Auswahl der wissenschaftlichen Literatur zum Thema „Tumor microenvironement“

Abstract Immunotherapy has proven clinical efficacy and tremendous potential. But clinical benefit is experienced by only a subset of patients, such that additional research is necessary to improve outcomes. In particular, analyze steady-state anti-tumor immunity and monitor the effects of therapy on the tumor microenvironement. However, TIL numbers can be very low. Flow cytometry phenotyping different cell populations requires dividing limited tumor material into multiple samples, reducing the number of cells available for analysis. Therefore, innovative tools and workflows are needed to maximize the quality of data obtained from limited tumor patient samples. We established workflows combining tissue storage, dissociation, cell isolation and analysis. We developed new reagents for magnetic cell sorting, incorporating novel technology enabling the removal of magnetic beads and antibody fragments (REAlease®). Tumor-infiltrating (TI) T-, B-, Myeloid cells, cancer-associated fibroblasts and tumor cells were sequentially isolated and characterized by flow cytometry, multi-parameter imaging and transcriptomics. Importantly, purification of TI T cells with high viability was essential for high-resolution immune profiling by single cell transcriptomics. In conclusion, we optimized workflows for the comprehensive analysis of the tumor microenvironement. Sequential isolation of the different cell types maximized the number of cells available for analysis. These workflows reduce experimental time and allow the performance of more complex experimental setups. We believe the use of these innovative tools and workflows can significantly increase the quality of the data obtained in immunooncology and immunotherapy research.

Ependymoma is a central nervous system tumor associated with a poor prognosis due to limited efficacy of current medical treatment modalities, often resulting in multiple surgical re-resections with each tumor recurrence. As traditional chemotherapeutic regimens have proved unsuccessful in long-term control of subtotally resected ependymoma, other agents targeting the tumor microenvironement including the angiogenic factors supplying neovascularization have recently been used. Anti-angiogenic agents such as bevacizumab are routinely used in adult patients with recurrent glioma. Selective intra-arterial cerebral infusion (SIACI) of biological agents within tumor-supplying cerebral vasculature has recently been re-examined as a means to avoid the systemic side-effects associated with intravenous use of bevacizumab. This technical paper describes the first reported use of SIACI for delivery of two targeted biologic agents, bevacizumab and cetuximab in a pediatric patient utilizing the basilar artery to selectively administer the drugs to the tumor microenvironment. We believe this method for therapeutic delivery will both broaden treatment options and better refine treatment methodology as the multi-modality treatment approach often required to treat patients with pediatric ependymomas and other intracranial malignancies evolves.

Abstract LAG3 is an immune checkpoint expressed on a variety of immune cells including a sub-population of 'exhausted' effector T cells and TREGs. Early-phase studies of anti-LAG3 mAb show promise in solid and haematological cancers. We have previously demonstrated LAG3 is enriched within the tumor microenvironment in Hodgkin Lymphoma (Gandhi et al. Blood 2006). Data in the aggressive B-cell lymphoma DLBCL is lacking. We used a conventional discovery/ validation approach in two population based Australian cohorts (discovery: Brisbane/Canberra; validation: Sydney) totalling 250 patients treated with R-CHOP with >4 year follow-up. Digital gene expression (NanoString) using a consistent LAG3 cut-off showed inferior 5-year overall survival (OS) in both cohorts (discovery: 54% vs. 82%, HR 3.13, p=0.003; validation: 63% vs. 86%, HR 2.95, p=0.025 respectively). In a multivariate model, LAG3HI(p=0.001) was a predictor of OS independent of R-IPI and Cell-of-Origin (by NanoString LST assay). PD-L1 expression was also a predictor of survival though to a lesser degree than LAG3. Notably, LAG3 expression stratified PD-L1HIpatients into two sub-groups with differential survival, with dual LAG3 and PD-L1 positivity conferring particularly poor OS (PD-L1HI/LAG3HI39% vs. 81% PD-L1HI/LAG3LO, HR 3.65, p=0.023). Next, the discovery/validation cohorts were combined with 129 additional DLBCL cases from the ALLG biobank (in whom tissue but no outcome data was available), to test for biological associations and correlations. In these 379 cases, LAG3HIwas enriched in the ABC/UC (66%) subtype vs. LAG3LO(p=0.003). LAG3 was positively correlated with numerous immune checkpoints/effectors including CD4, CD8, PD-1, PD-L1, PD-L2, TIM-3 and CD163 (all p<0.0001, r range 0.44-0.67) consistent with an adaptive immune response. High LAG3 expression was significantly more common in EBV+ DLBCL (p=0.037). LAG3 gene expression was highly correlated with protein expression by tissue microarray based immunohistochemistry (r=0.79, p<0.001). To determine which cells expressed LAG3, de-aggregated fresh-frozen tumor-infiltrating lymphocytes were interrogated by flow cytometry. CD4+ T-cells were ~2-fold higher than CD8+ T-cells. CD4+ T-cells were further sub-divided into 'classical' CD127LOCD25HITREGs, CD127LOCD25LOinducible-TREGs, and CD4 non-TREGs. This showed that LAG3 was highly expressed within the CD8 and both TREG populations, but there was minimal CD4 non-TREG expression. LAG3HICD8+ T-cells were frequently enriched in the inhibitory checkpoints PD-1 