressing monocytes inhibits tumor growth and metastasis[END_REF].

Prognostic significance of TAM in humans has been recently evaluated [START_REF] Zhang | Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature[END_REF]. Though there are evidences that in many cancers, such as breast, cervix, bladder, and gastric carcinoma, high numbers of TAM are significantly associated with poor prognosis, [START_REF] Qian | Macrophage diversity enhances tumor progression and metastasis[END_REF].

Nevertheless, other studies have reported that the prognostic significance of TAM can be controversial [START_REF] Zhang | Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature[END_REF]. For instance, in high-grade osteosarcoma high infiltration of CD68+ TAM (M1 and M2) are associated with reduced metastasis and improved survival [START_REF] Buddingh | Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clinical cancer research : an official journal of the[END_REF], whereas in classical Hodgkin's lymphoma and, large B-cell lymphoma this correlation was not found [START_REF] Kayal | CD68 tumor-associated macrophage marker is not prognostic of clinical outcome in classical Hodgkin lymphoma[END_REF][START_REF] Hasselblom | Expression of CD68+ tumor-associated macrophages in patients with diffuse large B-cell lymphoma and its relation to prognosis[END_REF]. However a recent study in patients with diffuse large B-cell lymphomas, reported that an increase of CD163+ (M2) cells predicts poor prognosis, while an increase in CD68+ cells was related to improved overall survival [START_REF] Nam | An increase of M2 macrophages predicts poor prognosis in patients with diffuse large B-cell lymphoma treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone[END_REF]. There is still a lot of work to clarify the role of TAM in cancer microenvironment, as TAM infiltration does not appear to have the same prognostic value for different cancer type.

B)

Natural killer cells have been initially identified as a lymphoid population able to lyse MHC class I (MHC-I) negative tumor and virus-infected cells and to orchestrate innate immunity of the organism. The majorities of NK cells are localized in peripheral blood, lymph nodes, spleen, and bone marrow but can migrate toward inflammation sites, such as primary or metastatic cancer cells [START_REF] Ferlazzo | The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic[END_REF][START_REF] Robertson | Role of chemokines in the biology of natural killer cells[END_REF]. The fact that NK cells constitutively express lytic machinery able to kill Unlike the innate immunity, the adaptive immune system is highly specific to a particular antigen. Adaptive immunity can also provide long-lasting protection, by the formation of a memory response. The adaptive immune response destroys invading pathogens, but can also be directed toward infected cells or defective cells like for cancer cells. Tumor cells express antigens that are recognized as foreign by the adaptive immune system, either by T or B lymphocytes. Depending on the lymphocytic population that recognize the antigen different adaptive immune response is initiated: -antibody responses and cell-mediated immune response-are also carried by two different lymphocytes (respectively B cells and T cells) [START_REF] Abbas | Cellular and molecular immunology[END_REF].

Even if the antibody response is important in the context of immunosurveillance of the tumors, we will focus mainly on the cell-mediated immune response.

Tumor associated antigens (TAAs) are expressed by tumor cells. These cells can be phagocytized by antigens presenting cells (mainly macrophages and DCs). APCs present these antigens with their class II MHC molecules to T cell receptor (TCR) expressed at the surface of T lymphocytes. ( 108).

TAA can also be presented by cancer cells with class I MHC molecules and can be directly recognized by the TCR of T lymphocytes(108).

Each T cell has a unique TCR that respond to a unique antigen. The principal TAAs found in cancer are recapitulated in table 2.2.

Table 2.2: Classification of tumor-associated antigens that are recognized by T cells (109).

The recognition of a specific antigen by lymphocytes leads to a clonal expansion and proliferation of the lymphocyte that depend on the strength of the signal. (111). The demonstration that CD4 T cell help was required for another specialized function of the immune system was the first example of cooperation between two cell populations in producing an immunological effect. A few years later it was also demonstrated that CD4+ T cells also help the activation and expansion of CD8 T cells (112). CD4 T cells can be divided into two main categories: T helper 1 (Th1) and T helper 2 (Th2) depending on the cytokines they produce in response to antigen activation. Th1 cells produce IL-2, IL-12 and IFN-γ. Th2 produce IL-4 and IL-5. This functional/phenotypic dichotomy influences the production of antibodies of the different isotypes and the types of CD8 T cells that originate after activation by antigen (113,114).

In cancer it has been shown that Th1 promote CD8+ T cells expansion, upregulation of the MHC class I at the surface of tumor cells. Presence of Th1 in the tumor microenvironment is considered good prognostic in the majority of cancer studies (115,116).

T H 2 cells contribute to the development of M2 macrophages through the production of the cytokine IL-10 and IL-4 and seem to be associated with aggressive tumors (116). However, this is not a general phenomenon, as T H 2 cells are also associated with favorable outcome in Hodgkin's lymphoma and breast cancer which suggests a protective effect of antibodies in these diseases (117,118). Immunohistochemical studies have concluded that tumor infiltrating CD8+ lymphocytes have antitumor activity as judged by their favorable effect on patients' survival in colorectal, ovarian esophageal, renal, lung and pancreatic tumors (122-129).

2) CD8+ cytotoxic T lymphocyte (CTL)

Cytotoxic

3) CD4+CD25+FoxP3+ Regulatory T lymphocyte (Treg)

Among T cells recruited to human solid tumors are CD4+FOXP3+CD25+ regulatory T cells (Treg). These T cells accumulate in tumors and in the peripheral blood of patients with cancer (130). The increased Treg frequency has been generally considered as a marker of poor prognosis in cancer presumably due to Treg-mediated suppression of anti-tumor immunity, which benefits the tumor (131-133). However, several lines of evidence suggest that Treg might play a dual role in carcinogenesis. On the one hand, Treg can down-regulate inflammation, which in some cancers has been linked to tumor progression ( 134

Principles of immunosurveillance and immunoediting

The idea that the immune system, might recognize and destroy tumor cells emerged more than 50 years ago (135 Thus, tumors formed in the absence of an intact immune system are, more immunogenic than tumors that arise in immunocompetent hosts. These results

show that the immune system not only protects the host against tumor formation, but also edits tumor immunogenicity. These new data prompted a refinement of the cancer immunosurveillance concept and led to the formulation of the cancer immunoediting hypothesis, which stresses the dual host-protective and tumorsculpting actions of immunity on developing tumors.

In cancer immunoediting all the immune populations that infiltrate tumors are involved and interact with tumor cells. This complex process can be separate into three phases (figure 2.8). That will be presented in the coming sections. Immunologic rejection of a developing tumor, likely requires an integrated response involving both the innate and adaptive arms of the immune system (section 2.2). Over the years, multiple strategies of the tumor cells to evade the immune response have been discovered. Since every strategies are very complex on their own, we focus on immune checkpoint upregulation as they are generating tremendous enthusiasms for immunotherapeutic approaches.

The myriad of genetic and epigenetic alterations that are characteristic of all cancers provide a diverse set of antigens that the immune system can use to distinguish tumor cells from their normal counterparts. In the case of T cells, the ultimate amplitude and quality of the response, which is initiated through antigen recognition by the T cell receptor (TCR), is regulated by a balance between costimulatory and inhibitory signals (that is, immune checkpoints) (163, 164) (Figure 2.8). Under normal physiological conditions, immune checkpoints are crucial for the maintenance of self-tolerance (that is, the prevention of autoimmunity) and also to protect tissues from damage when the immune system is responding to pathogenic infection. The expression of immune-checkpoint proteins can be deregulated by tumors as an important immune resistance mechanism. As shown in figure 2.10 there is a multitude of checkpoint inhibitor that regulates the quality of the TCR induction. Among them, two were the object of a multitude of study on cancer research: CTLA-4 and PD-1.

2) PD-1

In contrast to CTLA-4, the major role of PD-1 is to limit the activity of T cells in peripheral tissues at the time of an inflammatory response to infection and to limit autoimmunity (176, 177) (Figure 2.11b). This translates into a major immune resistance mechanism within the tumor microenvironment (178). PD-1 expression is induced when T cells become activated (179). When engaged by one of its ligands, PD-1 inhibits kinases that are involved in T cell activation through the phosphatase SHP2, although additional signaling pathways are also probably induced (177). Also, because PD-1 engagement inhibits the TCR 'stop signal', this pathway could modify the duration of T cell-APC or T cell-target cell contact(180). PD-1 is expressed on a large proportion of tumor-infiltrating lymphocytes (TILs) from many different tumor types (181).

The two ligands for PD-1 are PD-1 ligand 1 (PD-L1; also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC or CD273) (177, 182). Just as PD-1 is highly expressed on TILs in cancers, the PD1 ligands are commonly upregulated on the tumor cell surface from many different human tumors (164, 178). On cells from solid tumors, the major PD-1 ligand that is expressed is PD-L1. Forced expression of PD-L1 on mouse tumor cells inhibits local antitumor T cellmediated responses (178). The evident role of immune systems in restraining and/or promoting cancer growth makes it a target of choice for novel therapies. The next chapter will focus on these therapies now developed for the treatment of sarcomas.

Immunotherapies in sarcoma

Sarcomas have long been suspected to have a connection to the immune system. In 1891, physicians such as William Coley noted a relationship between infection and regression of sarcoma (183). Additionally, some patients with metastatic sarcomas can be cured with aggressive resection of all visible disease, unlike many other solid tumors. It is thought that immune surveillance and suppression of growth must play a role in the prevention of recurrence in these patients (184). Some sarcomas express highly immunogenic antigens, such as cancer testes antigens, microphthalmia transcription factor, and even the fusion proteins themselves. The presence of these antigens shows that immunotherapies could be operational for sarcomas treatments (table 2.3) (185, 186).

Over the past several years, immunotherapy has regained significant attention in solid tumors, among which sarcoma, even for tumors largely believed for having low immunogenicity, and novel targets and immunotherapeutic strategies are rapidly emerging. Here we review the different immunotherapies explored in sarcomas and chondrosarcomas and the future perspectives envisaged.

Cytokines therapies

Cytokines, including interleukins and growth factors, play an important role in the regulation of the immune system (187). Several studies have indicated that cytokine therapy could relieve immunosuppression in cancer patients (188) and therefore, cytokine therapy is a potential therapeutic approach for sarcoma patients. Two main cytokines therapies have been tested in sarcoma.

Interleukin 2 (IL-2) stimulates T cells proliferation, induces generation of CTLs, and facilitates the maintenance of NK cells (189,190). IL-2 is approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma, and

responses

Vaccines therapies

Vaccines directed against specific tumor antigens were some of the earliest targeted immunotherapies tested. The aim of tumor vaccines is to induce an antitumor response through exposure to tumor antigens. Multiple clinical trials using vaccines that target whole cells, lysates, proteins, and peptides have been investigated in patients with sarcomas (Table 3.2) (194)(195)(196). In these studies, patients with chondrosarcoma show high expression of tumor-associated antigens (TAAs) and show good response to vaccine. However there were very few patients with chondrosarcoma in these studies and follow-up need to be done. Vaccines therapies have been validated for bones and soft tissues sarcoma. Table 3.3 review the main clinical studies made for sarcomas. Currently, however, these agents have limited pediatric clinical data available;

several trials with these agents for relapsed or refractory pediatric solid tumors are currently ongoing.

Despite the overall successes of checkpoint inhibitors, only subsets of patients with melanoma, lung cancer, ovarian cancer, NHL, and Hodgkin lymphoma have responded. Two important studies have examined the tumors of responders versus non-responders, one in melanoma and one in non-small-cell lung cancer (212,213). In both cases, treatment efficacy was associated with a higher number of mutations in the tumors. In melanoma patients treated with ipilimumab, the investigators carefully examined the tumors of those who responded versus those who did not, and found that the responders had tumors with higher mutation rates and tumor antigens and in particular, those whose tumor neoantigens shared tetrapeptide sequences with viral antigens were most likely to be responders to checkpoint inhibition (213). To improve on the quality of response to immune checkpoint blockade, CTLA-4 and PD-1/PD-L1 antibodies are being tested in combination or when added to other anti-cancer agents such as chemotherapy, targeted therapy, radiotherapy, and other immunotherapy (209,214,215). Currently, the COG is conducting a phase I/II study (NCT02304458) of nivolimab alone or in combination with ipilimumab for relapsed and refractory solid tumors including sarcomas.

3.6 Futures directions for immunotherapies in sarcoma

Conventional treatment for bone and soft tissue sarcomas consists of surgical resection, chemotherapy, and radiotherapy. However, clinical outcomes by these therapeutic modalities have not significantly improved in recent decades. Under these circumstances, immunotherapy is expected to be a new therapeutic option for treatment. Cytokine therapies were initially regarded as a form of immunotherapy; however, their effectiveness was limited because of their toxicities. Only IFN-α-2 is used for maintenance therapy.

Although L-MTP-PE induces antitumor effects via macrophage activation, the FDA has not approved its use because of the limited effectiveness. In Europe, L-MTP-PE efficacy has been confirmed in an international multicenter study.

Vaccine therapy using tumor lysates or lysate-derived DCs has been investigated only in small-scale studies and in non-sarcoma patients. CTA peptide and fusion protein peptide therapies are expected to be novel sarcoma-effective vaccines.

Addition of L-MTP-PE as an adjuvant may improve the vaccine therapy outcome. As it was highlighted in the bibliographic section of this manuscript, chondrosarcoma is a complex malignancy with a spectrum of histological differentiation and a spectrum of malignant potential. Because of their extracellular matrix, low percentage of dividing cells, and poor vascularity, chondrosarcoma are relatively chemo-and radiotherapy resistant. To date, adequate surgical excision remains the most important principle of treatment for patients with chondrosarcoma. Improving chondrosarcoma clinical management is therefore a challenging problem and novel therapeutic approaches are needed to decrease tumor relapse and increase patient's survival and quality of life.

Recent scientific data in the microenvironment and molecular pathways involved in chondrosarcoma have highlighted the role of matrix metalloproteinases, bone niche, hypoxia-inducible factor (HIF), and mTOR (32)(33)(34). Treatments targeting tumoral microenvironment or signalling pathway have therefore been suggested

as complements to surgical excision (31,35,225). Some of these approaches are currently under investigation in clinical trials.

Immunotherapeutic approaches are other options that could be considered for Osteosarcoma represents a specific sarcoma in which immunotherapy has proven effective, such that regulatory authorities have approved the non-specific immune stimulator MTP (muramyl tripeptide) for human use in primary osteogenic sarcoma. New strategies based on clinically approved immune checkpoint inhibitors and specific targeting of tumor antigens using adoptive transfer and chimeric antigen receptor techniques have also been developed.

Keeping in mind/in view the promises and hopes generated by immunotherapeutic approaches targeting immune checkpoints like PD-L1, it appears that there is an immediate opportunity to assess the potential of such strategies in the spectrum of human chondrosarcomas.

Chondrosarcoma immune microenvironment and the implication of immune checkpoint in this group of tumors remaining largely unknown, my PhD project aimed at investigating chondrosarcoma immunological microenvironment, its implication in chondrosarcoma growth and response to therapy. Moreover, we planned to further explore the role played by immune checkpoint PD-1/PD-L1 in chondrosarcoma and determine whether targeting PD-1/PD-L1 pathway targeting alone or in combination with conventional/targeted therapies could be an innovative immune-based therapeutic strategy for CHS patients.

The results of this work will be presented in the coming paragraphs and the results will be divided into five main sections.

1.

Characterization of the immunological niche on human tumor biopsies at the cellular level (T lymphocytes, macrophages, PD-1) and the expression of PD-L1 on human chondrosarcoma cells

2.

Characterization of the immunological niche on Swarm rat chondrosarcoma model (SRC) and comparison to the human chondrosarcoma immune environment.

3.

Analysis of main immune populations (T lymphocytes and macrophages) involvement in chondrosarcoma progression. Given the difficulty to gain access to sufficient human CHS biological material, this study will be conducted in the SRC model.

4.

Effect of PI3K/Akt/mTOR pathway inhibition on chondrosarcoma immune response. Here we will investigate whether drugs targeting the PI3K/Akt/mTOR pathway-targeting agent could exert an immunostimulatory effect and initiate an immune response in chondrosarcoma. For this purpose, we will look at the changes in immune cells populations and cytokines secretion arising in response to the evaluated treatments.

5.

Development of preclinical studies to determine the role of the PD-1/PD-L1 pathway in relevant syngeneic CHS animal model, and determine whether a strategy using blocking mAb anti-PD-L1 is applicable in SRC.