and/or TIM3 consistent with a highly dysfunctional/exhausted phenotype. Finally, levels of soluble LAG3 (sol-LAG3) were quantified within paired plasma of patients enrolled into the ALLG NHL21 PET-adapted prospective DLBCL study. Samples taken pre-therapy and following 4 cycles of R-CHOP (at the time of interim-PET) were compared. Sol-LAG3 levels were higher in patients with DLBCL at diagnosis compared to healthy controls (p<0.0001). Interestingly patients that became interim-PET-ve had a significant drop in sol-LAG3 levels (p=0.008) between time-points, whereas no change was observed in those that remained interim-PET+, suggesting that sol-LAG3 has utility as a disease response biomarker. In conclusion, high expression of LAG3 in DLBCL is enriched in the non-GCB phenotype, and is associated with poor outcome independent of clinical and biological prognosticators. Dual PD-L1HI/LAG3HIexpression confers particularly poor outcome after conventional front-line immuno-chemotherapy. Intratumoral LAG3 expression is high on PD-1+ CD8+ and TREG subsets. Sol-LAG3 appears to a circulating disease response biomarker. The results combined indicate a key role for LAG3 within the immunobiology of DLBCL and provide a strong rationale for early phase clinical trials utilising anti-LAG3 and anti-PD1 mAb combinations. Disclosures Keane: BMS: Research Funding; Roche: Other: Education Support, Speakers Bureau; Celgene: Consultancy, Research Funding; Takeda: Other: Educational Meeting; Merck: Consultancy. Gould:Novo Nordisk: Other: Educational Travel. Abro:Novartis: Consultancy; Amgen: Other: education support congress attendance; Janssen: Other: education support congress attendance; Bristol-Myers Squibb: Speakers Bureau; Celgene: Other: education support congress attendance. Tobin:Celgene: Research Funding; Amgen: Other: Educational Travel. Birch:Medadvance: Equity Ownership. Talaulikar:Novartis: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding; Roche: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria. Bird:Amgen, Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hertzberg:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees. Gandhi:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Takeda: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Background: Primary myelofibrosis (PMF) is a clonal stem cell disorder associated with somatic mutations in three genes: Janus kinase 2 (JAK2), calreticulin (CALR) and thrombopoietin receptor (MPL). Although, our understanding of the microenvironment in PMF is limited, in PMF levels of Treg, cytotoxic T-cells, B-cells, macrophages and megakaryocyte cell populations have been reported to be elevated in either peripheral blood or bone marrow (BM) (Barosi Curr Hematol Malig Rep 2014). In addition, various cellular pathways including JAK/STAT, TGFβ1, and cytokine pathways (CXC family, hematopoietin family, PDGF family and TGF family), have been reported to play an important role in the dysregulation of hematopoietic cell proliferation and disease progression. Here-in we characterize the tumor microenvironment in formalin fixed paraffin embedded (FFPE) BM biopsies obtained from PMF patients and correlate these findings with mutational status. Methods: We applied the enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) to identify and assess immunological function in the microenvironement of archival FFPE bone marrow samples from patients with PMF. Twelve archival bone marrow FFPE biopsies from PMF patients along with clinical information and 5 normal controls were analyzed using upto 500ng of RNA (at 100ng/ul) for digital expression profiling. The panel included 109 genes that define 24 immune cell types and populations and forty housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 3.0 and the Advanced Analysis Module (v.1.0.84). Results: Gene expression profiles for cellular immune pathways were analyzed for global changes based mutation. Globally, cellular functions involving immune cell development and cellular responses/functions were dramatically decreased in myelofibrotic marrow (chemokines, complement, cytokines, cytotoxicity) when compared to normal marrow. However, only in areas of adhesion, antigen processing, transporter function and senescence genes were transcription levels elevated over normal controls. Differential expression analysis of JAK2V617F+ marrow showed decreased expression of genes involved in cell regulation, NK cell function, T-cell functions and pathogen defense and increased expression of genes involved in inflammation, chemokines and transporter functions over normal marrow. Whereas CALR+ bone marrow biopsies showed fewer genes down regulated and an increased number of genes up regulated, particularly involved in fibrosis, inflammation, chemokines, adhesion, antigen processing and regulation. Pathway analysis suggested a particular role for FLT3 ligand in myeloid stem cell regulation, thrombospondin (THBS1) which has been reported to promote the activation of the latent forms of TGFβ1, and mitogen-activated protein kinases (JNK1, ERK) in PMF cell proliferation and differentiation. Conclusions: Digital immune expression profiling reveals a distinct PMF tumor microenvironment and illustrates potential transcriptional differences based on their mutational status. (JAK2+ or CALR+). These transcriptional changes in myelofibrotic marrow are reflected in global changes in immune cells and pathway activation These data provide for the first time in situ evidence of the importance of the immune system in PMF pathogenesis. Barosi G, 2014 An immune dysregulation in MPN. Curr Hematol Malig Rep 9:331-339. Disclosures No relevant conflicts of interest to declare.

2019 Background: Gliomagenesis is regulated by dynamic epigenetic modifications of DNA methylation, deregulation of histones and alteration of the human Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes. These epigenetic genes are responsible for treatment resistance by inducing stemness of glioma cells and immune cells with in the tumor microenvironement (TME). We evaluated the key chromatin remodeling (CR) genes and their interactions with other regulatory genes that are of prognostic importance. Methods: 1856 HGGs underwent molecular profiling at Caris Life Sciences (Phoenix, AZ). Analyses included next-generation sequencing of DNA (592 Genes, NextSeq or WES, NovaSeq) and RNA (WTS, NovaSeq). Cell infiltration in the TME was estimated by quanTIseq. X2/Fisher’s-exact/Mann-Whitney U tests were used for comparison, and significance was determined as p-value adjusted for multiple comparison by the Benjamini-Hochberg method (q < 0.05). Overall survival (OS) was calculated from the start of temozolomide (TMZ) to last contact using insurance claims data. Results: In a cohort of 1856 HGGs, 181 (9.8%) displayed >=1 mutation of 19 CR genes considered, including mutations (mt) of histone methyltransferases (HM) comprising SETD2 (62, 3.4%), KMT2D (18, 1.0%), KMT2C (11, 0.6%); SWI/SNF complexes (SSNF) including ARID1A (32, 1.74%), ARID2 (15, 0.82%), SMARCA4 (14, 0.76%) and ARID1B (12, 0.66%); and others including ( DNMT3A, 0.94%, ASLXL1: 13, 0.98%). When compared to CR-WT, CR-mt HGGs showed higher prevalence of Tumor Mutational Burden-High (TMB-H) (23% vs. 1.3%), MSI-H/dMMR (13% vs. 0.2%), gLOH (9.5% vs 4.3%), and mts in IDH1/2 (29% vs. 14%), TP53 (55% vs. 36%), MSH6 (8.8% vs. 0.6%), and PIK3CA (18.8% vs. 8.3%) (all q<0.05). Investigation of CR-mt subgroups showed that SSNF mt had the highest MGMT-promoter methylation (68%) and IDH1/2 mt (46%), while HM and others showed similar prevalence. In IDH-WT and MSS HGGs, the CR association with TMB-H, MSH6, TP53 and PIK3CA persisted (q<0.05). When studying the immune profile, CR-mt HGGs showed significantly lower expression of immune-related genes including PD-L1 (Fold change: 0.76), PD-L2 (0.72), IDO1 (0.65), TIM3 (0.76) and CD86 (0.77) and colder TME as manifested by lower infiltrations of M2 (0.87) and higher Treg (1.27, all q<0.05); such effects were not observed in the subgroup of IDH WT/MSS tumors. Among TMZ-treated HGG tumors, CR mt was associated with improved OS (33 months vs 22m, HR: 0.713, 95% CI: 0.581-0.876, p =.001). In the IDH WT/MSS subgroup this effect was also observed (31.6m vs 19.2m, HR: 0.764, 95% CI: 0.59-0.99, p = 0.041). Conclusions: Nearly 10% of HGGs carry mts in CR genes. CR-mt HGGs possess significantly more favorable genetic alterations and colder TME compared to the CR-WT HGGs and showed better OS when treated with TMZ. Multivariate modeling and analysis of associations with specific targeted therapies is underway.