This study will allow the characterization to better understanding of the chondrosarcoma immunological niche and determine its potential role in tumor progression and response to targeted therapy. Understanding this relation between the chondrosarcoma and its immune environment will help us to better characterize and treat this complex tumor. This project would open the way to novels approaches of combined therapies associating immunotherapy and targeted therapy for the treatment of resistant or refractory bone sarcoma

Materials and methods

Cell cultures

The human chondrosarcoma (SW1353), rhabdomyosarcoma (SJCRH30), bone marrow (K-562), Natural Killer (NK-92) cell lines, and rat osteosarcoma cell line (UMR-106) were all obtained from the ATCC (LGC Standards, Molsheim, France) and were cultured at 37°C, under 5% CO 2 , using DMEM or RPMI medium (for NK-92 cell line and SJCRH30) supplemented with 10% FBS, 2% Lglutamin, and penicillin/streptomycin solution (10,000 U/ml; 10,000 μg/ml) (GIBCO ThermoFisher Scientific, Waltham, USA). Primary rat chondrosarcoma cells and chondrocytes were isolated from SRC or healthy animals by collagenase digestion ( 228) and cultured in DMEM medium supplemented with 10% FBS, 2% L-glutamine, and penicillin/streptomycin solution (10,000 U/ml; 10,000 μg/ml).

The induction of PD-L1 in vitro

The induction of PD-L1 expression in cultured cell lines was performed using 20 ng/ml INFγ (Sigma Aldrich, St-Louis, USA) in culture medium over night. Cells were incubated with PI3K inhibitor GDC-0941 (100 mM, Genentech, San

Fransisco, Ca, USA), Akt inhibitor G-594 (100 mM, Genentech), mTOR inhibitor Everolimus (10 mM, Novartis, Bâle, Switzerland) or DMSO over night. The next day, cells were used to performed immunoblotting or FACs analysis. and its corresponding 95% confidence interval (CI) were calculated.

In vivo assay

At the completion of treatment for all experimentations, rats were sacrificed, tumors and spleens harvested. Part of tumors and spleen were used for chondrosarcoma cells isolation and splenocytes harvesting while the remaining samples were fixed in 10% formalin for further analyses. Blood was collected by cardiac puncture and allowed to clot at room temperature to obtain serum for ELISA analysis.

Immunoblotting

Immunoblotting was performed to evaluate PD-L1 expression in bone sarcoma cell lines. Proteins extraction and immunoblotting procedures have been previously reported [START_REF] Perez | Inhibition of chondrosarcoma growth by mTOR inhibitor in an in vivo syngeneic rat model[END_REF]

Splenocytes harvesting

Spleens were mashed on 40-μm-cells strainer (BD Bioscience, San Jose, USA) placed on top of a 50 mL Falcon tube, using a syringe plunger. The collected cells were centrifuged (250 g, 5 min, 4°C) after addition of 10 mL RPMI medium supplemented by 10% FBS. To remove red blood cells, pellet was incubated 3 min at room temperature in ACK red blood cell lysis buffer (GIBCO) before being centrifuged (250 g, 5 min) and resuspended in complete RPMI medium.

Splenocytes were either used immediately for cytotoxicity assays or frozen in RPMI medium supplemented with FBS (50%) and DMSO (10%) and stored in liquid nitrogen for further analyses.

Cytotoxicity assay

A nonradioactive, fluorometric cytotoxicity assay using calcein-acetoxymethyl (calcein AM; Thermofisher C3099) was performed to measure the lysis of rat chondrosarcoma cells and chondrocytes (further named target cells) by splenocytes. Target cells (1 × 10 6 /ml) were labeled with Calcein-AM (10 μmol/L) at 37°C for 1 h before being washed twice in complete culture medium and plated at 10 4 cells/wells in round bottom 96-well microtiter plates (Nunc, P8116, Sigma Aldrich, St-Louis, USA). Target cells where then incubated with 400 μmol/L Sulfinpyrazone (Sigma-Aldrich) in complete medium (1h, 37°C) to avoid spontaneous calcein release.

Splenocytes isolated from animals of all treatment group were used as effector cells and added to targets cells at E:T ratios ranging from 100:1 to 12,5:1.

Effectors and targets were incubated 6h at 37 °C under normal culture conditions. Targets cells spontaneous and maximum release were assessed respectively by plating target cells alone in 96 wells or by incubating target cells in presence of 2% of Triton X-100 for 6h under normal culture conditions.

Supernatants were then collected (100 µl) by centrifugation ( 250

Phenotypic analyses of chondrosarcoma immune microenvironment

Chondrosarcoma immune microenvironment was analyzed from 52 SRC and 26

conventional grade 1 to grade 3 human chondrosarcoma samples treated in our institution between 2004 and 2012. All samples were fixed in 4% formalin (pH 7.4) and embedded in paraffin. Deparaffination and rehydration were done under standard procedures using xylene (VWR international S.A.S, Fontenay sous Bois; France) followed by ethanol gradient (VWR).

For SRC, 5 µm sections from formalin-fixed, paraffin-embedded (FFPE) tumor blocks were stained with the following primary antibodies: CD3 (Ventana, Tucson, AZ, USA), CD8 (1/100), CD163 (1/300), PD-1 (1/100), PD-L1 (1/300) (respectively OX-8; ED2; AHP1706; AHP1703; AbD SeroTech, Oxford, UK). CD8

and CD163 primary antibodies were revealed using an unconjugated goat antimouse secondary antibody and CD3, PD-1, PD-L1 by unconjugated goat antirabbit antibody (respectively AI-9200, AI-1000; Vector lab, Burlingame, CA, USA; dilution 1:100) followed by avidin-biotin complex and DAB peroxidase (VECTASTAIN Elite ABC Reagent, ImmPACT reagent; Vector Lab).

Counterstaining was performed with hematoxylin (Sigma-Aldrich).

For human chondrosarcoma samples, 5 μm sections from FFPE blocks were deparaffinized and rehydrated. Immunostaining for CD3, CD8, CD4 and CD163 was performed on automated Ventana Discovery XT staining system. Primary antibodies to CD3 CD8, CD4, and CD163 were all from Ventana (respectively clone 2GV6, SP57, SP35, MRQ-26) (PD-1 and PD-L1 antibodies (AHP1706 and AHP1703 from Abd Serotec) were applied at 1/100. All primary antibodies were revealed with a biotinylated anti-Rabbit secondary antibody and a DABMap Kit followed by counterstaining with hematoxylin (all reagents from Ventana).

All slides were analyzed using a Nikon microscope (EclipseNi-E Nikon Corp)

Quantitative analysis of chondrosarcoma immune infiltrates

Quantitative analysis of chondrosarcoma immune infiltrates was similarly conducted on SRC and human chondrosarcoma slides. Chondrosarcoma's immune cells were counted in five representative areas with highest density of infiltrates cells located at the periphery of the tumors. Sections were scored based on the intensity and the extent of the staining using the following grading:

significant if the Student t-test gives a significance level (P value <0.05. Multiple comparisons were performed using a univariate analysis of variance (ANOVA).

All statistical analysis was performed using GraphPad Prism version 6.00 (GraphPad Software, La Jolla, CA, USA, www.graphpad.com).

Results

Human chondrosarcoma immune microenvironment

The localization, lineage and density of immune cells present in chondrosarcoma microenvironment were analyzed in a cohort of 26 grade 1 to 3 conventional chondrosarcoma.

On each tumor, positive cells for each immune marker considered (CD4, CD8, CD163) were counted on 5 different areas located at the margin of cartilaginous nodules and surrounding muscles. • Lymphocytes and macrophages are found in the marginal area of human conventional chondrosarcoma.

• Lymphocyte infiltration is of good prognostic while macrophages is of bad prognostic for human conventional chondrosarcoma

•CD8 infiltration is inversely correlated to the CD163 infiltration. As in human chondrosarcoma, CD8+ cells count was found to be inversely correlated with tumor size (measured at the time of animal's sacrifice) whereas CD163+ cells count was correlated with it (Figure 6.9). In the SRC model the CD8/CD163 ratio was inversely correlated with tumor size (Figure 6.10). The effects of each treatment on tumor progression and immune environment were analyzed and are reported in the following paragraphs.

All treatments affected SRC immune environment.

In animals treated by clodronate or mifamurtide, a lower infiltration of CD163+ cells in tumor microenvironment was observed. In azathioprine treated animals, CD8+ and CD3 T cells were significantly decreased in tumor's environment (Fig 6.11). Both clodronate and mifamurtide affected tumor progression. Animals treated with these agents had significantly smaller tumors compared to control animals (respectively 1127 ± 177 mm 3 and 1294 ± 195 mm 3 versus 1913 ± 202 mm 3 P < 0,05 on day 18). In rats treated with clodronate or mifamurtide, the tumor growth rate was lower than in the control group: tumor proliferation rate was respectively 16,23 (± 2,19) in the clodronate treated group and 24,32 (± 3,42) in the mifamurtide treated rats versus 63,17 (± 14,32) in the control group at day 18 (P < 0,005) (Fig 6 .12A). These results confirmed the possibility to use PI3K pathway inhibitors to slow down chondrosarcoma progression, as we previously shown for mTOR inhibitor [START_REF] Perez | Inhibition of chondrosarcoma growth by mTOR inhibitor in an in vivo syngeneic rat model[END_REF].

To further investigate the effects of these inhibitors on chondrosarcoma immune microenvironment and the immune response, we analysed composition of SRC immune environment with a focus on CD163+, CD8+ and CD3+ cells.

The three PI3K pathway inhibitors evaluated did not affect SRC immune microenvironment composition. Similar densities of CD3+, CD8+ T cells and CD163+ cells (monocytes/macrophages) were observed in all tumors and were mostly located in the peripheral area of the tumor (Fig 6.17). We also studied the effect of these targeted therapies on cytotoxic immune populations and production of pro-inflammatory cytokines. These studies are presented in the following paragraphs.

The cytotoxicity of splenocytes isolated from healthy animals (n=6), progressive SRC (n=6), PI3K pathway inhibitors treated SRC (n=6 for each treatment) were evaluated against primary chondrosarcoma cells isolated from progressive untreated SRC.

As we found previously, splenocytes isolated from healthy animals did not have a cytotoxic activity against chondrosarcoma cells even at the E:T ratio of 100:1 (specific lysis of 1,3 % (± 0,3)) (Fig 6.18). On the opposite, chondrosarcoma cells were lysed by splenocytes isolated from animals bearing a progressive tumor: a significant lysis of 19,5 (± 0,8%) was obtained at the E:T ratio 100:1.

The splenocytes isolated from all the PI3K pathway inhibitors treated groups showed a significant lesser cytotoxic action on chondrosarcoma cells than the one of the control group (p <0,005). A specific lysis of respectively 10,84 % (± 1,3), 12,7% (± 0,9), 10,3 (± 1,5) was observed at the E/T ratio of 100 for the GDC-0941, G-594 and everolimus treated rats (Fig 6 .18). • The three inhibitors did not affect tumor immune infiltration.

• The cytotoxic assay and the seric cytokines level showed that the PI3K pathway inhibitors caused a decrease in the immune response toward chondrosarcoma.

• The immunosuppressive effects of PI3K pathway inhibitors abolished the idea to combine an agent targeting this pathway with immunotherapy.

6.4 Immune Checkpoint targeting

As they may also allow immune tolerance to tumors, immune checkpoint inhibitors against cytotoxic T lymphocyte antigen 4 (CTLA-4) or programmed cell death protein 1 (PD-1) or PD-1 Ligand (PD-L1) are showing promise in the clinic.

They increase endogenous antitumor activity and might increase the tumour immunogenicity that is induced by chemotherapy, radiotherapy or targeted therapies. So there are opportunities to combine immune checkpoint inhibitors with first-line therapies.

Thus in a first step we evaluated the expression of the immunosuppressive ligand PD-L1 and its receptor PD-1 by tumor cells and tumor infiltrating cells were assessed both in human chondrosarcoma samples and in SRC. Then we tested the possibilities to modulate and/or block this immune checkpoint. PD-L1 expression was evaluated in the same series of 26 human conventional chondrosarcoma presented in paragraph 6.1.

In this series, we found that PD-L1 was expressed in all 26 human chondrosarcoma tumors both on tumor cells and on tumor-infiltrating immune cells (Fig 6 .20a, Table 6.1). PD-L1 expression differed among patients and was not correlated with tumor aggressiveness (Table 6.1 Fig 6 .20B) but tended to be correlated with patient's survival and relapse: a higher expression of PD-L1 was associated with better survival and fewer relapse (Fig 6 .20C). These results need to be confirmed on a larger cohort.

PD-1 was expressed in all human chondrosarcoma and its expression did not significantly vary among patients and was not associated with higher immune infiltrates or with superior PD-L1 expression (Fig 6 .20a). This pattern of expression associated with the other characteristics of the SRC model (tumor progression, immunological tumor microenvironment) validates it as accurate preclinical model for chondrosarcoma immunological therapeutics approaches.

We analyzed the impact of PI3K pathway inhibitors (GDC-0941: PI3K inhibitor;

G-594: Akt inhibitor and Everolimus: mTOR inhibitor) on PD-L1 and PD-1 expression. Surprisingly, all three inhibitors caused a decrease in PD-L1 expression. Fewer PD-L1 positive cells were found in these groups of treatment and the expression level of PD-L1 was also lower than in the normal progressive SRC (Fig 6.22). Interestingly none of the PI3K pathway treatments affect PD-1 expression. These results indicate that PD-L1 is expressed on human chondrosarcoma cells, in the SRC model and sarcoma cell lines. We observe PD-1 expression on infiltrating cells both in human and in SRC. Moreover, our data show that PI3K/Akt/mTOR inhibitors downregulate PD-L1 expression in SRC and sarcoma cell lines. The next section will investigate further on the role of PD-L1 in chondrosarcoma.

To further investigate the role of PD-1/PD-L1 checkpoint in chondrosarcoma, it is necessary to block the interaction of PD-L1 with its receptor PD-1 in vivo and study the consequences of this blocking on tumor progression and immune environment. Nevertheless, there is a huge pitfall. No PD-L1 blocking antibody crossreacting with the rat species is yet available.

Therefore we examined the possibility to block rats PD-L1 using a murine PD-L1 These results indicate that a murine PD-L1 antibody could be used in the SRC model to evaluate the effect of PD-L1 blocking strategy on chondrosarcoma progression. These assays are currently ongoing.

• PD-L1 is expressed at different level in human chondrosarcoma patients, in SRC and in human sarcoma cell lines.

• PD-1 is expressed on tumor infiltrating cells both in human and SRC.

Discussion

Chondrosarcomas (CHS) is a rare and heterogeneous family of mesenchymal tumors, accounting for 20% of bone malignancies, whose treatment has remained largely unchanged over the last 3 decades ( 4). The phenotypic features of this tumor; i.e. a dense extracellular matrix, a low percentage of dividing cells, and a poor vascularity, contribute to CHS chemo-and radio-resistance (4).

However, cytokines and chemokines produced by CHS can cross the barrier formed by the extracellular matrix (ECM) ( 4), attract immune cells and can induce the secretion of metalloproteases which degrade the ECM a step essential for CHS invasion (232).

As new therapeutic approaches are being preclinically evaluated for chondrosarcoma, the hypothesis emerges that the best approaches could probably be to take advantages of phenotypic microenvironmental features of CHS instead of fighting them. Chondrosarcoma's immune microenvironment is one of the least known microenvironmental features of this tumor. Given the hopes and results generated by immunotherapies based on immune checkpoint inhibitors in different tumors types, it seems crucial for chondrosarcoma clinical management to assess the potential of such strategies. Thus, the aim of this study was: i) to describe the immune cell infiltrates associated with chondrosarcoma in human samples and in a syngeneic rat model recapitulating the human disease; ii) assess their functional role in CHS progression; iii) analyze the effects of targeted therapy on chondrosarcoma's immune environment and iv) determine the role of the PD1/PDL1 immune checkpoint on chondrosarcoma progression and evaluate the possibility to use blocking antibody anti-PDL1 in chondrosarcoma.

In this section of the manuscript, we will discuss of the prognostic value of T lymphocytes and macrophages infiltrates in chondrosarcoma, the opposite role of CD8+ and CD163+ populations in chondrosarcoma, the immunogenicity of chondrosarcoma cells, the immunomodulation of chondrosarcoma.

7.1 Prognosis value of T lymphocytes infiltrates in chondrosarcoma

As it was highlighted in the introduction of this manuscript, lymphocytes play an important role in the progression and control of cancer (142). Notably they are essential in the mechanism of tumor immunosurveillance (142).

So far, no study reporting the presence and implication of this immune population in chondrosarcoma microenvironment has been published. The following paragraphs resume and discuss our data relevant to T lymphocytes localization, implication and potential prognosis value for chondrosarcoma.