Abstract Current chemotherapy of pediatric T cell acute lymphoblastic leukemia (T-ALL) efficiently reduces the tumor mass with, however, undesirable long term consequences and remains ineffective in adolescent and adult T-ALL. Furthermore, relapse can be caused by pre-leukemic stem cells (pre-LSCs) that were spared by current protocols and evolved to malignancy. A distinctive characteristic of pre-LSCs is their critical dependence on interactions with the microenvironment for survival, which guided our strategy to target pre-LSCs using niche-based screening assays. Using transgenic mouse models that closely reproduce the human disease, we showed that the SCL/TAL1 and LMO1 oncogenic transcription factors establish a pre-leukemic state by reprogramming normal pro-T cells into aberrantly self-renewing pre-LSCs (Gerby et al. PloS Genetics, 2014). We now provide direct evidence that pre-LSCs are much less chemosensitive than leukemic blasts to current drugs, due to a distinctive lower proliferative state as assessed by real-time imaging in a competitive assay. We therefore designed a robust protocol for high-throughput screening (HTS) of compounds targeting primary pre-LSCs that are maintained on stromal cells engineered for optimal NOTCH1 activation to mimick the thymic microenvironement. The multiparametric readout takes into account the intrinsic complexity of primary cells to specifically monitor pre-LSCs. We screened a targeted library of 1904 compounds and identified UM0119979 that disrupts both cell autonomous and non-cell autonomous pathways: UM0119979 abrogates pre-LSC viability and self-renewal activity in vivo by specifically inhibiting the translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remain functional. Moreover, in vivo administration of UM0119979 efficiently reduced the leukemia propagating activity of primary human T-ALL samples in xenografted mice. Finally, in addition to SCL-LMO-induced T-ALL, our results reveal a novel possibility of therapeutic intervention in MYC-dependent hematologic malignancies. In summary, our screening assay, built on the genetic dependencies of pre-LSCs, revealed their vulnerabilities to compounds that inhibit both the primary oncogenes and non-cell autonomous pathways triggered by the microenvironment. The results illustrate how recapitulating tissue-like properties of primary cells in high throughput screening is a promising avenue for innovation in cancer chemotherapy. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 1601 Background: The prognosis of follicular lymphoma (FL) remains a challenge despite stratification on the FLIPI scoring, histological grading, and microenvironement cell type infiltration. Thus, new biomarkers are needed to identify patients (pts) who require a more aggressive therapeutic strategy or targeted therapies. Transferrin receptor (TfR) is highly expressed in proliferating cells including non Hodgkin lymphoma cells. High TfR level has been associated with disease progression in chronic lymphocytic leukaemia and is observed at relapse in mantle cell lymphoma. Murine anti human TfR (CD71) antibody has been validated for histological and flow cytometric evaluation of TfR expression. The aim of this work was to assess expression and prognosis value of TfR expression on FL cells. Material and Methods: We retrospectively analyzed the expression level of CD71 in neoplastic B cells of FL lymph node biopsies at diagnosis of 45 pts who received first-line treatment with Rituximab and anthracyclines based chemotherapy at the hematology department of Necker Hospital, Paris, France, and assessed its prognosis value (second treatment free survival (TFS) and overall survival (OS)). High CD71 and Ki67 expression in FL lymph nodes biopsies were defined as upper than 25% of FL cells. CD71 expression was compared with FLIPI at diagnosis and the expression and prognosis value of Ki67 expression. Results: Characteristics of the cohort are shown in table 1. FLIPI risk was low in 7 pts (16%), intermediate in 19 (43%) and high in 18 (41%). FLIPI stratification did not show correlation with both OS and TFS in this small cohort of pts. Among 44 evaluable pts, CD71 expression was heterogeneous ranging from 0% to 90% of FL cells. T cell labeling was used as a quality control. CD71 low expression (225% of tumor cells) was observed in 31 samples (69%) and KI67 low expression in 27 (60%). After a median follow-up of 40 months (range 1 – 94) for the all cohort, TFS was 68.9 % (95 % CI 0.55–0.82) and OS was 91.1% (95% CI 0.83 – 0.99). The mean TFS after R-chemotherapy was 86 months in the low CD71 level group vs. 18 months in the high CD71 level group (p=0,075). The mean estimated OS after first R-chemotherapy was 89 months in the low CD71 group vs. 54 months in the high CD71 group (p=0,006) (figure 1). By univariate analysis, high Ki67 expression was associated with significant lower OS (p=0.035) but not TFS (p=0,162). In multivariate analysis including CD71 level, FLIPI risk, FLIPI parameter groups, CD71 was the only factor independently associated with OS (p=0,019) and TFS (p=0,037). Furthermore, Cox regression analysis did not reveal any significant correlation between Ki67 and TFS or survival. Conclusion: CD71 expression is an important independent histological prognostic factor in this monocentric retrospective study predicting OS in FL patients after first-line treatment with R anthracyclin based regimen. Ki 67 was also associated with OS in univariate analysis, but CD71 was the only independent factor associated with OS and TFS in multivariate analysis. These promising results warrant further studies in a larger prospective cohort and provide a rationale to target Tfr in relapsing refractory FL. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Gene expression profiling studies have demonstrated aberrant expression of inflammatory response genes in myeloproliferative neoplasm (MPN) granulocytes and/or CD34+ cells. Our understanding of the immune response to primary myelofibrosis (PMF) hematopoietic stem cells and tissue-specific microenvironments is not complete due to a limited availability of bone marrow (BM) aspirates and fresh spleen samples from PMF patients. In order to overcome this obstacle, we utilized a novel approach with mRNA enrichment analysis which utilizes formalin fixed, paraffin embedded (FFPE) specimens of BM and spleen from PMF patients to identify immune and other microenvironment cell types and to construct pathway activation patterns. Methods: We applied enzyme-free NanoString nCounter® PanCancer Immune Profiling Panel system (NanoString Technologies, Inc., Seattle, WA) consisting of 770 standard gene panel and 20 custom gene panel for identification of immune cells and assessing immunological milieus in the microenvironement of matched, archival FFPE spleen and BM samples from MPN patients.. Up to 500ng of RNA (at 100ng/ul) isolated from FFPE BM and/or spleen specimens from PMF patients was used for digital expression profiling in accordance with the manufacturer's protocol. The panel included 109 genes that define 24 immune cell types and populations, and 40 housekeeping genes that facilitate sample-to-sample normalization. Data analysis was performed using nSolver software 2.5 and the PanCancer Immune Profiling Advanced Analysis Module (v.1.0.22). Findings identified from the digital expression profiles on cells types were confirmed via immunohistochemical evaluation. Results: Twenty-six archival FFPE tissue samples (13 BM and 13 spleen) obtained from PMF patients who had undergone therapeutic splenectomy and BM biopsy at the same time, and normal tissue controls, were analyzed as described previously (Liew et al 2015). Following data normalization, genes were selected based on P < 0.05 (unpaired t -test) and fold change > 2.0 differentially expressed mRNA levels in the BM (n=208) and spleen (n=108). These genes were distributed across several functional categories including: TNF superfamily (e.g. TNFRS13C, CD70, LTB), signal ligands (cytokine, chemokine) (e.g. JAK3, IFI16, SPP1), B and T cell functions (TIGIT, CXCR5, CXCL14), and cell adhesion (e.g. ITGB3). In supervised clustering of the significantly expressed genes, the first bifurcation of the dendrogram separated controls from PMF samples in both BM and spleen. Twenty-seven genes were significantly differentially expressed by both PMF BM and spleen, compared to control specimens. Interestingly, the PMF BM samples were further separated in a second bifurcation of the dendrogram into 3 subgroups, indicating immune transcriptional diversity within PMF samples (Figure 1). Further characterization of these subgroups and potential clinical relevance are being studied in a larger number of specimens in order to achieve statistical power. Cell type analysis indicated a significant (P =<0.05) difference in activated CD4 T-cells, T helper-1 cells, CD8 T-cells, and B-cells across all BM and spleen samples. Macrophages (P =<0.001) were increased in the spleen, and neutrophils (P =0.01) were increased only in BM samples. A decrease in CD8-positive T-cells in PMF samples (p =0.009) was confirmed using immunohistochemistry with computer assisted image analysis. Heterogeneity of Tregs in PMF spleen samples (n=10) was further confirmed by immunohistochemistry (n-3). Conclusions: Digital immune expression profiling coupled with immunohistochemistry is a novel approach for characterization of tumor microenvironment in fibrotic PMF marrow and spleen. Our preliminary findings indicate a consistent decrease in cytotoxic CD8 T-cells but varying expression of Tregs. In addition, we identified several genes in various immune functional categories within PMF patients that could potentially serve as therapy targets. Disclosures Hoffman: All Cells, LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding; Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees. Salama:Promedior: Consultancy.