Our study was conducted on a series of 26 human conventional chondrosarcoma. In this series, we report the presence of T lymphocytes located in the marginal area of tumor on human CHS samples. This l infiltration is composed of CD3+, CD4+ and CD8+ T cells. We show that the infiltration of CD8+ T cells is a marker of good prognostic (section 6.1.3).

Indeed, patients with above the mean CD4+, CD8+ and CD3+ T cells infiltrates had better progression free survival than patients with below the median T cells counts. We also find that CD8+ T cells infiltration is correlated with the size of the tumor at the time of surgery (section 6.1.3). ; a high infiltration of CD8+ T cells being encountered in smaller tumor whereas a low CD8+ T cells count was associated with tumors of higher agressivity infiltrating the surrounding tissues.

These data established on a small cohort of chondrosarcoma need to be conformed on a larger series of CHS. Nevertheless, they suggest that like for other types of cancer (melanoma, osteosarcoma and pleural mesothelioma),

CD8+T cells infiltrations could be a prognosis factors for CHS outcome(233-235).

To fully elucidate the implication of T lymphocytes in chondrosarcoma, the CD4+ T cells, more particularly the Treg subset need to be studied. By gaining insight into the CD4+ T cells infiltration and its potential correlation with chondrosarcoma patients evolution we will then be able to have a full picture of T cell's role in chondrosarcoma.

Chondrosarcoma is a rare tumor and few biological materials are available to further study the implication of immune populations in this pathology and a relevant animal model is necessary to address this question. The Swarm rat chondrosarcoma (SRC) model is well suited for such studies as; unlike most sarcoma models established in immunocompromised animals, (236, 237) the tumors are established on immunocompetent animals, and grow orthotopically.

In the SRC model, the same immune populations (CD3+, CD8+ T lymphocytes were identified in the same location as in the human samples. As in human conventional chondrosarcoma, a high CD8+ T cells infiltration is correlated with less aggressive tumor (as indicated by the measure of the tumor proliferative rate). These observations indicate that SRC model is accurate to study chondrosarcoma immunological environment. Thus further analyses conducted in SRC to gain insight into the implication of immune populations in chondrosarcoma could be translated to human chondrosarcoma.

The CD4+ T cells populations could not be analyzed by IHC staining in rats, as no reliable marker is yet available.

Azathioprine, an agent blocking the proliferation of lymphocytes and preventing the differentiation of naïve and memory T cells in effectors T cells (230), was used to perform T cell depletion in SRC. Azathioprine caused a net decrease in T cells infiltrating SRC. This decrease was associated with a significant accrual in tumor progression and a dramatic decrease in animal's survival (P < 0,0005).

These data further support the implication of CD8+ T cells as agents restraining tumor growth.

Azathioprine did not solely inhibits CD8+ T cells; it affects all lymphocytes populations. Thus to gain further indications into the exact implication of each lymphocytic population on chondrosarcoma progression selective depletion using CD3 or CD8 blocking antibodies should be done. Those experiments are quite expensive to complete in rat models.

The use of azathioprine as depletion agent could be discussed as in addition to its immunosuppressive action this agent was initially develop as chemotherapy but didn't appear to have antitumor efficacy (238,239). To date, the exact mechanisms of action of azathioprine in promoting cancer progression as not been reported but from our observations we could hypothesize that the decrease this agent causes in T cells will result in a decrease of immunosurveillance mechanisms which in turn will promote tumor progression.

One point we should take into consideration is the possible role of B lymphocytes, as their implication in tumor progression is more and more documented (240-242). The lack of reagents available for rat studies prevented us from studying this immune population. The following paragraphs resume and discuss our data relevant to macrophages localization, implication and potential prognosis value for chondrosarcoma.

In the series of 26 human conventional chondrosarcoma, we reported the presence at the peripheral area of the tumor of macrophages expressing the CD163 antigen in all patients. In contrast to T lymphocytes infiltration, no significant correlation was found between CD163+ cells infiltration and overall survival or relapse of CHS patients (section 6.1).

However, CD163+ cells infiltration was correlated with tumor size and aggressivity, a higher CD163+ cells infiltration being associated with bigger tumors at the time of surgery and with tumors infiltrating surrounding tissues.

All these data suggest that CD163+ macrophages play a pro-tumoral role in human conventional chondrosarcoma.

These data established on a small cohort of chondrosarcoma need to be confirmed on a larger series of chondrosarcoma. Nevertheless, they suggest that like for other types of cancer (melanoma, osteosarcoma and pleural mesothelioma), CD163+ cells infiltrations could be a prognosis factor for CHS outcome (233)(234)(235).

This observation suggesting that in chondrosarcoma CD163+ TAMs role is protumoral is similar to other made in melanoma or breast cancer (11). Surprisingly, these data differ from TAM activity reported in osteosarcoma [START_REF] Buddingh | Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clinical cancer research : an official journal of the[END_REF], in which TAM are of good prognosis.

Like for the observation made on lymphocytes population, our data on macrophages infiltration are still incomplete.

The CD163+ maker we used is expressed by all macrophagic populations M1 and M2, even though its expression is higher on M2 population. Thus we can conclude that TAMs in general and not just M2 are present in chondrosarcoma microenvironment and are involved in this tumor's progression (245).

We need to investigate the role of each M1 and M2 subpopulation in chondrosarcoma. This could be done on a larger cohort of chondrosarcoma patients using specific markers for M1 (CD14/HLA-DRα) and M2 (CD14/CD163) TAMs as it was done in osteosarcoma [START_REF] Buddingh | Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clinical cancer research : an official journal of the[END_REF].

The implication of macrophages in chondrosarcoma was further evaluated in SRC model.

Similarly as in human chondrosarcoma an infiltration of CD163+ macrophages located in the marginal area of tumor was found in SRC. Like in human a high CD163+ infiltration correlated with the slowest growing tumor. Of note: CD163+ is actually the sole antibody available to identify macrophages in paraffin embeds rats tissues.

In our study, two agents were used to modulate macrophage population: i) clodronate which induces macrophage's death by apoptosis (231); ii) mifamurtide, a specific ligand of NOD2, which causes M2 to M1 macrophages conversion (203).

Clodronate caused a significant decrease in CD163+ cells associated with a significantly increased survival of the animals (P <0,05) (section 6.2.3).

Mifamurtide also reduced the density of CD163+ cells in chondrosarcoma microenvironment, slowed down tumor progression and consequently improved animal's survival. These data confirm that phagocytic immune cells and mainly CD163+ macrophages play a role in chondrosarcoma progression, and can be modulated to induce tumor regression.

Mifamurtide has already been investigated in other bone tumor. This agent has proven effective in osteogenic sarcoma, such that regulatory authorities have approved the non-specific immune stimulator MTP (muramyl tripeptide) for human use in primary osteosarcoma (200). MTP activates macrophages and monocytes as a potent activator of immune response. It also improves the overall survival from 70 to 78% and results in a one-third reduction in the risk of death from osteosarcoma (200).

The choice of these two agents can be discussed:

-Clodronate does not specifically target macrophages, but causes apoptosis of all phagocytic populations (DC, neutrophils, monocytes) ( 246). Nevertheless, it was shown that in long-term depletion experimentation, similar to the one we conducted here, macrophages populations were the most affected by clodronate ( 246).

-Mifamurtide caused a net decrease in CD163+ macrophages infiltration without affecting the lymphocytes population. This agent did not cause depletion in macrophages but acts by favoring the conversion of M2 pro-tumoral macrophages to M1 pro-inflammatory macrophages (203). So we could say that mifamurtide had induce a M2 (CD163+) to M1 (CD163-) conversion in our model

The action of mifamurtide, the consequences of mifamurtide treatment on chondrosarcoma progression, indicates that M2 macrophages are normally present in progressive chondrosarcoma and that these M2 macrophages play a pro-tumoral role.

Nevertheless, this point needs to be confirmed by a clear identification of M1 population present in SRC microenvironment, point for which the tools are still lacking. 7 7.3 The opposite role of CD8+ and CD163+ populations in chondrosarcoma

As we have discussed in the previous sections, both CD8+ T cells and CD163+

populations play a role in the progression of chondrosarcoma, the first ones restraining it while the later ones promote it.

We also reported that the ratio CD8+ T cells/CD163+ cells are correlated with tumor aggressivity both in human chondrosarcoma and SRC model and that this ratio has a prognosis value in human chondrosarcoma. Indeed, a high CD8/CD163 ratio (i.e. more lymphocyte than macrophages) is correlated to better progression free survival, and smaller tumor at the time of surgery. A high CD8/CD163 is encountered in grade II non-invasive chondrosarcoma and I whereas a low CD8/CD163 ratio is found in grade II and III invasive tumors. This indicates that CD8/CD163 ratio could be used as a marker of tumor aggressivity as it is done in pleural mesothelioma (5).

The presence of each of these populations in chondrosarcoma environment is inversely proportional: indeed in the same sample concomitant high CD8+ T cells and high CD163+ infiltration could never be found. It seems like these two populations interfere with each other in term of recruitment to the tumor site.

Given that the depletion of lymphocytes did not affect macrophagic composition of tumor environment and vice versa, it seems like these two populations do not directly interact. This indicates that another factor/element from the tumor microenvironment plays a role in inducing the recruitment of each population.

7.4 Immunogenicity of chondrosarcoma cells

Two elements indicate that chondrosarcoma induces an immune response and is susceptible to immunosurveillance:

-the fact that splenocytes isolated from chondrosarcoma-bearing rats have a specific cytotoxic action against chondrosarcoma cells while splenocytes from healthy rats didn't show any cytotoxic action.

-the increase of pro-inflammatory cytokines that was detected in in sera of tumor bearing rats and absent in the sera of healthy animals.

In the splenocytes, two immunes cells population could induce the specific lysis of the chondrosarcoma cells that we observe: The Natural Killer (NK) cells and the CD8 cytotoxic T Lymphocytes (CTL). We believe that if both populations are involved in the cytotoxic response we observed, this one is more a result of the action of CD8+ T cells.

Indeed, The Natural Killer (NK) cells belong to the innate immune cells. They are known to cause cell death to cells that have lost the expression of "self" proteins at their surface (majorly trough the lost of MHC class I) (247). NK cells cannot be responsible of the totality of the cytotoxic action that we observe. Since we would also observe cytotoxicity with splenocytes coming from healthy rats.

We base this hypothesis on the fact that NK cells in the spleen are all on a naïve state of activation and they need only a danger signal (like the lost of MHC class I) at the surface of their target cell to induce their cytotoxic action (247). NK cells isolated from the spleen are all on a naïve state of activation and need a danger signal (like the lost of MHC class I) at the surface of their target cell to induce their cytotoxic action (247

7.5 Immunomodulation and chondrosarcoma

In the course of our project, we demonstrated that inhibitors of the PI3K pathway affect chondrosarcoma progression and immune environment. These agents proved efficient in significantly slowing down tumor progression (section 6.3) as it was previously shown by our group for an mTOR inhibitor [START_REF] Perez | Inhibition of chondrosarcoma growth by mTOR inhibitor in an in vivo syngeneic rat model[END_REF].

As we envisioned applying a combination of this targeted approaches with immune-modulatory strategy (PD1/PD-L1 blocking strategy), we first needed to analyse the effect of these inhibitors on chondrosarcoma immune response.

None of these inhibitors caused changes in immune population infiltration in chondrosarcoma. Moreover these agents caused a decrease in PD-L1 expression both in vivo and in vitro. By downregulating PD-1/PD-L1 on chondrosarcoma cells, the PI3K pathway inhibitors disrupt the checkpoint inhibitor that could enhance T cells response toward chondrosarcoma. Such an effect of PI3K pathway inhibitors (one Pan PI3K inhibitor, two dual PI3K/mTOR inhibitors and two mTOR inhibitors) was reported in glioblastoma (24). Authors discuss about the possibility to use these inhibitors in combination with a T cells activating immunotherapy. They also discuss on the possibility that the PI3K pathway inhibitor could have an immunosuppressive effect without confirming it.

One drawback of these inhibitors is the effect they have on the cytotoxic activity of splenocytes and on the pro-inflammatory seric cytokines. The three agents tested in our study caused a decrease in cytotoxic activity of SRC splenocytes associated with a decrease in the seric pro-inflammatory cytokines. These data suggest that these agents in addition to their anti-tumoral action have also an immunosuppressive activity.

These observations correlate with other studies which reported the immunosuppressive action of the PI3K/Akt/mTOR inhibitors (250). The authors have shown that PI3K inhibitors and mTOR inhibitors affect both innate and adaptive immune response. In the end, it all conducted to a decrease of the immune response toward infected/cancerous cells (250).

The PI3K signaling pathway is involved in multiple processes, including cancer progression (251) and immune suppression (250). The implication of this pathway in these processes and the data we obtained raise questions regarding the use of PI3K pathway inhibitors in combination with immunotherapy.

On one hand, PI3K pathway inhibition would slow down chondrosarcoma progression and down regulate PD1/PDL1 immune checkpoint. On the other hand, PI3K pathway inhibitors could repress the cytotoxic immune response thus leading to a decrease in antitumor immune response.

Our data describing chondrosarcoma immune environment show that this tumor can be submitted to immunotherapeutic approaches, notably to the ones targeting immune checkpoints.

With all the observations we made, we though that a vaccine therapy using cancer cells lysate therapy could be the most effective to induce a stronger immune response in chondrosarcoma. Unfortunately, such an approach was already tested in clinic with sarcoma and chondrosarcoma patients and didn't prove effective (194)(195)(196). This lack of effectiveness of vaccine therapy in chondrosarcoma could be related with expression of PD-L1 by this tumor. A

strong PD-L1 expression can abolish a specific T cells response in cancer (178).

Chondrosarcoma have all the criteria to be reactive to an immunotherapy blocking the immunocheckpoint PD-1/PD-L1 (252):

Chondrosarcoma express PD-L1.

PD-1+ cells are present in the microenvironment of the tumors CD8+ T cells are presents in the microenvironment.

Moreover, strategies to reactivate antitumor immune responses with drugs targeting CTLA-4 and PD-1/PD-L1 have led to striking advances in the therapy of melanoma, some carcinomas, and hematopoietic neoplasia (252). For bone sarcoma, it has been shown that PD-1 is expressed on CTLs that infiltrate osteosarcoma and osteosarcoma cells express PD-L1 (211). However, very few papers report the beneficial effect of blocking PD-1/PD-L1 on osteosarcoma growth in vivo (253,254). Thus we wanted to apply a PD-1/PD-L1 blocking strategy in SRC model.

Our biggest concern is that no PD-1 or PD-L1 blocking antibody crossreacting with the rat protein is yet available. Thus we had to built chimera to establish in in vitro cell models that a murine anti-PD-L1 antibody could block PD-1/PD-L1 interaction in rat bone sarcoma cell line.

Based on this interesting and promising data we are currently evaluating in vivo in the SRC model the possibility to block PD-1/PD-L1 by using this antibody. The consequences of PD-1/PD-L1 blocking on tumor progression and immune environment will be soon reported.

7.6 Conclusion

This work shows that CHS is infiltrated with CD8+ lymphocytes and CD163+ M2 macrophages in the marginal zones of the cartilaginous nodules, both in human and SRC model. We also show that the infiltration of those two populations correlates with the outcome of the disease. The contribution of lymphocytes and macrophages to the tumor progression was demonstrated by selective depletion.

We also show that chondrosarcoma could be susceptible to immunotherapy like PD-1/PD-L1 blocking antibody and mifamurdide. We also, observe that murine PD-L1 blocking antibody could be used in vivo in our SRC model to evaluate the role of PD1/PD-L1 in chondrosarcoma and whether an immune checkpoint blocking strategy is relevant for chondrosarcoma.

We find that SRC is a perfect model to study immunotherapy for o The possible role of B lymphocytes will also be addressed o M1 and M2 subpopulation will be better characterized using specific markers for M1 (CD14/HLA-DRα) and M2 (CD14/CD163) TAMs as it was done in osteosarcoma [START_REF] Buddingh | Tumor-infiltrating macrophages are associated with metastasis suppression in high-grade osteosarcoma: a rationale for treatment with macrophage activating agents. Clinical cancer research : an official journal of the[END_REF].

o The identification of tumor associated antigens expressed is chondrosarcomas is also one of the research axis investigated by our team in a collaborative program (Immunosarc project)

The rat SRC model will be used to:

o Evaluate in vivo the possibility to block PD-1/PD-L1 by using murine PD-L1 blocking antibody as well as the consequences of

Background

Chondrosarcoma (CHS) is the second most common skeletal malignancy after osteosarcoma, with a worldwide incidence of 1/200 000 per year [START_REF] Gelderblom | The clinical approach towards chondrosarcoma[END_REF]. CHS is a heterogeneous group of tumors arising preferentially in long bones or in the pelvis and characterized by the capacity of tumor cells to synthetize cartilage. Because of their dense extracellular matrix, low percentage of dividing cells, and poor vascularity, CHS are known to be highly chemo-and radio resistant (2,3). The only curative treatment for CHS still remains surgery with large en bloc tumor resection or amputation to ensure local control of tumor and to prevent future metastases [START_REF] Gelderblom | The clinical approach towards chondrosarcoma[END_REF]4). Chondrosarcoma's 10-year survival rate range from 29-83% depending on CHS subtype and grade: novel therapeutic approaches are therefore urgently needed [START_REF] Gelderblom | The clinical approach towards chondrosarcoma[END_REF]5,6). cyclin-dependent kinase 4 (CDK4), matrix metalloproteinases (MMP), SRC, AKT, hypoxia-inducible factor (HIF), mTOR are possible relevant targets in chondrosarcoma (7)(8)(9). (10)(11)(12)(13).

Little is known regarding chondrosarcoma microenvironment, besides its major proteoglycane (PG) contents and its hypoxic nature compared to normal tissues or benign cartilaginous tumors (14). These characteristics are considered the main limitations of therapeutic agents efficiency in CHS (15). CHS immunological microenvironment also remains largely unexplored, including its description and its potential involvement in CHS progression. The crosstalk between tumor cells and immune cells has been extensively studied in many tumors over the last 20 years (16), showing that tumor immune microenvironment is involved in tumor control and progression. Indeed, CD8+ T cells and pro-inflammatory M1 macrophages play in most cancers an antitumoral role, while Treg and M2 macrophages may promote tumor progression in most cancers (17,18), with the possible exception of metastatic osteosarcoma (19). The expression of PD-1 and its corresponding ligands (PD-L1 and PD-L2) are demonstrated mechanisms of tumor tolerance and escape. PD-1 is a cell surface receptor expressed on subsets of T and B lymphocytes, as well as other immune cells (20). In melanoma, ovarian and colorectal cancer PD-L1 is constitutively upregulated and the ligation of PD-1 by PD-L1 expressed on tumor cells inhibits CTL proliferation, cytokine production and cytotoxicity, thereby leading to immune escape and tumor proliferation (21). The understanding of the role of immune checkpoints CTLA-4 and PD-1, and their therapeutical manipulation has led to novel immune-targeted therapies with major successes in diseases as various as metastatic malignant melanoma (MMM), non small cells lung cancer (NSCLC), bladder cancer, head and neck cancer (HNC) and Hodgkin's lymphoma (22).

To date, the analyses of the immune environment of bone sarcoma have led to the following conclusion. For osteosarcoma, the presence of Tumor Infiltrating Lymphocytes (TIL) is of good prognosis ( 23) and the presence of M2 Tumor Associated Macrophages (TAM) is associated with metastases suppression (19). So far, one study has reported the expression of PD-1/PD-L1 in osteosarcoma samples ( 24) and PDL1-directed therapy in combination with CTLA-4 or PD-1 blocking antibody was efficient in a metastatic murine osteosarcoma model (25,26).

In this study, we report on the description and role of immune infiltrates in chondrosarcoma. CD8+ T cells and CD163+ cells were observed at the margins of chondrosarcoma and their presence was found associated tumor's progression. We also show that the PD-1 is present at the surface of TIL, that PD-L1 is expressed by chondrosarcoma cells and TIL.

Materials and methods

Cell cultures

The human chondrosarcoma (SW1353), rhabdomyosarcoma (SJCRH30), bone marrow (K-562), Natural Killer (NK-92) cell lines, and rat osteosarcoma cell line (UMR-106) were all obtained from the ATCC (LGC Standards, Molsheim, France) and were cultured at 37°C, under 5% CO 2 , using DMEM or RPMI medium (for NK92 cell line) supplemented with 10% FBS, 2% L-glutamin, and penicillin/streptomycin solution remaining samples were fixed in 10% formalin for further analyses. Blood was collected by cardiac puncture and allowed to clot at room temperature to obtain serum for ELISA analysis.

Splenocytes harvesting

Spleens were mashed on 40-μm-cells strainer (BD Bioscience, San Jose, USA) placed on top of a 50 mL Falcon tube, using a syringe plunger. The collected cells were centrifuged (250 g, 5 min, 4°C) after addition of 10 mL RPMI medium supplemented by 10% FBS.

To remove red blood cells, pellet was incubated 3 min at room temperature in ACK red blood cell lysis buffer (GIBCO) before being centrifuged (250 g, 5 min) and resuspended in complete RPMI medium.

Cytotoxicity assay

A nonradioactive, fluorometric cytotoxicity assay using calcein-acetoxymethyl (calcein AM; Thermofisher C3099) was performed to measure the lysis of rat chondrosarcoma cells and chondrocytes (further named target cells) by splenocytes. Target cells (1 × 10 6 /ml) were labeled with Calcein-AM (10 μmol/L) at 37°C for 1 h before being washed twice in complete culture medium and plated at 10 4 cells/wells in round bottom 96-well microtiter plates (Nunc, P8116, Sigma Aldrich, St-Louis, USA). Target cells where then incubated with 400 μmol/L Sulfinpyrazone (Sigma-Aldrich) in complete medium (1h, 37°C) to avoid spontaneous calcein release.

Splenocytes isolated from animals of all treatment group were used as effector cells and added to targets cells at E:T ratios ranging from 100:1 to 12,5:1. Effectors and targets

were incubated 6h at 37 °C under normal culture conditions. Targets cells spontaneous and maximum release were assessed respectively by plating target cells alone in 96 wells or by incubating target cells in presence of 2% of Triton X-100 for 6h under normal culture conditions.

Supernatants were then collected (100 µl) by centrifugation (250 g; 5 min) and transferred in transparent 96 wells flat bottom plates (Nunc, P7366, Sigma Aldrich). NK-92 and K-562 cells lines were used as positive control (29). For each sample, calcein release was measured on Tecan Infinite F500 (Tecan group ltd. Switzerland) (excitation filter: 485 ± 9 nm; band-pass filter: 530 ± 9 nm). Specific lysis induced by the effectors was calculated according to the formula: Specific sample lysis= [(sample releasespontaneous release)/(maximal releasespontaneous release)] × 100. All tests were run in triplicate.

Measurement of Cytokines secretion

Levels of seric IL-10, IL-1β, TNFα and IFNγ from animals of the different treatments groups were determined by ELISA assays as per the manufacturer's instructions. IL-1β, TNFα and INFγ ELISA kits were from Peprotech (Rocky hill, NJ, USA); Il-10 ELISA kit was from Sigma-Aldrich.

Phenotypic analyses of chondrosarcoma immune microenvironment

Chondrosarcoma immune microenvironment was analyzed from 52 SRC and 26 conventional grade 1 to grade 3 human chondrosarcoma samples treated in our institution between 2004 and 2012. All samples were fixed in 4% formalin (pH 7.4) and embedded in paraffin. Deparaffination and rehydration were done under standard procedures using xylene (VWR international S.A.S, Fontenay sous Bois; France) followed by ethanol gradient (VWR).

For SRC, 5 µm sections from formalin-fixed, paraffin-embedded (FFPE) tumor blocks were stained with the following primary antibodies: CD3 (Ventana, Tucson, AZ,

Statistical analysis

Tumor growth inhibition (TGI) was defined as the difference between the mean tumor volume (MTV) of the control group and the MTV of the drug-treated group, expressed as percentage of the MTV of the control group at the end of the experiment:

TGI = [1 -(MTV drug-treated /MTV control )] × 100.
All the data are reported as the sample mean ± the standard deviation (SD). Pairwise comparisons between means of different groups were performed using a Student t-test (two tailed, unpaired) where, for each couple of normally distributed populations, the null hypotheses that the means are equal were verified. The difference between two subsets of data is considered statistically significant if the Student t-test gives a significance level (P value <0.05. Multiple comparisons were performed using an univariate analysis of variance (ANOVA).

Kaplan-Meier analysis was carried out to estimate the survival probability of different treatment groups at a given time. The median time to endpoint (TTE) and its corresponding 95% confidence interval (CI) were calculated.

All statistical analysis was performed using GraphPad Prism version 6.00 (GraphPad Software, La Jolla, CA, USA, www.graphpad.com).

Results

Chondrosarcoma immune microenvironment and tumor progression

The localization, lineage and density of immune cells present in chondrosarcoma microenvironment were analyzed in a cohort of 26 grade 1 to 3 conventional chondrosarcoma.

CD4+, CD8+ T cells and CD163+ cells (monocytes/macrophages) were observed in all the 26 tumors and were mostly located in the peripheral area of the tumor (Fig. 1a).

Density of lymphocytes and macrophages varied among patients and ranged from minor to dense (Supplementary table S1). the less aggressive tumors had the lowest CD8/CD163 ratio while G3 and G2 infiltrating tumors had the highest CD8/CD163 ratio. The same observation was made with CD8+ infiltration while the opposite effect was found with the CD163+ infiltration (Fig 2d).

SRC immune microenvironment recapitulates human chondrosarcoma

Immune infiltrates were then analyzed on rat chondrosarcoma from 17 untreated progressive SRC. As for human chondrosarcoma, CD3+, CD8+ T cells and CD163+ cells The opposite effect was observed when lymphocytes were depleted using azathioprine. In this case, tumor growth rate was increased (91,90 ± 21,48 for azathioprine treated group These two sets of data suggest that macrophages play a pro-tumoral action while lymphocytes repress chondrosarcoma growth possibly by specific cytotoxic activity directed against chondrosarcoma cells. To elucidate this point, cytotoxicity of splenocytes from different treatment groups was analysed and production of pro-inflammatory cytokines was measured in rat sera.

The cytotoxic activity of splenocytes from progressive SRC (n=6), azathioprine treated SRC (n=6), mifamurtide treated SRC (n=8), clodronate-treated SRCs (n=6) and healthy animals (n=6) was evaluated against primary chondrosarcoma cells isolated from SRC tumors.

Splenocytes isolated from healthy animals did not have a cytotoxic activity against chondrosarcoma cells ((E:T ratio of 100:1, specific lysis of 2,5 % ± 1,7). Conversely, chondrosarcoma cells were lysed by splenocytes isolated from chondrosarcoma bearing rats: (27,0 ± 6,5% was obtained at the E:T ratio 100:1). The splenocytes from azathioprine, treated rats had a lower cytotoxic activity on chondrosarcoma cells. (11,3 ± 3,5 % at the same E:T ratio of 100:1 (P < 0,0005). Splenocytes isolated from treatment targeting macrophages had a non-significantly lower cytotoxic activity (P > 0,05)(Fig S1). PD-1 was expressed on immune infiltrate cells in all human chondrosarcoma its expression did not significantly vary among patients and was not associated with higher immune infiltrates or with superior PD-L1 expression (Fig 6a, Supplementary table S1). The expression of PD-L1 and its receptor PD-1 were also found in SRC model. As in human chondrosarcoma, PD-L1 was detected both on tumor cells and on infiltrating immune

Discussion

Chondrosarcomas (CHS) is a rare and heterogeneous family of mesenchymal tumors, accounting for 20% of bone malignancies, whose treatment results have remained largely unchanged over the last 3 decades (1). The phenotypic features of this tumor; i.e. a dense between CD163+TAM CD8+ lymphocytes infiltration, we show that the presence of TAM could be a barrier to CD8+ infiltration in the tumors. Such results have also been reported in gastric carcinoma (35). These observations conducted in 26 chondrosarcoma patients need to be validated on a larger cohort.

Chondrosarcoma is a rare tumor and biological materials available to study the implication of immune populations in this pathology are scarce. A relevant animal model is necessary to address this question. Given the similar features of Swarm rat chondrosarcoma (SRC) with the human counterpart, this model is well suited for such studies as; unlike in most sarcoma models established in immunocompromised animals, (36,37) the tumors are established on immunocompetent animals, and grow orthotopically.

The SRC model mimics human grade II chondrosarcoma in term of growth and therapeutic resistance (38). Analyses of SRC showed that SRC immune microenvironment also mimics its human counterpart. In this model, CD8+ T cells and CD163+ macrophages infiltrations were correlated with tumor aggressiveness: smaller tumors had a high CD8+T cells count and low CD163+ infiltrate, whereas larger tumors were characterized by low CD8+ T cells and high CD163+ infiltrates. These results confirmed that chondrosarcoma's immunological microenvironment plays a role in the tumor progression: the CD8+T cells repressing tumor progression and CD163+ cells having a pro-tumoral action. Both cell components act independently since the depletion of each population in the SRC model affects tumor growth.

Azathioprine, an agent that blocks the proliferation of T cells and prevents the differentiation of naïve and memory T cells in effectors T cells (39) was used to perform T cell depletion in SRC. Azathioprine affected tumor's immunological microenvironment causing a net decrease in T cells associated with faster growing tumors and a dramatic decrease in animal's survival. Splenocytes from progressive SRC have a specific cytotoxic action directed against chondrosarcoma cells that is absent in azathioprine treated rats, indicating that T cells play an anti-tumoral role in this chondrosarcoma model, as in other tumor models (16).

M2 tumor associated macrophages (TAMs) (40,41) has been shown to present a protumoral activity ( 18) (42). Clodronate which induces macrophage's death by apoptosis (43), caused a significant decrease in CD163+ cells associated with a significantly increased survival of the animals (P <0,05). Mifamurtide, a ligand of NOD2, which drives M2 to M1 macrophages conversion (44), reduced the density of CD163+ cells in chondrosarcoma microenvironment and resulted in an increase in animal's survival, confirming that phagocytic immune cells and mainly CD163+ macrophages may play a role in chondrosarcoma progression, and can be modulated to induce tumor regression.

Mifamurtide, has already shown its potential therapeutic value in osteosarcoma (45).

In metastatic osteosarcoma model, blockade of PD-1/PD-L1 showed potent antimetastatic action (26). We evaluated the expression of PD-1/PD-L1 immune checkpoint both in human samples and SRC. PD-L1 was found expressed both in human samples and SRC model. Surprisingly, in human samples, high level of expression of PD-L1 was associated with an absence of relapse and death. Although non significant, the functional significance of this observation is being tested in the SRC model.

.

In conclusion, this work shows that CHS is infiltrated with CD8+ lymphocytes and CD163+ M2 macrophages in the marginal zones of the cartilaginous nodules, both in human and SRC model. We also show that the infiltration of those two populations is correlated with the presentation, and outcome of the disease. The contribution of Dans le model SRC, la déplétion sélective de lymphocytes T a entrainé une accélération de la progression tumorale, tandis que la déplétion de macrophages l'a ralenti. Les splénocytes isolés de rats porteurs de CHS ont montré une cytotoxicité spécifique dirigée contre les cellules de chondrosarcome (27%), qui a diminué de manière significative avec des rats appauvrie en CD3 (11%). Les inhibiteurs de la voie de signalisation PI3K/mTOR ne peuvent pas être associés à une immunothérapie car ils induisent une action immunosuppressive in vivo.

L'environnement immunitaire contribue à la progression du CHS à la fois chez l'homme et chez le rat, ce qui suggère que une approche immunomodulatrice avec des anticorps bloquant PDL1 pourrait être testée pour le CHS.

Mots Clés : Chondrosarcome, lymphocyte, PD-1, PD-L1, macrophage, immunothérapie, PI3K/Akt/mTOR, immunosurveillance Chondrosarcoma is highly resistant to chemotherapy and radiation and there is an urgent need in developing new therapeutic strategies for this malignancy; among these, some immunotherapy approaches could be of great interest. We are currently investigating the immune system implication in chondrosarcoma progression and therapeutic response both on human samples and in rat chondrosarcoma model (SRC).

In human and rat chondrosarcoma, immune infiltrates composed of lymphocytes and macrophages were identified in the peritumoral area. Immune infiltrates composition was found correlated with tumors characteristics and evolution (grade, invasiveness and size). Expression of PD-1 and PD-L1 was detected in CHS immune infiltrates, both in human and rat (and on tumor cells). PD-L1 expression level correlated with patients survival and relapse rate. In SRC, T lymphocytes depletion resulted in an accelerated tumor progression, while CD163+ macrophages depletion slowed down tumor progression. Splenocytes isolated from CHS bearing SRC showed a specific cytotoxicity directed against chondrosarcoma cells (27%), which significantly decreased in CD3 depleted SRC (11%). The PI3K/mTOR pathway inhibitors can't be associated to an immunotherapy since they induce an immunosuppressive action in vivo.

Faculté

  le microenvironnement immunitaire du chondrosarcome à la fois dans des échantillons de chondrosarcome humains (26 échantillons) et dans le modèle de rat de chondrosarcome Swarm (SRC).