Les cancers du poumon sont responsables de plus de 1,8 million de décès dans le monde chaque année, dont le cancer bronchique non à petites cellules (CBNPC) représentant 85% des cas. Aujourd'hui, l'immunothérapie est le traitement majeur du CBNPC et vise à inverser l'anergie des LTs intra-tumoraux et restaurer leur action antitumorale. Malgré leur efficacité, seulement 15 à 20% des patients sont répondeurs et de fait, une meilleure compréhension du micro- environnement tumoral (TME) est requise. Le compartiment vasculaire est une composante majeure du TME et favorise la croissance tumorale par l'angiogenèse, et en module l'infiltration du système immunitaire. Cependant, la capacité immunorégulatrice des cellules endothéliales (ECs) reste largement sous- étudiée. Dans cette étude nous avons cherché à caractériser les ECs issues de biopsies et de modèles d’études in vitro alternatifs. Ces derniers visent à mimer les communications entre les cellules de CBNPC et des ECs. Notre hypothèse de travail était de déterminer si l’endothélium associé à la tumeur joue un rôle dans l’anergie ou l’activation des cellules immunitaires. Nos résultats montrent que les cocultures peuvent affecter la façon dont les ECs régulent l’inflammation et l’activité des LTs à travers la sécrétion de cytokines et l’expression de récepteurs de costimulation. En effet, les ECs ayant été conditionnées par les cellules tumorales diminuent la prolifération des LT CD4+ et participent à la mise en place d’un environnement immunosuppresseur en promouvant les LTs régulateurs et la polarisation des macrophages vers un phénotype pro-tumoral M2-like. En conclusion, cette étude souligne l’importance du compartiment vasculaire dans l’immunomodulation du TME et l’importance de le prendre en considération pour l’optimisation de traitements dans le CBNPC. Les cancers du poumon sont responsables de plus de 1,8 million de décès dans le monde chaque année, dont le cancer bronchique non à petites cellules (CBNPC) représentant 85% des cas. Aujourd'hui, l'immunothérapie est le traitement majeur du CBNPC et vise à inverser l'anergie des LTs intra-tumoraux et restaurer leur action antitumorale. Malgré leur efficacité, seulement 15 à 20% des patients sont répondeurs et de fait, une meilleure compréhension du micro- environnement tumoral (TME) est requise. Le compartiment vasculaire est une composante majeure du TME et favorise la croissance tumorale par l'angiogenèse, et en module l'infiltration du système immunitaire. Cependant, la capacité immunorégulatrice des cellules endothéliales (ECs) reste largement sous- étudiée. Dans cette étude nous avons cherché à caractériser les ECs issues de biopsies et de modèles d’études in vitro alternatifs. Ces derniers visent à mimer les communications entre les cellules de CBNPC et des ECs. Notre hypothèse de travail était de déterminer si l’endothélium associé à la tumeur joue un rôle dans l’anergie ou l’activation des cellules immunitaires. Nos résultats montrent que les cocultures peuvent affecter la façon dont les ECs régulent l’inflammation et l’activité des LTs à travers la sécrétion de cytokines et l’expression de récepteurs de costimulation. En effet, les ECs ayant été conditionnées par les cellules tumorales diminuent la prolifération des LT CD4+ et participent à la mise en place d’un environnement immunosuppresseur en promouvant les LTs régulateurs et la polarisation des macrophages vers un phénotype pro-tumoral M2-like. En conclusion, cette étude souligne l’importance du compartiment vasculaire dans l’immunomodulation du TME et l’importance de le prendre en considération pour l’optimisation de traitements dans le CBNPC
Lung cancer is responsible for more than 1.8 million deaths worldwide each year, with non-small cell lung cancer (NSCLC) accounting for 85% of cases. Today, immunotherapy is the leading treatment for NSCLC, aiming to reverse the anergy of intra- tumoral LTs and restore their anti-tumor activity. Despite their efficacy, only 15-20% of patients respond, thus a better understanding of the tumor microenvironment (TME) is needed. The vascular compartment is an important component of the TME. It promotes tumor growth through angiogenesis and modulates immune infiltration. However, immune regulatory capacity of endothelial cells (ECs) remains largely unstudied. In this study, we characterized ECs from biopsies and alternative in vitro models. These are designed to mimic the communication between NSCLC cells and ECs. Our working hypothesis was to determine whether tumor-associated endothelium plays a role in immune cell anergy or activation. Our results show that cocultures can influence the way ECs regulate inflammation and LT activity through cytokine secretion and costimulatory receptor expression. Indeed, ECs conditioned by tumor cells decrease CD4+ LT proliferation and participate in the establishment of an immunosuppressive environment by promoting regulatory LTs and macrophage polarization toward a pro-tumor M2-like phenotype. In conclusion, this study highlights the importance of the vascular compartment in the immunomodulation of TME and the need to take it into account when optimizing treatments for NSCLC