- Figure 6 . 22 :Figure 6 . 23 :

 622623 Figure 1.1:Histological aspect of conventional chondrosarcoma ------------------------------------------Figure 1.2:Histological grading of chondrosarcomas ---------------------------------------------------------Figure 2.1:Evolution of the hallmarks of cancer over the past decade -----------------------------------Figure2.2:Innate and adaptive immunity -------------------------------------------------------------------------Figure 2.3:The two type of adaptive immune response -------------------------------------------------------Figure 2.4:Direct cancer recognition by the innate immune system --------------------------------------Figure 2.5:"Signal-strength model" of T cell differentiation --------------------------------------------------Figure 2.6:Granzyme/Perforin mediated cytotoxic action of CTL ------------------------------------------Figure 2.7:The prognostic impact of T cells in tumor ---------------------------------------------------------Figure 2.8:The three phases of cancer immunoediting. ------------------------------------------------------Figure 2.9:A proposed model for the elimination phase of the cancer immunoediting process ----Figure 2.10: Co-stimulatory and inhibitory signal of TCR ----------------------------------------------------Figure 2.11: Immune checkpoint in cancer ----------------------------------------------------------------------Figure3.1:Immunotherapy against tumor cells -----------------------------------------------------------------Figure5.1:Transplantable orthotopic Swarm rat chondrosarcoma ----------------------------------------Figure 5.2:The targets and effects of each immunomodulator treatment --------------------------------Figure6.1:Immune infiltrate in human chondrosarcoma -----------------------------------------------------Figure 6.2:Immune infiltrates correlation with chondrosarcoma's survival ------------------------------Figure6.3:Immune infiltrates correlation with chondrosarcoma's progression -------------------------Figure6.4:Correlation between CD8+, CD163+ cells counts and tumor size --------------------------Figure6.5:Correlation between CD8+, CD163+ cells counts and tumor agressivity -----------------Figure6.6:Correlation between CD8+ lymphocytes and the CD163+ macrophages in tumor's microenvironment -----------------------------------------------------------------------------Figure6.7:CD8/CD163 ratio and tumor agressivity -----------------------------------------------------------Figure6.8:Immune infiltrate in SRCmodel ----------------------------------------------------------------------Figure6.9:Correlation between CD8+, CD163+ cells counts and tumor size -------------------------Figure 6.10:CD8/CD163 ratio and tumor agressivity ---------------------------------------------------------Figure 6.11:Effects of selective depletion on SRC immune infiltrates -----------------------------------Figure6.12:Effects of selective depletion on SRC progression -------------------------------------------Figure 6.13:Cytotoxic response in chondrosarcoma ---------------------------------------------------------Figure 6.14:Inflammatory response in chondrosarcoma ----------------------------------------------------Figure6.15:PI3K, Akt and mTOR inhibitors affect SRC progression ------------------------------------Figure6.16:PI3K, Akt and mTOR inhibitors increase SRC survival -------------------------------------Figure6.17:Effect of PI3K pathway inhibitors on SRC immune environment -------------------------Figure6.18:Effect of PI3K pathway inhibitors on cytotoxic response in chondrosarcoma ---------Figure6.19:Effect of PI3K pathway inhibitors on inflammation in chondrosarcoma -----------------Figure6.20:Expression of PD-L1 and PD-1 in human conventional chondrosarcoma -------------Figure6.21:Expression of PD-L1 and PD-1 in SRC ---------------------------------------------------------Figure 6.22:Expression of PD-L1 and PD-1 in PI3K pathway inhibitors treated SRC ---------------Figure6.23:Expression of PD-L1 and PD-1 in PI3K pathway inhibitors treated sarcoma cell lines -------------------------------------------------------------------------------------------------Figure6.24:Blocking of rat PD-1 immune checkpoint using a murine PD-L1 blocking antibody -

Figure 1 . 1 :

 11 Figure 1.1: Histological aspect of conventional chondrosarcoma. a: Grade 1 chondrosarcoma; b: Grade 2 chondrosarcoma; c and d: Grade 3 chondrosarcoma (2)

Figure 1 . 2 :

 12 Figure 1.2: Histological grading of chondrosarcomas (2).

1 1 . 4

 14 Survival and Prognosis factorsSurvival of chondrosarcoma varies considerably across the large number of subtypes and histologic grades of these tumors. Patients with adequately resected low-grade chondrosarcomas have an excellent survival rate. In general the survival of patients with high-grade tumors varies depending on the location, subtype, effectiveness of surgical treatment, size and stage of the tumor (4).

Figure 2 . 1 :

 21 Figure 2.1: Evolution of the hallmarks of cancer over the past decade. A) Cancer hallmarks in 2000. B) Cancer hallmarks in 2011 with novel additions notably of hallmarks of the involvement of the immune system (tumor -promoting inflammation and avoiding immune destruction) (79).

Figure 2 . 2 :

 22 Figure 2.2: Innate and adaptive immunity. The mechanisms of innate immunity provide the initial defense against infections. Adaptive immune responses develop later and consist of activation of lymphocytes. The kinetics of the innate and adaptive immune responses are approximations and may vary in different infections.[START_REF] Abbas | Cellular and molecular immunology[END_REF]

.

Figure 2 . 3 :

 23 Figure 2.3: The two types of adaptive immune response. Humoral immunity (left) and cellmediated immunity (right) are resume on this figure. (81)

Figure 2 . 4 :

 24 Figure 2.4: Innate immunity and Cancer. NK, macrophages, DC, neutrophils, eosinophils, and mast cells are effectors of the innate immunity: NK cells can cause tumor cell death through the interaction of receptors expressed by NK cells with ligand expressed on tumor cells (like NKG2D and MICAB) or ADCC mechanism. NK cells can also stimulate adaptive immunity or induce tumor cell proliferation. Macrophages phagocytes apoptotic cancer cells via interaction of receptors expressed on macrophages (TAM TIM CD14) and ligand expressed on tumor cells (PS ICAM3). DC phagocyte apoptotic tumor cells. Cytokine secreted by DC promote antigen presenting cell activation, leading to cellular and humoral adaptive immune response (82).

  target cells independently from any previous activation suggests a role for NK cells in the control of tumor growth and metastasis diffusion in vivo. Some of the mechanisms are aborted below. NK cells respond to the absence of class I MHC. Some tumors lose expression of class I MHC molecules, maybe as a result of selection against CTL. This loss of class I MHC molecules by tumors make them particularly good targets for NK cells. In addition NK cells can be targeted to IgG antibody coated cells by Fc receptors (CD16). The tumoricidal capacity of NK cells is increased by cytokines including interferon and interleukins. (97-99). There are clinical and experimental evidence demonstrating the implication of NK cells in tumor immunity. Over the last few years, studies have revealed the phenotypic status and functionality of NK cells in tumor site and also in peripheral blood of cancer patients. First studies on tumor immune infiltration have shown that the presence of NK cells represents a positive prognostic marker in different colorectal, gastric carcinomas and squamous cell lung cancer (100-102). C) DCs are potent professional antigen presenting cells, and bridge between innate and adaptive immune system. The two major groups of DC are known as the myeloid DC and the plasmacytoid DC. Functional subsets of myeloid DC in the skin, epidermal Langerhans cells and dermal interstitial DC are also characterized with distinct immune induction potentials. Activated epidermal Langerhans cells secret interleukin 15 (IL-15) and induce CD4+ and CD8+ T cell priming to elicit cellular immunity. Dermal interstitial DC stimulates B cell priming to produce humoral immunity (103, 104). Engaging DC via different receptors and subpopulations may stimulate different inflammation responses, producing multiple T cell outcomes including T helper cells of type 1 (Th1), Th2, Th17, Th21 and T regulatory cells. In tumor microenvironment, DCs will phagocyte apoptotic cancer cells; a reaction mediated by the interaction of αvβ5 integrin (expressed by tumor cells) with CD36 receptors (expressed by DC) (105). Similar to macrophages, DC can recognize the so-called "eat me" signals expressed on apoptotic cells through endocytotic receptors, scavenger receptors, and TIM receptors. Phagocytosis of apoptotic tumor cells by DC in the absence of danger signals generally leads to immune tolerance (106). Antibody-coated tumor cells are internalized into DC through activating FcγR. This internalization leads to more efficient MHC class I and II-restricted antigen presentation and induces tumor-specific effector and memory T cells (107).

Figure 2 .

 2 Figure 2.5 recapitulates this clonal expansion that lead to anergic T cells (if the signal is too weak) or effector T cells and memory T cells.

Figure 2 . 5 :

 25 Figure 2.5: "Signal-strength model" of T cell differentiation. T cells receive antigenic, costimulatory, and inflammatory signals from DC during priming. In concert, these of stimulations determine the fate of T cell clonal expansion and differentiation. Greater proliferation correlates with stronger signal. However, insufficient stimulation results in death by neglect, while excessive stimulation causes activation induced cell death. Stronger stimulation also drives T cells toward terminal differentiation and reduces their memory-forming potential (110).

  CD8+ T lymphocytes are the keystone of the cancer elimination process. They are crucial components of tumor-specific cellular adaptive immunity. They directly attack tumor cells presenting tumor-associated antigen peptide with MHC class I on their surface. Activated CTL release perforin and granzyme that lead to the formation of pore within the cytoplasmic membrane and activate the caspase pathway of the target cell (119) (fig 2.6).

Figure 2 . 6 :

 26 Figure 2.6: Granzyme/Perforin mediated cytotoxic action of CTL. The cytotoxic lymphocyte recognizes its target cell and forms an immunological synapse (part a). The microtubuleorganizing center (MTOC) of the cytotoxic lymphocyte polarizes and secretory granules traffic towards the presynaptic membrane (part b). The secretory granules then fuse with the presynaptic membrane and release performing and granzymes into the synaptic cleft (parts c and d). At the postsynaptic membrane, perforin forms large transmembrane pores that enable the diffusion of granzymes into the target cell cytosol. Granzymes then initiate apoptosis of the target cell, and the cytotoxic lymphocyte detaches from the dying cell (indicated by the arrow; part e) and can interact with another target cell to carry out serial killing (not shown). (119)

  ) and thus Treg benefit the host. On the other hand, Treg accumulations in cancer predict poor outcome (131-133). It appears that Treg can benefit the host by controlling inflammation but can also be harmful by suppressing anti-tumor immunity and promoting cancer progression. As mentioned above, the function of lymphocytes in cancer is very variable between subtypes. Figures 2.7 resume the prognostic factors of all subtypes of population in 124 published articles.

Figure 2 . 7 :

 27 Figure 2.7: The prognostic impact of T cells in tumor. The analysis of 124 published articles studying the impact of cytotoxic T cells, memory T cells, regulatory T (T Reg ) cells and T helper (T H ) cell subpopulations with regard to prognosis of cancer patients (20 different cancer types were analyzed) is represented. 'Good' means that the cell type is associated with a good prognosis, 'none' means that there was no correlation and 'poor' means that the cells are associated with a poor prognosis (116).

2

 2

Figure 2 . 8 :

 28 Figure 2.8: The three phases of cancer immunoediting. Cancer immunoediting is the result of three processes that function either independently or in sequence. Once normal cells are transformed into tumor cells by the combination of acquired oncogenes and failed intrinsic tumorsuppressor mechanisms, the immune system may function as an extrinsic tumor suppressor by eliminating tumor cells or preventing their outgrowth. In the first phase, elimination, previously known as cancer immunosurveillance, innate and adaptive immune cells and molecules recognize transformed cells and destroy them, resulting in a return to normal physiological tissue. However, if antitumor immunity is unable to completely eliminate transformed cells, surviving tumor variants may enter into the equilibrium phase, where cells and molecules of adaptive immunity prevent tumor outgrowth. These variants may eventually acquire further mutations that result in the evasion of tumor cell recognition, killing, or control by immune cells and progress to clinically detectable malignancies in the escape phase. (Abbreviations: CTLA-4, cytotoxic T lymphocyte associated protein-4; IDO, indoleamine 2,3-deoxygenase; IFN, interferon; IL, interleukin; MΦ, macrophage; MDSC, myeloid-derived suppressor cells; NK, natural killer; NKG2D, NK group 2, member D; PD-L1, programmed cell death 1 ligand 1; TGF-β, transforming growth factor-β; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Treg, regulatory T cell; VEGF, vascular endothelial growth factor.)(142)

Figure 2 . 9 :

 29 Figure 2.9: A proposed model for the elimination phase of the cancer immunoediting process. The events underlying this process are described in the text. Tumor cells are in blue; non-transformed cells in gray; lymphocytes, dendritic cells (DC), and macrophages (Mac) are marked and colored appropriately. Dead tumor cells are identified as white to gray gradient circles surrounded by a dashed black line, and tumor antigens are in blue squares. Panel (a) represents the initiation of the response, wherein cells of innate immunity recognize the nascent tumor. In panel (b), the initial amount of IFN-produced starts a cascade of innate immune reactions that result in some tumor cell death by both immunologic and non-immunologic mechanisms. In panel (c), events of innate immunity charge the adaptive response; tumor cells killed due to the increased cytocidal activities of NK cells and activated macrophages are ingested by DCs, which migrate to the draining lymph node and present antigen to naive CD4+ and CD8+ T cells. In panel (d), tumor-specific CD4+ and CD8+ T cells home to the tumor along a chemokine gradient where they recognize and destroy tumor cells expressing distinctive tumor antigens (136).

Figure 2 . 10 :

 210 Figure 2.10: Co-stimulatory and inhibitory signal of TCR. T cell response to antigen (which is mediated by peptide-major histocompatibility complex (MHC) molecule complexes that are recognized by the T cell receptor (TCR)). These responses can occur at the initiation of T cell responses in lymph nodes (where the major APCs are dendritic cells) or in peripheral tissues or tumors (where effector responses are regulated). In general, T cells do not respond to these ligand-receptor interactions unless they first recognize their cognate antigen through the TCR.Many of the ligands bind to multiple receptors, some of which deliver co-stimulatory signals and others deliver inhibitory signals. In general, pairs of co-stimulatory-inhibitory receptors that bind the same ligand or ligands such as CD28 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) display distinct kinetics of expression with the co-stimulatory receptor expressed on naive and resting T cells, but the inhibitory receptor is commonly upregulated after T cell activation. One important family of membrane-bound ligands that bind both co-stimulatory and inhibitory receptors is the B7 family (165).

1) CTLA- 4 CTLA- 4 ,

 44 the first immune-checkpoint receptor to be clinically targeted, is expressed exclusively on T cells where it primarily regulates the amplitude of the early stages of T cell activation. Primarily, CTLA-4 counteracts the activity of the T cell co-stimulatory receptor, CD28 (166, 167). CD28 and CTLA-4 share identical ligands: CD80 (also known as B7.1) and CD86 (also known as B7.2) (168). Although the exact mechanisms of CTLA-4 action are under considerable debate, it has been proposed that its expression on the surface of T cells dampens the activation of T cells by outcompeting CD28 in binding CD80 and CD86, as well as actively delivering inhibitory signals to the T cell (169. The specific signalling pathways by which CTLA-4 blocks T cell activation are still under investigation, although a number of studies suggest that activation of the protein phosphatases, SHP2 (also known as PTPN11) and PP2A, are important in counteracting kinase signals that are induced by TCR and CD28 {Rudd, 2009 #4115). Moreover, CTLA-4 also confers 'signaling-independent' T cell inhibition through the sequestration of CD80 and CD86 from CD28 engagement, as well as active removal of CD80 and CD86 from the antigen-presenting cell (APC) surface (170). The central role of CTLA-4 in inactivation in T cell is demonstrated by the lethal systemic immune hyperactivation phenotype of Ctla4-knockout mice (171). Even though CTLA-4 is expressed by activated CD8 + effector T cells, the major physiological role of CTLA-4 seems to be through distinct effects on the two major subsets of CD4 + T cells: downmodulation of helper T cell activity and enhancement of regulatory T (Treg) cell immunosuppressive activity (172, 173). CTLA-4 blockade results in a broad enhancement of immune responses that are dependent on helper T cells and, conversely, CTLA-4 engagement on T Reg cells enhances their suppressive function. CTLA-4 is a target gene of the forkhead transcription factor FOXP3 (174), the expression of which determines the T Reg cell lineage (175), and T Reg cells therefore express CTLA-4 constitutively. Although the mechanism by which CTLA-4 enhances the immunosuppressive function of T Reg cells is not known, T Reg cell-specific CTLA-4 knockout or blockade significantly inhibits their ability to regulate both autoimmunity and antitumor immunity(172, 173)(Figure 2.11a).

Figure 2 . 11 :

 211 Figure 2.11: Immune checkpoint in cancer. a | The cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)-mediated immune checkpoint is induced in T cells at the time of their initial response to antigen. The level of CTLA4 induction depends on the amplitude of the initial T cell receptor (TCR)-mediated signaling. High-affinity ligands induce higher levels of CTLA4, which dampens the amplitude of the initial response. The key to the regulation of T cell activation levels by the CD28-CTLA4 system is the timing of surface expression. CTLA4 functions as a signal dampener to maintain a consistent level of T cell activation in the face of widely varying concentrations and affinities of ligand for the TCR. b | By contrast, the major role of the programmed cell death protein 1 (PD-1) pathway is not at the initial T cell activation stage but rather to regulate inflammatory responses in tissues by effector T cells recognizing antigen in peripheral tissues. Activated T cells upregulate PD-1 and continue to express it in tissues. Inflammatory signals in the tissues induce the expression of PD-1 ligands, which downregulate the activity of T cells and thus limit collateral tissue damage in response to a microorganism infection in that tissue. The best characterized signal for PD-1 ligand 1 (PD-L1; also known as B7-H1) induction is interferonγ (IFNγ), which is predominantly produced by T helper 1 (T H 1) cells, although many of the signals have not yet been defined completely. Excessive induction of PD-1 on T cells in the setting of chronic antigen exposure can induce an exhausted or anergic state in T cells. MHC, major histocompatibility complex (165).

3. 3

 3 Adoptive Cell TransferAdoptive cell transfer (ACT) means the transfer of immune cells with antitumor activity. This can be done by expansion and infusion of autologous or allogeneic tumor infiltrating lymphocytes, the use of genetically modified lymphocytes. Most recently scientist have generated chimeric antigen receptors (CAR) that will be explained below.By using gamma retroviruses or lentiviruses, lymphocytes can be genetically modified to encode T cell receptors (TCR) that recognize specific tumor antigens or encode molecules that can enhance their antitumor activity(197). These lymphocytes then acquire antitumor activity.Historically, these are conventional TCR that have alpha and beta chains that form heterodimers to recognize cancer antigens that are presented on the surface of MHC molecules on tumor cells(197). T cells genetically engineered to target NY-ESO-1 expressing synovial sarcoma have shown some promise (185).In a small study, patients with NY-ESO-1 expressing synovial sarcomas were treated with a lymphodepleting chemotherapy regimen consisting of cyclophosphamide and fludarabine, followed by infusions of autologous T lymphocytes designed to recognize a specific NY-ESO-1 antigen. Four of 6 patients with synovial sarcoma had evidence of partial response lasting from 5-18 months. Limitations of conventional TCR are that they are restricted to antigen presentation on specific MHC molecules (197). In addition, downregulation of class I MHC molecules has been described as a mechanism of avoiding TCR recognition(198).

3 3 . 4

 34 Modulation of macrophage response in sarcoma Mifamurtide, liposomal muramyl tripeptide phosphatidylethanolamine (L-MTP-PE), is a new agent that is a synthetic analog of a muramyl dipeptide (MDP) (199). Although its pharmacological behavior is similar to that of MDP, L-MTP-PE has a longer half-life than MDP (200). The intracellular pattern recognition molecule NOD2 detects MDP and enhances NF-κB signaling (201). Therefore, recognition of L-MTP-PE by NOD2 stimulates the production of IL-1β, IL-6, and TNF-α via the activation of NF-κB signaling in monocytes and macrophages (202) and M2 to M1 conversion in macrophages (203). The efficacy of L-MTP-PE treatment for osteosarcomas has been examined in dogs. Dogs with postoperative osteosarcomas were treated by intravenous L-MTP-PE injections. The median survival time of dogs treated by L-MTP-PE (222 days) was longer than that of non-treated dogs (77 days) (204). In human, intergroup study 0133 (INT 0133) began in 1993. 662 patients with osteosarcoma were recruited in this study. The aim of the study was to evaluate the efficacy of supplementation with ifosfamide (IFO) and L-MTP-PE in basic adjuvant chemotherapy (cisplatin, doxorubicin, and high-dose methotrexate (MAP)). Patients were randomly assigned to receive MAP alone, MAP + IFO, MAP + L-MTP-PE, and MAP + IFO + L-MTP-PE. It was observed that the addition of L-MTP-PE to chemotherapy improved the six-year overall survival rate from 70% to 78%. The hazard ratio for overall survival with the addition of MTP was 0.71 (95% CI: 0.52-0.96) (205). Therefore, L-MTP-PE has been approved in Europe for the treatment of osteosarcoma with chemotherapy. However, it has not been approved by FDA in the United States (202).

3 3 . 5

 35 Immune Checkpoint BlockadeRecently, there has been much excitement about the potential of the immune checkpoint inhibitors in solid tumors including sarcomas following their clinical successes and approvals for treatment of metastatic melanoma and metastatic squamous non-small cell lung cancer(206)(207)(208).Ipilimumab is a human IgG4 monoclonal antibody that blocks the anti-cytotoxic Tlymphocyte-associated protein 4 (CTLA-4) and was the first of the new generation of checkpoint inhibitors to gain FDA approval (206). CTLA-4 is a member of the immunoglobulin superfamily; after T cell activation, CTLA-4 is expressed on the plasma membrane of cells where it acts to inhibit T cell function through a variety of mechanisms, allowing tumor cells to escape immune surveillance(209, 210). The experience of ipilimumab in pediatric patients is limited; GI toxicity was the major concern. A small phase II study in adults with synovial sarcoma had no clinical or immunological responses (207).Antibodies targeting the programed cell death protein 1 pathway (PD-1/PD-L1) (nivolumab, pembrolizumab) function in a similar manner to ipilimumab by removing the brakes on T cells which then can perform active anti-tumor immune surveillance(209, 210). Preclinical studies have demonstrated expression of PD-1L in OS and suggest that high expression levels may correlate with worse clinical outcomes (211); In vivo studies using murine sarcoma models with anti-CTLA-4 And PD-1 antibodies have also shown promise for these agents (208).

  Novel microparticle-based drug delivery systems, such as microemulsion, nanoemulsion, nanoparticles, liposomes, and others, can load many kinds of various drugs and improve the drug delivery to target sites(216)(217)(218). It has been reported that these systems improve the efficacy of vaccine and reduce adverse effects of cytokines(219, 220). Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg) fraction of immunoglobulin G molecule, binds to neutrophils and macrophages (221).Tuftsin stimulates their phagocytic activity and enhances expression of nitric oxide synthase in macrophages. It has been demonstrated that tuftsin improves the efficacy of antibiotics against protozoan, bacterial, and fungal infections.Besides, tuftsin-bearing liposomized etoposide enhanced the therapeutic efficacy in murine fibrosarcoma models (222).Immune checkpoint mechanism inhibits CD8 T cell function in tumor microenvironment(223). Although immune checkpoint blockade molecules, anti-CTLA-4 antibody and anti-PD-1 antibody, have not been proven currently to have the effectiveness, there is too little information to decide efficacy of ipilimumab and nivolumab in sarcomas. Thus, immune checkpoint blockade medicines should be evaluated in the future. Adoptive cell transfer approaches are also the subject of new sarcoma treatment trials. Overall, these trials and successes suggest that immunotherapy is moving to the forefront of therapy for bone and soft tissue sarcomas. All immunotherapies are reviewed in figure3.1.

Figure 3 . 1 :

 31 Figure 3.1: Immunotherapy against tumor cells. A number of strategies exist for induction of antitumor immunity against hepatocellular carcinoma cells. Antibody therapy using CTLA-4, adoptive cell of CTL specific to the tumor, cytokine therapy using INF or IL-2 to stimulate a CTL response and peptide/DC vaccine. Tumor-specific cytotoxic T lymphocytes (CTLs) activated by various immunotherapies are capable of recognizing and eliminating cancer cells. (224)

  chondrosarcoma. Immunotherapy has regained interest over the last few years and cancer immunotherapy was Science magazine's 2013 advance of the year {Couzin-Frankel, 2013 #4596}. Passive immunotherapy (i.e., monoclonal antibodies) has proven remarkably successful in selected cancer subtypes, such as lymphoma. Strategies to reactivate antitumor immune responses with drugs targeting CTLA-4 and PD1/PD-L1 have led to striking advances in the therapy of melanoma, some carcinomas, and hematopoietic neoplasias. Nevertheless, immunotherapy strategies for people with sarcomas (recently reviewed (214)) are comparatively less advanced. Evidence from mouse models (226) and the observation that immunosuppressed patients triple their incidence of (non-Kaposi) sarcomas (227) support a major role for the immune system in preventing the development and progression of these cancers.

  Experiments were conducted in accordance with the European and French laws and were validated by the local animal ethical evaluation committee C2EA-UCBL55, protocol number: DR2014-55. Animals were maintained and experiments performed in the pathogen-free animal facility "SCAR" at the Rockefeller Medicine faculty (Agreement # A 69 388 10 01).The transplantable orthotopic Swarm rat chondrosarcoma model (SRC) has been previously described(38) (Fig 5.1). This model is a grade II chondrosarcoma with mild cellular atypia. It mimics its human counterpart in terms of aggressivity and chemo resistance. Tumors were grafted on 25-days-old Sprague-Dawley rats (Charles River Laboratories, L'Arbresle, France). Briefly, tumor fragments (10mm 3 ) were transplanted on the right posterior tibia of the rats after periostal abrasion. Upon establishment of palpable tumors, i.e. 10 days after tumor implantation, animals were randomly divided into treatments groups. Two kinds of treatments were performed and will be detailed in the following paragraphs:

Figure 5 . 1 :

 51 Figure 5.1: Transplantable orthotopic Swarm rat chondrosarcoma. The figure represent the implantation procedure of SRC and the time lapse of the of the model

Figure 5 . 2 :

 52 Figure 5.2: The targets and effects of each immunomodulator treatment.

  g; 5 min) and transferred in transparent 96 wells flat bottom plates (Nunc, P7366, Sigma Aldrich). NK-92 and K-562 cells lines were used as positive control (229). For each sample, calcein release was measured on Tecan Infinite F500 (Tecan group ltd. Switzerland) (excitation filter: 485 ± 9 nm; band-pass filter: 530 ± 9 nm). Specific lysis induced by the effectors was calculated according to the formula: Specific sample lysis= [(sample releasespontaneous release)/(maximal releasespontaneous release)] × 100. All tests were run in triplicate.5 5.7 Measurement of cytokines secretion Levels of seric IL-10, IL-1β, TNFα and IFNγ from animals of the different treatments groups were determined by ELISA assays as per the manufacturer's instructions. IL-1β, TNFα and INFγ ELISA kits were from Peprotech (Rocky hill, NJ, USA); Il-10 ELISA kit was from Sigma-Aldrich.

  CD4+, CD8+ T cells and CD163+ cells (monocytes/macrophages) were observed in all the 26 tumors and were mostly located in the peripheral area of the tumor (Fig 6.1).

Figure 6 . 1 :

 61 Figure 6.1: Immune infiltrate in human chondrosarcoma. IHC staining for CD8, CD3, CD4, CD163 indicated the presence of CD8+, CD3+, CD4+, CD163+ cells (black arrow) at the periphery of tumors in human chondrosarcoma (original magnification 200X).

Figure 6 . 2 :

 62 Figure 6.2: Immune infiltrates correlation with chondrosarcoma's survival. Each graph presents the correlation between one immune population and patients survival. Patients with above the median CD3+, CD4+ CD8+ cells have better survival than patients with below the median T cell counts. Interestingly CD163+ cells count did not correlate with patients survival.

Figure 6 . 3 :

 63 Figure 6.3: Immune infiltrates correlation with chondrosarcoma's progression.CHS relapse was evaluated based on immune cell counts. Patients with above the median CD3+, CD4+ CD8+ cells tended to relapse later than patients with below the median T cell counts; whereas CD163+ cells count did not correlate with tumor relapse.

Figure 6 . 4 :

 64 Figure 6.4: Correlation between CD8+, CD163+ cells counts and tumor size. In this series of 26 conventional chondrosarcoma, an inverse correlation between CD8+cells count (red dots) and tumor size (in mm3) was found; whereas a correlation between CD163+ cells (black dots) and tumor size was encountered.

Figure 6 . 5 :B

 65 Figure 6.5: Correlation between CD8+, CD163+ cells counts and tumor agressivity. The correlation between CD8+ cells infiltrate (A) or CD163+ cells infiltrate (B) and tumor agressivity (expressed as tumor grade) was evaluated. Chondrosarcoma are classified as follow: + grade I chondrosarcoma; ++ non-invasive grade II chondrosarcoma; +++ invasive grade II chondrosarcoma; ++++grade III chondrosarcoma

Figure 6 . 6 :

 66 Figure 6.6: Correlation between CD8+ lymphocytes and the CD163+ macrophages in tumor's microenvironment. Each dot represents one of the 26 conventional chondrosarcoma of this study.

Figure 6 . 7 :

 67 Figure 6.7: CD8/CD163 ratio and tumor agressivity. Correlation between CD8/CD163 ratio tumor size at the time of resection (A) and tumor grade (B) was evaluated. A; Each dot represents one of the 26 conventional chondrosarcoma of this study. B: Chondrosarcoma are classified as follow: + grade I chondrosarcoma; ++ non-invasive grade II chondrosarcoma; +++ invasive grade II chondrosarcoma; ++++grade III chondrosarcoma

  The analyses of chondrosarcoma immunological environment realized on 26 conventional chondrosarcoma samples indicate that the immune populations could be involved in chondrosarcoma progression. The implication of each immune population in chondrosarcoma progression cannot be conducted on human chondrosarcoma samples, partly because of the rarity of this tumor. Thus to conduct such analyses, an animal model recapitulating the human pathology and its immunological environment is necessary.The Swarm rat chondrosarcoma model (SRC) mimics human chondrosarcoma in term of evolution, thus we the characterized its immunological environment and conducted further analyses in this model.As in human chondrosarcoma, tumor immune infiltrate was analyzed on a "cohort" of 17 progressive SRC. On each tumor, positive cells for each immune marker considered (CD4, CD8, CD163) were counted on 5 different areas located at the margin of cartilaginous nodules and surrounding muscles.CD4+, CD8+ T cells and CD163+ cells (monocytes/macrophages) were observed in all the 17 tumors and were mostly located in the peripheral area of the tumor(Fig 6.8).

Figure 6 . 8 :

 68 Figure 6.8:Immune infiltrate in SRC model. IHC staining for CD8, CD3, CD4, CD163 indicated the presence of CD8+, CD3+, CD4+, CD163+ cells (black arrow) at the periphery of tumors in rat chondrosarcoma (original magnification 200X).

Figure 6 . 9 :

 69 Figure 6.9: Correlation between CD8+, CD163+ cells counts and tumor size. In this series of 17 progressive, an inverse correlation between CD8+cells count (red dots) and relative tumor size (Size at day 21/size at day 0) was found; whereas a correlation between CD163+ cells (green dots) and tumor size was encountered.

Figure 6 .

 6 Figure 6.10: CD8/CD163 ratio and tumor agressivity. Correlation between CD8/CD163 ratio and tumor size at the time of sacrifice. Each dot represents one of the 17 progressive SRC of this study

Figure 6 . 11 :

 611 Figure 6.11: Effects of selective depletion on SRC immune infiltrates. Average immune cells infiltration (CD3+, CD8+, CD163+) in each treatment groups at the end of the experimentation. Azathioprine caused a decrease in CD3 and CD8+ cells whereas mifamurtide and clodronate affected CD163+ cells. Counts were made on 5 areas / tumor (400X magnification).

Figure 6 . 12 :Figure 6 . 13 :

 612613 Figure 6.12: Effects of selective depletion on SRC progression. A: SRC evolution in control, CD3/CD8 depleted group (azathioprine treated), macrophages depleted (clodronate) or macrophages modulated (mifamurtide) groups expressed as relative tumor size. (*= p < 0,05; ***= p < 0,005). B: animal's survival in control and immune depleted groups (*= p < 0,05; ***= p < 0,005).