Le microenvironnement tumoral, et notamment le stress hypoxique, joue un rôle immunosuppressif permettant l’échappement des cellules tumorales à la surveillance du système immunitaire. Des études récentes ont montré que l’échange de microvésicules (MVs) entre les cellules tumorales et les cellules du système immunitaire peut être responsable de l’établissement d’un microenvironnement immunosuppressif. Dans ce contexte, nous avons étudié l’effet des MVs issues des cellules tumorales hypoxiques sur la cytotoxicité des cellules «Natural Killer» (NKs). Nos résultats démontrent clairement que les cellules NKs sont capables d’internaliser les MVs issues des cellules tumorales normoxiques et hypoxiques. Cependant, seules les MVs hypoxiques sont capables de diminuer significativement la cytotoxicité des cellules NKs. Ainsi, nous avons déterminé que les MVs dérivées des cellules tumorales hypoxiques séquestrent deux immunomodulateurs, le TGF- et le miR-23a. Nous avons montré que le transfert de TGF- et miR-23a aux cellules NKs était responsable de la diminution respective de l’expression du récepteur activateur NKG2D à leur surface et de la protéine membranaire associée aux lysosomes (LAMP-1/CD107a) impliquée dans la dégranulation des granules cytotoxiques. Dans la deuxième partie de cette étude nous avons montré que les cellules tumorales soumises à un stress hypoxique étaient capables de déjouer un système immunitaire fonctionnel et d’échapper ainsi à la surveillance immunitaire des cellules NKs. En effet, nos résultats ont clairement démontré que la résistance des cellules tumorales hypoxiques à la lyse par les cellules NKs n’était pas liée à un défaut de reconnaissance, mais plutôt à l’activation d’un mécanisme de résistance intrinsèque dans les cellules tumorales. Ce mécanisme de résistance implique l’activation de l’autophagie qui opère dans les cellules tumorales pour dégrader le granzyme B, une protéase à sérine secrétée par les cellules NKs dont l’internalisation par les cellules tumorales cibles est nécessaire pour induire leur mort. Les expériences d’imagerie cellulaire combinées à des approches biochimiques ont confirmé que le niveau de granzyme B dans les cellules tumorales hypoxiques était significativement mois élevé par rapport à celui des cellules tumorales normoxiques. Ces résultats suggèrent fortement que le granzyme B est destiné à être dégradé par autophagie dans les cellules tumorales hypoxiques. En effet, l’inhibition génétique et pharmacologique de l’autophagie dans les cellules tumorales hypoxiques était suffisante pour contrecarrer la dégradation de granzyme B et ainsi restaurer la sensibilité des cellules tumorales hypoxiques à la lyse par les cellules NKs. Nos résultats ont clairement établi que l’inhibition de l’autophagie pouvait améliorer la réponse immunitaire antitumorale dépendante des cellules NK. Nous avons validé ce concept in vivo chez la souris en utilisant deux modèles syngéniques de cancer du sein et de mélanome. L’ensemble de nos travaux indiquent clairement que le stress hypoxique, qui est une caractéristique majeure du microenvironnement tumoral, peut favoriser l’établissement d’un microenvironnement immunosuppressif par plusieurs mécanismes qui ne s’excluent pas mutuellement. En effet, le stress hypoxique modifie les caractéristiques des cellules tumorales et active des mécanismes de résistance à la surveillance immunitaire. De plus, les cellules tumorales modifiées peuvent éduquer et exporter leur phénotype hypoxique aux cellules immunitaires présentes dans le microenvironnement afin d’affaiblir leur pouvoir cytotoxique. Nos résultats ouvrent ainsi la voie à la mise en place de nouvelles applications cliniques en immunothérapie anticancéreuse basées sur la réactivation des lymphocytes cytotoxiques et l’inhibition simultanée de l’autophagie
The tumor microenvironment, including hypoxic stress plays an immunosuppressive role in tumor cell escape from immune surveillance. Recent studies have shown that the exchange of microvesicles (MVs) between tumor cells and cells of the immune system could be responsible for the establishment of an immunosuppressive microenvironment. In this context, we investigated the effect of MVs derived from hypoxic tumor cells on the cytotoxicity of Natural Killer (NK) cells. Our results clearly demonstrated that NK cells are able to internalize MVs derived from both normoxic and hypoxic tumor cells. However, only hypoxic MVs are able to significantly reduce the cytotoxicity of NK cells. Thus, we revealed that MVs derived from hypoxic tumor cells sequester two immunomodulators, TGF- and miR-23a. We have shown that the transfer of TGF- and miR-23a to NK cells was responsible for the respective reduction of the expression of NKG2D activating receptor on their surface and lysosomal-associated membrane protein (LAMP-1 / CD107a) involved in degranulation of cytotoxic granules.In the second part of this thesis we have shown that tumor cells subjected to hypoxic stress were able to outmaneuver a functional immune system and thus escape NK-mediated immune surveillance. Indeed, our results clearly demonstrated that the resistance of hypoxic tumor cells to NK-mediated lysis was not related to the impairment of recognition by NK cells, but rather to the activation of an intrinsic resistance mechanism in tumor cells. We showed that the resistance mechanism involves the activation of the autophagy which operates in the tumor cells to degrade the granzyme B, a serine protease secreted by NK cells and internalized by target tumor cells to induce cell death. Cell imaging experiments combined to biochemical approaches have confirmed that the level of granzyme B in hypoxic tumor cells was significantly higher compared to normoxic tumor cells. The analysis of the subcellular distribution of granzyme B reveals that it is predominantly present in the endosomes and autophagosomes of hypoxic tumor cells. These results strongly suggest that granzyme B is subjected to be degraded by autophagy in hypoxic tumor