Figure 6 . 14 :

 614 Figure 6.14: Inflammatory response in chondrosarcoma. Seric levels of IFNγ (B) and TNF-α (B) in normal animals, progressive chondrosarcoma CD163 or CD3/CD8 depleted chondrosarcoma bearing rats (*= p < 0,05). . Data are given as the mean of each marker measured ± SE. Chs: progressive chondrosarcoma; Chs Aza: azathioprine treated SRC; Chs clod: clodronate treated SRC; Chs Mifa: mifamurtide treated SRC

Figure 6 . 15 :

 615 Figure 6.15: PI3K, Akt and mTOR inhibitors affect SRC progression. SRC evolution expressed as relative tumor size in control, GDC-0941 (PI3K inhibitor), G594 (Akt inhibitor) and Everolimus (mTOR inhibitor) treated SRC. (**: p < 0,005; ***: p < 0,005) (n=12).Ctr: progressive chondrosarcoma; PI3K: GDC-0941-treated SRC; Akt: G-594treated SRC; mTOR: Everolimus-treated SRC

Figure 6 . 16 :

 616 Figure 6.16: PI3K, Akt and mTOR inhibitors increase SRC survival. (*: p < 0,05; **: p < 0,05) (n=12).Ctr: progressive chondrosarcoma; PI3K: GDC-0941-treated SRC; Akt: G-594 treated SRC; mTOR: Everolimus-treated SRC

Figure 6 . 17 :

 617 Figure 6.17: Effect of PI3K pathway inhibitors on SRC immune environment. Average Immune cells density for each treatment groups at the end of the experimentation. Immune infiltrate were counted in 5 areas (magnification x400). None of the inhibitor caused a change in immune infiltrate composition and density. Ctr: progressive chondrosarcoma; PI3K: GDC-0941-treated SRC; Akt: G-594-treated SRC; mTOR: Everolimus-treated SRC

Figure 6 . 18 :Figure 6 . 19 :•

 618619 Figure 6.18: Effect of PI3K pathway inhibitors on cytotoxic response in chondrosarcoma. Splenocytes from healthy animals did not show a specific cytotoxic activity directed against chondrosarcoma cells. This activity was present only in animals with progressive tumor and was decreased by the PI3K pathway inhibitors used (*** = p < 0,0005). Chs: progressive chondrosarcoma; Chs PI3K: GDC-0941-treated SRC; Chs Akt: G-594treated SRC; Chs mTOR: Everolimus-treated SRC

Figure 6 . 20 :

 620 Figure 6.20: Expression of PD-L1 and PD-1 in human conventional chondrosarcoma. A) Representative IHC of PD-L1 and PD-1 expression in human chondrosarcoma showing diffuse membranous PD-L1 expression by tumor cells (green arrow) and tumor infiltrating immune cells (black arrow). PD-1 is highly expressed on tumor infiltrating lymphocytes (black arrow). B) Correlation between PD-L1 and tumor aggressiveness C) Correlation between PD-L1 and PD-1 expression and survival and relapse.

Figure 6 . 21 :

 621 Figure 6.21: Expression of PD-L1 and PD-1 in SRC. Representative IHC of PD-L1 and PD-1 expression in rat chondrosarcoma. Tumor cells and tumor infiltrating immune cells showed an intense staining corresponding to a high expression of PD-L1 (green arrow), whereas PD1 expression was found on the membrane of tumor infiltrating lymphocytes (black arrow).

Figure 6 . 22 :

 622 Figure 6.22: Expression of PD-L1 and PD-1 in PI3K pathway inhibitors treated SRC. Representative IHC of PD-L1 and PD-1 expression in SRC model after 3 weeks of PI3K pathway inhibitor treatment. A decrease in PDL1 expression was observed for all treatments.

Figure 6 . 23 :

 623 Figure 6.23: Expression of PD-L1 and PD-1 in PI3K pathway inhibitors treated sarcoma cell lines MNNG/HOS (osteosarcoma), SW-1353 (chondrosarcoma), RMS SJRH30 (Rhabdomyosarcoma) were treated by INFγ known to upregulate PDL1 expression, then submitted to either PI3K, Akt or mTOR inhibitors (RAD) and expression of PDL1 was then analyzed by western blot (n=3) PI3K inh: GDC-0941; Akt inh: G-594; mTOR inh: Everolimus

Figure 6 . 24 :

 624 Figure 6.24: Blocking of rat PD-1 immune checkpoint using a murine PD-L1 blocking antibody. Expression of PD-L1 by rat UMR-106 osteosarcoma cell line is detected by flow cytometry using a murine PD-1Fc chimera coupled to FITC dye. Cells were pre-treated with INFγ (bottom part of the quadrant) or not (upper part of the quadrant) and with PD-L1 blocking antibody (right side of the quadrant) or the isotype control (left side of the quadrant).

7 7 . 2

 72 Prognosis value of macrophages in chondrosarcomaTumor-associated macrophages (TAMs) have a dual role in cancer depending on their pro-inflammatory (M1) or pro-tumoral (M2) phenotype (243, 244). Generally M2 macrophages secrete anti-inflammatory cytokines (IL-10, TGF-b) and are mainly considered pro-tumoral (243). M1 macrophages secrete pro-inflammatory cytokines (TNF-α, IL-12) and are of good prognostic in certain tumors (244), even though a study showed that both M1 and M2 TAMS could be of good prognostic in osteosarcoma (243).

  chondrosarcoma. The only problem remains the lack of tools available for the studies of the rat immune populations compared to what is available for the mouse. Our group is currently working on developing new tools (flow cytometry antibodies panels, detection of markers by qRT-PCR on microdissected tumors) to evaluate the immune response in the SRC model. This will allow better understanding of the immune microenvironment of chondrosarcoma and the evaluation of new therapeutic approaches in a model closely related to the human disease.

7 7 . 7

 77 Future directionsA more complete description of human chondrosarcoma environment is going to be performed in a large cohort of human chondrosarcoma.o To fully elucidate the implication of T lymphocytes in human chondrosarcoma, the CD4+ T cells, more particularly the Treg subset will be analyzed.

(

  monocytes/macrophages) were detected at the periphery of all tumors (Fig 3a). On each tumor, positive cells for each immune marker were counted on 5 different areas located at the margin of cartilaginous nodules and surrounding muscles (magnification 200X) (fig 3a). In the SRC model the CD8/CD163 ratio and the CD8+ count were inversely control group, p < 0,05) (Fig 4d). At day 34, 3 animals remained alive in the clodronate and mifamurtide treated groups (Fig 4d).

versus 46 ,

 46 33 ± 7,36 for the control group P < 0,05 at day 14) (Fig 4c) with a significantly higher tumor volume than the control groups (respectively 2349 ± 86 mm 3 versus 1434 ± 169mm 3 at d14 P < 0,0005) (Fig 4b). Azathioprine-treated rats reached the maximum tumor volume (2000 mm 3 ) sooner than the control group (12,33 ± 0,41 days versus 16,92 ± 0,70 days p < 0,0005) (Fig 4d). At 14 days after initiation of the treatments all animals from the azathioprine treated group were dead whereas 10 animals remained alive in the control group (Fig 4d).

  5a). These results confirm the hypothesis that effectors CD8+ T cells are located in chondrosarcoma microenvironment and play an antitumoral role and maintain chondrosarcoma growth. Two key cytokines INFγ and TNF-α were analyzed on all animal sera. IFNγ and TNF-α, were detectable in the sera of all chondrosarcoma animals while undetectable in healthy animals (Fig 5b,c,d). The three modulatory agents used (azathioprine, clodronate, mifamurtide) increased the level of seric IFNγ compared to untreated SRC animals (P<0,05) (Fig5b,d). None of the immunomodulator treatments caused changes in TNF-α level (Fig 5c,d). Expression of PD-1/PD-L1 in chondrosarcoma Expression of the immunosuppressive ligand PD-L1 and its receptor PD-1 by tumor cells and tumor infiltrating cells were finally assessed both in human chondrosarcoma samples and in SRC.PD-L1 expression was observed in all 26 human chondrosarcoma both on tumor cells and on tumor-infiltrating immune cells (Fig 6a, Supplementary table

  cells (Fig 6b) when PD-1 was identified on tumor infiltrating immune cells (Fig 6b).

Figure 1 :

 1 Figure 1: Immune infiltrate in human chondrosarcoma. A) From top to bottom representative IHC staining of CD8+, CD3+, CD4+, CD163+ cells (black arrow) in human chondrosarcoma (original magnification 200X). B) Immune populations are correlated with patients survival's. CHS patients OS was compared in two groups based on the median of the immune cell counts. C) Immune populations and CHS relapse. Patients were split in two based on the median of immune cell counts.

Figure 2 :Figure 3 :

 23 Figure 2: Impact of immune infiltrate on human chondrosarcoma progression. A) Correlation between CD8+cells (red dots), CD163+ cells (black dots) and tumor size. B) Correlation between the CD8/CD163 ratio and tumor size at the time of resection. C) Correlation between the infiltration of CD8+ lymphocytes and the CD163+ macrophages in the microenvironment of the tumors D) Correlation between CD8+ counts, CD163+ counts and CD8/CD163 ratio and tumor grade. (+ grade I chondrosarcoma; ++ noninvasive grade II chondrosarcoma; +++ invasive grade II chondrosarcoma; grade III chondrosarcoma) Figure 3: Immune infiltrate in rat chondrosarcoma model. A) From top to bottom representative IHC staining of CD8 + , CD3 + , CD163 + (black arrow) from SRC (original magnification 400X). B) Correlation of CD8/CD163 ratio with tumor growth index measured at day 21 in progressive SRC. C) Correlation between CD8+cells (red dots), CD163+ cells (black dots) and tumor size at day 21 in progressive SRC.

Figure 4 :

 4 Figure 4: Effects of selective depletion on tumor's immune environment. A) Average immune cells infiltration (CD3+, CD8+, CD163+) in each treatment groups at the end of the experimentation. Azathioprine caused a decrease in CD3 and CD8+ cells whereas mifamurtide and clodronate affected CD163+ cells. Counts were made on 5 areas / tumor (400X magnification). B) Tumor growth evolution in control and CD163 (mifamurtide or clodronate treated) or CD3/CD8 (azathioprine treated) depleted chondrosarcoma. C)

Figure 5 :

 5 Figure 5: Cytotoxic and inflammatory responses in chondrosarcoma. A) Specific cytotoxicity of splenocytes isolated from tumor-bearing animals. Non tumor-bearing animals did not show a specific cytotoxic activity directed against chondrosarcoma cells. This activity was present only in chondrosarcoma bearing rats and was decreased by selective depletion of CD163 or CD3/CD8 cells (*** = p < 0,0005). B) Serum IFNγ levels in normal animals, progressive chondrosarcoma CD163 or CD3/CD8 depleted chondrosarcoma bearing rats (*= p < 0,05). C) Serum TNF-α level in normal animals, progressive chondrosarcoma CD163 or CD3/CD8 depleted chondrosarcoma-bearing rats. Data are given as the mean of each marker measured ± SE. All values are in pg/ml. D) Serum levels of INFy, TNF-α in normal animals, progressive chondrosarcoma, CD163 or CD3/CD8 depleted chondrosarcoma-bearing rats. Data are given as the mean of each marker measured ± SE. All values are in pg/ml (N=7).

Figure 6 :

 6 Figure 6: Expression of PD-L1 and PD-1 in human and rat chondrosarcoma. A) Representative images of PD-L1 and PD-1 expression in human chondrosarcoma showing diffuse membranous PD-L1 expression by tumor cells (green arrow) and tumor infiltrating immune cells (black arrow). PD-1 is highly expressed on tumor infiltrating lymphocytes (black arrow). B) Representative figure of PD-L1 and PD-1 expression in rat chondrosarcoma. Tumor cells and tumor infiltrating immune cells showed an intense staining demonstrating a high expression of PD-L1 (green arrow) and on tumor infiltrating immune cells (black arrow), whereas PD1 expression was found on the membrane of tumor infiltrating lymphocytes (black arrow).

Table of contents 1. Chondrosarcoma: A rare tumor and a therapeutic challenge -

Table 1 .1: 10 years survival of chondrosarcoma patients in function of the histological grade

 1 The 10 years survival rate of this tumor has not evolved in more than 20 years as indicated by the 2 studies conducted in 1977 by Evans and in 2001 by Anract et al (Table1.1).

	10 years survival	Evans 1977(6)	Anract, de Pinieux and al. 2001(7)
	Grade I chondrosarcoma	83%	83%
	Grade II chondrosarcoma	64%	60%
	Grade III chondrosarcoma	29%	5%

  to IL-2 have been reported in several other cancers including lung and breast cancers(191). High-dose IL-2 was given in relapsed pediatric patients among which 4 patients with osteosarcoma and 2 patients with Ewing sarcoma.Two of the four osteosarcoma patients had complete responses, while the other two and both Ewing sarcoma patients had progressive disease(191). These results associated to the toxicity of high-dose IL-2 make the administration of IL-2 as a single agent unlikely in sarcoma. Several studies are ongoing in sarcomas combining IL-2 given in a variety of different routes and dosages with antibody therapy, vaccines, and adoptive cell therapy(192).Interferons are a complex family of molecules that bind to IFN receptors; IFNα and IFNβ activate type I receptors, while IFNγ activates type II receptors(193).

Both IFNα and IFNβ activate immune cells and increase antigen presentation to T cells. INFα is approved for use in melanoma and has also been studied in sarcomas. Interferons used in bones sarcomas are resume in table

3.1.

Table 3 .1: Interferons studies in bone sarcoma (192).

 3

Table 3 .2: TAAs expression in sarcoma tumor samples and cell lines

 3

(194)

.

Table 3 .3: Vaccines immunotherapy in sarcomas (192)

 3

  ). So, if chondrosarcoma cells by themselves could generate a signal strong enough to activate NK cells we would observe cytotoxicity with splenocytes from healthy rats in co-culture with chondrosarcoma cells. As we didn't observe such an activity we believe that if NK cells have a cytotoxic action in chondrosarcoma this activity is almost negligible.

	The second population is the CD8 cytotoxic T Lymphocytes (CTL). In cancer,
	naïve CD8 lymphocytes must undergo maturation to become CTL expressing a
	TCR specific for a Tumor Associated Antigen (TAA) (248). These specific CTL
	are found not only in the microenvironment of the tumors but also in the spleen
	(248). The fact that splenocytes of progressive SRC have a specific cytotoxic
	action directed toward chondrosarcoma cells, makes us believe that CTLs
	present in the spleen of the chondrosarcoma bearing animals are responsible for
	this response. Another indication in favor of CTL implication in SRC immune
	surveillance is the decrease in cytotoxic action observed in azathioprine treated
	rats (i.e. in T lymphocytes depleted animals) (230).
	The tumor-associated antigen against recognized by CTL still remains to be
	identified. It is known that normal chondrocytes can induce specific T
	lymphocytes response directed against membrane proteins that give rise to
	rheumatoid arthritis (249). Moreover, it was demonstrated that human
	chondrosarcoma express high level of diverse TAAs of the cancer-testis antigens
	(CTA) family (194). The expression of these CTAs in other sites than the testis
	gives rise to a strong immune response (108). These are two potential families of
	antigens which expression could be investigated in the SRC model. The
	identification of potential antigenic targets harbored by chondrosarcomas is
	currently one of the research program conducted by our group in association with
	the Immunosarc program consortium.

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	PD-1/PD-L1 blocking on tumor progression and immune 9.1 Manuscript submitted identify potential therapeutic targets. We analyzed chondrosarcoma immunological
	environment. microenvironment in human chondrosarcoma samples and in the Swarm rat
	o Clarify the impact of clodronate and mifamurtide on M1 chondrosarcoma model. We report here that CD8+ lymphocytes and CD163+
	macrophages. We are currently designing flow cytometry antibodies macrophages are involved in chondrosarcoma progression, and that PD-1/PD-L1 is
	panel and q RT PCR primers to analyze M1 and M2 macrophages expressed in chondrosarcoma. Our data indicate that immunomodulatory approaches can
	in tumor microenvironment. Dear Mr. Simard, be envisioned for chondrosarcoma.
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  On each tumor, positive cells for each immune marker were counted on 5 different areas located at the margin of cartilaginous nodules and surrounding muscles (magnification 200X) (fig 1a). The highest density of T cells was observed in invasive grade II and III chondrosarcoma.

	Immune infiltrate density was correlated with tumor agressiveness. Indeed, patients with
	above the median of CD8+, CD4+ and CD3+ immune infiltrates density had a better
	overall survival than patients with lower peritumoral CD8+, CD4+ and CD3+
	counts.(p=0.04), (Fig 1b) and tended to have better PFS (P > 0,05). An inverse
	correlation between CD8 + T cells count and tumor size was observed (Fig 2a).
	The density of CD163+ cells ranged from negligible to important (supplementary table
	S1) ; the highest density of CD163+ cells was found in grade III chondrosarcoma
	(Supplementary table S1). A correlation between high CD163+ cell count and tumor size
	was observed (Fig 2a). CD163+ infiltrations do not correlated with overall survival and
	relapse (Fig 1b,c).
	CD8+ and CD163+ were found inversely correlated in this series of 26 tumors (R 2 =0.2,
	p=0.035) (Fig 2c). The CD8/CD163 ratio, i.e. the number of CD8+ T cells over M2
	related macrophages (30) count is inversely correlated with tumor size (Fig 2b) and tumor
	grade (Fig 2d). CD8/CD163 ratio, correlated to tumor grade (Supplementary table S2):

The immune environment contributes to CHS progression in both human and animal models, this associated with expression of immune checkpoint PD1/PDL1 suggest that immunomodulatory approaches with PD-L1 blocking antibody could be applied in CHS; this approach is currently being tested in SRC.

Remerciements

Acknowledgements: This research was supported by The Liddy Shriver Sarcoma Initiative and La Ligue contre le Cancer comité de Saône et Loire

Aim of the thesis

1: when 1 to 25% of positive cells were encountered in the field; 2 when 26 to 50 % positive in the field; 3 when we encounter 50% and more positive cells. 5 5.10 In vitro blocking of PD-L1

To investigate the role of PD-1/PD-L1 checkpoint in chondrosarcoma, it is necessary to block the interaction of PD-L1 with its receptor PD-1 in vivo and study the consequences of this blocking on tumor progression and immune environment. Nevertheless, there is a huge pitfall. No PD-L1 blocking antibody crossreacting with the rat species is yet available. Therefor we examine the possibility to block rats PD-L1 using a mouse PD-L1 antibody.

UMR-106 rat osteosarcoma cell line was used for the blocking of PD-L1 in vitro.

Cells were treated or not with 20 ng/ml of INFγ over night for the upregulation of PD-L1. We then add 10 µg of PD-L1 in vivo ready blocking antibody (10 µg BioXcell, West Lebanon, NH, USA) or rat in vivo ready IgG2 isotype control ( 10µg LTF-2, BioXcell) was added to the cell culture for 1h on ice. After being washed with PBS, cells were marked with a mouse PD-1Fc chimera (10 µg RnDsytems, Minneapolis, MN, USA) chimera or isotype control human IgG1 isotype control (10 µg,RnDsystems), both coupled to CF488a (Mix-n-stain antibody labeling kits, Biotium, Hayward, Ca,USA). The analyses were performed on 10 000 events on a 4 color Facscalibur cell analyzer (BD Biosciences, San Jose, Ca, USA) and cellquest program (BD Biosciences).

Statistical analysis

All the data are reported as the sample mean ± the standard deviation (SD).

Pairwise comparisons between means of different groups were performed using a Student t-test (two tailed, unpaired) where, for each couple of normally distributed populations, the null hypotheses that the means are equal were verified. The difference between two subsets of data is considered statistically The density of lymphocytes and macrophages varied among patients and ranged from minor to dense (Table 6.1). The highest density of T cells was observed in grade II chondrosarcoma. The density of CD163+ cells ranged from negligible to important (Table 6.1); the highest density of CD163+ cells was found in grade III chondrosarcoma (Table 6.1). 6.12B). At day 34, when the assay was stopped, 3 animals remained in both of the "macrophage modulation" groups, whereas all the control animals had died.

B)

T lymphocytes depletion by azathioprine caused an increase in tumor progression. In this case, tumor growth rate was increased (91,90 ± 21,48 for azathioprine treated group versus 46,33 ± 7,36 for the control group P < 0,05 at day 14) (Fig 6 .12A). Animals from the azathioprine-treated group had a significantly higher tumor volume than the control groups at day 14 (respectively 2349 ± 86 mm 3 versus 1434 ± 169mm 3 (P < 0,0005).

As a consequence of the increase in tumor progression, azathioprine-treated rats reached the endpoint (i.e. tumor volume of 2000 mm 3 ) sooner than the control group (12,33 ± 0,41 days versus 16,92 ± 0,70 Fourteen days after initiation of the treatments all animals from the azathioprine treated group were gone whereas ten animals remained in the control group (Fig

6.12B).

These data suggest that macrophages play a pro-tumoral action while lymphocytes repress chondrosarcoma growth possibly by specific cytotoxic activity directed against chondrosarcoma cells.

To gain more insight into the involvement of effector T cells and inflammation mechanisms in chondrosarcoma progression, the cytotoxicity of splenocytes from different treatment groups was analysed and production of pro-inflammatory cytokines was measured in rat sera.

The cytotoxic activity of splenocytes from progressive SRC (n=6), azathioprine treated SRC (n=6), mifamurtide treated SRC (n=8), clodronate-treated SRCs (n=6) and healthy animals (n=6) was evaluated against primary chondrosarcoma cells isolated from SRC tumors. Splenocytes isolated from healthy animals did not have a cytotoxic activity against chondrosarcoma cells (E:T ratio of 100:1, specific lysis of 2,5 % ± 1,7) (Fig 6.13). Conversely, chondrosarcoma cells were lysed by splenocytes isolated from chondrosarcoma bearing rats: (27,0 ± 6,5% was obtained at the E:T ratio 100:1). The splenocytes from azathioprine, treated rats had a lower cytotoxic activity on chondrosarcoma cells. (11,3 ± 3,5 % at the same E:T ratio of 100:1 (P < 0,0005). Splenocytes isolated from treatment targeting macrophages had a non-significantly lower cytotoxic activity (P > 0,05) (Fig

6.13).

IL-1β and IL-10 were not detected either in healthy or chondrosarcoma bearing animals (Table 6.2). Table 6.2: Inflammatory cytokines in chondrosarcoma. Serum levels of INFγ, TNF-α, IL10 and IL1β in normal animals, progressive chondrosarcoma, CD163 or CD3/CD8 depleted chondrosarcoma-bearing rats. Data are given in pg/ml as the mean of each marker measured ± SE, in each group N=7.Ctr: progressive chondrosarcoma; Azathioprine: azathioprine treated SRC; Clodronate: clodronate treated SRC; Mifamurtide: mifamurtide treated SRC Two inflammatory cytokines INFγ and TNF-α were detected in the sera of all chondrosarcoma animals while undetectable in healthy animals (Table 6.2;Fig 6.14).

The three modulatory agents used (azathioprine, clodronate,mifamurtide) increased the level of seric IFNγ compared to untreated SRC animals (P < 0,05) (Table 6.2;Fig 6.14A). None of the immunomodulator treatments caused changes in TNF-α level (Table 6.2; Fig 6 .14B).

• In SRC we showed by IHC and depletion/modulation that CD163 macrophages are associated with a faster tumor growth • We show by cytotoxic assay and measure of cytokine that immunosurveillance is active in SRC.

The results in human and SRC model show that we could apply an immunotherapy to chondrosarcoma to boost the immune response toward the tumor. Before, we wanted to see if the inhibitors of the PI3k/mTOR pathway, that had given good results to slow down chondrosarcoma growth in the SRC model [START_REF] Perez | Inhibition of chondrosarcoma growth by mTOR inhibitor in an in vivo syngeneic rat model[END_REF], could synergize with an immunotherapy.

M Modulation of immune response in chondrosarcoma by PI3K pathway inhibitor

We evaluate the effect of agents targeting the PI3K/Akt/mTOR pathway on chondrosarcoma immune environment and assess whether a PI3K/mTOR pathway-induced immune response could contribute to the therapeutic efficiency of one of this pathway's inhibitors.

The three inhibitors tested G-594: Akt inhibitor; GDC-0941: PI3K inhibitor; everolimus: mTOR inhibitor were used at concentrations previously determined as being the most effective.

There was evidence of antitumor efficacy for all the inhibitors tested; the animals receiving these treatments showed smaller tumors after 21 days of treatment in comparison to the control animals (P < 0,05) (Fig 6.15). Among these agents, PI3K inhibitor (GDC-0941) appeared to be the most efficient agent as the tumor growth rate of this group was 13,56 (± 3,39) at day 21. The second most effective agent was the Akt inhibitor (G-594) which

The three PI3K pathway inhibitors used GDC-0941 (PI3K), G-594 (Akt), Everolimus (mTOR) all significantly decreased the level of seric IFNγ and TNF-α compared to untreated SRC animals (P < 0,05 for GDC-0941 and P < 0,005 for G-594 and Everolimus) (Fig 6.19). The fact that none of these cytokines were found in the sera of healthy animals indicate clearly that inflammation is caused by the tumor itself and that the variations observed happen in response to the corresponding treatments.

As we previously found, no IL-1β and Il-10 were detectable in the sera of healthy animals, progressive tumor or either one of the PI3K pathway treated tumor (data not shown).

Statement of Translational Relevance

New immune-targeted therapies have recently generated tremendous enthusiasm with the development of immune checkpoint inhibitors. As they may increase tumor immunogenicity induced by chemotherapy or targeted therapies, they seem to be of great interest for highly resistant tumors like chondrosarcoma. Chondrosarcoma immunological microenvironment remaining largely unknown, we designed a translational project aiming at describing it and deciphering its implication in chondrosarcoma growth and Abstract BACKGROUND: Chondrosarcoma (CHS) is a rare bone malignancy characterized by its resistance to conventional systemic and radiation therapies. Whether immunotherapy targeting immune checkpoints like PD-1/PD-L1 may be active in these tumors remains unknown. To explore the role of the immune system in this tumor, we analyzed the immune environment of chondrosarcomas both in human sample, and in a syngeneic rat model, and tested the contribution of T lymphocytes, macrophages and immune checkpoint expression in chondrosarcoma progression. METHODS: Immunohistochemical stainings were performed on human chondrosarcoma samples and on Swarm rat chondrosarcoma model (SRC). Selective immunodepletion assays were performed in SRC to evaluate immune population's involvement in tumor progression.

RESULTS: In human and rat chondrosarcoma, immune infiltrates composed of lymphocytes and macrophages were identified in the peritumoral area. Immune infiltrates composition was found correlated with tumors characteristics and evolution (grade, invasiveness and size). Expression of PD-1 and PD-L1 was detected in CHS immune infiltrates, both in human and rat (and on tumor cells). PD-L1 expression level correlated with patients survival and relapse rate. In SRC, selective depletions of T lymphocytes resulted in an accelerated growth rates, whileCD163+ macrophages depletion slowed down tumor progression.. Splenocytes isolated from CHS bearing SRC showed a specific cytotoxicity directed against chondrosarcoma cells (27%), which significantly decreased in CD3 depleted SRC (11%).

CONCLUSION: The immune environment contributes to CHS progression in both human and animal models, suggesting that immunomodulatory approaches could be tested in bone chondrosarcoma.

Key words

Chondrosarcoma, immune infiltrates, CD163 macrophages, PD-1/PD-L1, immune checkpoint.

(10,000 U/ml; 10,000 μg/ml) (GIBCO ThermoFisher Scientific, Waltham, USA).

Primary rat chondrosarcoma cells and chondrocytes were isolated from SRC or healthy animals by collagenase digestion (27) and cultured in DMEM medium supplemented with 10% FBS, 2% L-glutamine, and penicillin/streptomycin solution (10,000 U/ml; 10,000 μg/ml).

Animal protocols

Experiments were conducted in accordance with the European and French laws and were validated by the local animal ethical evaluation committee C2EA-UCBL55, protocol number: DR2014-55. Animals were maintained and experiments performed in the pathogen-free animal facility "SCAR" at the Rockefeller Medicine faculty (Agreement # A 69 388 10 01).

The transplantable orthotopic Swarm rat chondrosarcoma model (SRC) has been previously described (28). This model is a grade II chondrosarcoma with mild cellular atypia. It mimics its human counterpart in terms of aggressivity and chemo resistance.

Tumors were grafted on 25-days-old Sprague-Dawley rats (Charles River Laboratories, L'Arbresle, France). Briefly, tumor fragments (10 mm 3 ) were transplanted on the right posterior tibia of the rats after periostal abrasion. Upon establishment of palpable tumors, i.e. 10 days after tumor implantation, animals were randomly divided into treatments groups: PBS (1 ml; n=14) Azathioprine (2mg/kg; n=14; Sigma Aldrich, St-Louis, USA), Mifamurtide (0,1 mg/kg; n=14; Adooq bioscience, Irvine, USA), Clodronate (50 mg/kg; n=14; Clodronateliposomes.com, Amsterdam, The Netherland).

Treatments were administered IP 5 days a week for azathioprine and twice a week for the two other groups. Rats were treated over a period of 5 weeks or till tumors reached 2000 mm 3 .

Tumor growth was monitored by regular visual inspection and tumor dimensions were measured every 2-3 days. Tumor volume was calculated using the formula: Volume = longest tumor diameter x (shortest tumor diameter) ²/2. At the completion of treatment, rats were sacrificed, tumors and spleens harvested. Part of tumors and spleen were used for chondrosarcoma cells isolation and splenocytes harvesting while the USA), CD8 (1/100), CD163 (1/300), PD-1 (1/100), PD-L1 (1/300) (respectively OX-8; ED2; AHP1706; AHP1703; AbD SeroTech, Oxford, UK). CD8 and CD163 primary antibodies were revealed using an unconjugated goat anti-mouse secondary antibody and CD3, PD-1, PD-L1 by unconjugated goat anti-rabbit antibody (respectively AI-9200, AI-1000; Vector lab, Burlingame, CA, USA; dilution 1:100) followed by avidin-biotin complex and DAB peroxidase (VECTASTAIN Elite ABC Reagent, ImmPACT reagent;

Vector Lab). Counterstaining was performed with hematoxylin (Sigma-Aldrich).

For human chondrosarcoma samples, 5 μm sections from FFPE blocks were deparaffinized and rehydrated. Immunostaining for CD3, CD8, CD4 and CD163 was performed on automated Ventana Discovery XT staining system. Primary antibodies to CD3 CD8, CD4, and CD163 were all from Ventana (respectively clone 2GV6, SP57, SP35, MRQ-26) (PD-1 and PD-L1 antibodies (AHP1706 and AHP1703 from ABD Serotec) were applied at 1/100. All primary antibodies were revealed with a biotinylated anti-Rabbit secondary antibody and a DABMap Kit followed by counterstaining with hematoxylin (all reagents from Ventana).

All slides were analyzed using a Nikon microscope (EclipseNi-E Nikon Corp).

Quantitative analysis of chondrosarcoma immune infiltrates

Quantitative analysis of chondrosarcoma immune infiltrates was similarly conducted on SRC and human chondrosarcoma slides. Chondrosarcoma's immune cells were counted in five representative areas with highest density of infiltrates cells located at the periphery of the tumors. Sections were scored based on the intensity and the extent of the staining using the following grading: 1: when 1 to 25% of positive cells were encountered in the

T lymphocytes and macrophages in chondrosarcoma progression

To further investigate the involvement of each immune population in chondrosarcoma progression, selective depletions of T lymphocytes or macrophages were performed in SRC model.

Macrophages were targeted using either clodronate (which deplete macrophages population) or mifamurtide (which enable the conversion from pro-tumoral M2 macrophage to pro-inflammatory M1 macrophage). T cell depletion was obtained using azathioprine. In animals treated by clodronate or mifamurtide, a lower infiltration of CD163+ cells in tumor microenvironment was observed. In azathioprine treated animals, CD8+ and CD3 T cells were significantly decreased in tumor's environment (Fig 4a (31). The aim of this study was to describe the immune cell infiltrates associated with chondrosarcoma, and determine their functional role in CHS progression, both in human samples and in a syngeneic rat model recapitulating the human disease.

Immune infiltrate composed of lymphocytes and macrophages were found located in the peritumoral area both on human CHS samples and in a rat chondrosarcoma model (SRC).

A high density of CD163 + macrophages was associated with a more invasive tumor whereas a high CD8+ tumor infiltrate was associated with less aggressive tumors in both models as well. This is noteworthy, considering that the SRC tumors are isogenic in these animals, suggesting that the parameters governing infiltration are not only related to the genomic alteration of the tumor. Similar results were observed in malignant pleural mesothelioma where high amounts of CD163+ and low count of CD8+ cells were associated with local tumor outgrowth (32).

In a series of 26 samples, patients with infiltrates above the median for CD4+, CD8+ and CD3+ counts had a better overall survival and were correlated with smallest tumors. The presence of CD8, CD4, or CD3 lymphocytes at the periphery of the tumor is of good prognosis in human chondrosarcoma. Similar observations, on the role of lymphocytes population, have been reported in other cancers (16) among which melanoma (33) and osteosarcoma (23).

No significant correlation was found between CD163+ infiltration and overall survival or relapse of CHS patient and was associated with biggest tumors. In osteosarcoma, tumor associated macrophages (TAM) (both M1 and M2 subtypes) are associated with a good prognostic compared to carcinomas where TAM are often considered to be pro-tumoral and are associated with bad prognosis (34). We also observed an inverse correlation lymphocytes and macrophages to the tumor progression was demonstrated respectively by azathioprine and clodronate/mifamurtide in the SRC model.

Competing interests

All authors have declared no conflicts of interest.

Authors' contributions

FAS conceived the study, draft the manuscript and carried out every experiment.

IR and AV helped on in vivo and in vitro experimentation AVD and JPM carried out the chondrosarcoma patient's selection and evaluated IHC staining.

CC, JYB and AD participated in its design and coordination, and critically revised each draft of the manuscript.

All authors read and approved the final manuscript.