cells. Genetic and pharmacological inhibition of autophagy in hypoxic tumor cells was sufficient to block the degradation of granzyme B and thus restore the sensitivity of hypoxic tumor cells to NK-mediated lysis. Our results clearly demonstrated that inhibition of autophagy could improve NK-mediated antitumor immune response. We validated this concept in vivo using two syngeneic mice model of breast cancer and melanoma.Taken together, our work clearly shows that hypoxic stress, which is a major feature of the tumor microenvironment, can promote the establishment of an immunosuppressive microenvironment by several mechanisms which are not mutually exclusive. Thus, hypoxic stress changes the characteristics of tumor cells and activates the mechanisms of resistance to immune surveillance. In addition, tumor cells can educate and export their hypoxic phenotype to the immune cells in the microenvironment in order to impair their cytotoxicity. Our findings pave the way for the development of new clinical applications in cancer immunotherapy based on the reactivation of cytotoxic lymphocytes and simultaneous inhibition of autophagy

Les LT γδ contribuent à l'immunité anti-tumorale au sein du microenvironnement tumoral dans divers cancers. Malgré leurs fonctions effectrices bien décrites, de récentes études ont corrélé leur présence dans le microenvironnement tumoral à une meilleure progression des tumeurs solides, suggérant que les LT γδ peuvent présenter des activités pro-tumorales. Mon projet visait à caractériser ces LT γδ présentant des activités tumorales et à déchiffrer leur rôle dans le cancer. Nous avons démontré in vitro que les signaux inflammatoires favorisent le développement d'une sous-population de LT γδ régulateurs caractérisée par l'expression de CD73 et affichant des fonctions immunosuppressives par la production de molécules immunosuppressives telles que l'IL-10, l'adénosine et le facteur angiogénique et chimiotactique IL-8. Le défi associé à la caractérisation des LT γδ CD73+ réside dans l'évaluation de leur existence in vivo ainsi que de leur pertinence dans les cancers humains. Nous avons montré dans le cancer du sein que ~20% des lymphocytes γδ infiltrant la tumeur (γδ TILs) expriment CD73 et présentent les mêmes fonctions immunosuppressives que celles décrites in vitro, suggérant qu'ils pourraient favoriser le développement tumoral via ces mécanismes. En accord avec ces observations, nous avons montré que la présence de γδ TILs est associée à des grades tumoraux tardifs dans le cancer du sein. Nous avons étendu ces observations au cancer ovarien et montré que la densité des CD73+ γδ TILs est négativement corrélée à la survie des patients, suggérant que la densité des γδ TILs CD73+ pourrait être utilisée comme facteur pronostic en clinique. En utilisant l'imagerie par cytométrie de masse, nous avons étudié les réseaux cellulaires des γδ TILs régulateurs (CD73+) et de leurs homologues effecteurs (CD73-) dans les tumeurs d’ovaire afin de mieux comprendre leur rôle dans le cancer. Nos données montrent des écosystèmes immédiats différents pour les γδ TILs CD73+ par rapport aux CD73 renforçant l'idée que les LT γδ CD73+ et CD73- sont fonctionnellement différents.Dans l'ensemble, ces données améliorent nos connaissances sur l'immunobiologie des LT γδ dans le développement du cancer chez l’Homme, avec notamment la caractérisation approfondie du ce nouvel sous-ensemble de LT γδ régulateurs, leur localisation et leurs fonctions au sein du microenvironnement tumoral
Γδ T cells contribute to the anti-tumor immunity within the tumor microenvironment in various cancers. Despite their well-described effector functions, recent studies correlated their presence in the tumor microenvironment with solid tumor progression suggesting that γδ T cells may display pro-tumor activities. My project aimed to characterize those regulatory γδ T cells and decipher their role in cancer.We demonstrated in vitro that inflammatory signals promote the development of a regulatory γδ T cell sub-population characterized by the expression of CD73 and displaying immunosuppressive functions through the production of immunosuppressive molecules such as IL-10, adenosine and the angiogenic and chemotactic factor IL-8. The challenge associated with the characterization of CD73+ γδ T cells resides in assessing their existence in vivo as well as their relevance in human cancers. We showed in human breast cancer that ~20% of tumor infiltrating γδ T lymphocytes (TILs) expressed CD73 and displayed the same immunosuppressive functions as described in vitro, suggesting that they could promote tumor development via these mechanisms. In line with these observations, we showed that the presence of γδ TILs is associated with late tumor grades in breast cancer. We extended such observations to ovarian cancer and showed that the density of CD73+ γδ TILs negatively correlates with patient survival, suggesting that CD73+ γδ TILs density could be used as a prognosis factor. Using Imaging by Mass Cytometry, we investigated the cellular networks of regulatory γδ TILs (CD73+) and their effector counterpart (CD73-) in breast and ovarian tumors to better understand their role in cancer. Our data show different ecosystems for CD73+ compared to CD73- γδ TILs reinforcing the idea that CD73+ and CD73- γδ T cells are functionally different.Altogether, these data improve our knowledge on human γδ T cell biology during cancer development, with the in-depth characterization of the new regulatory γδ T cell subset, their localization and their functions within the tumor microenvironment