UNCOVERING IMMUNE EVASION MECHANISMS FOR THE PROGNOSIS AND THERAPY OF ENDOMETRIAL AND OVARIAN CANCERS

Gynecologic malignancies are a heterogeneous group of solid tumors which include endometrial cancer (EC) and ovarian cancer (OC). EC is the most common gynecological neoplasm and the only gynecologic cancer with increasing incidence and mortality. It is generally diagnosed at an early stage and is well managed with surgery, radiation, and/or chemotherapy. However, EC patients with advanced/recurrent disease have poor outcomes and respond poorly to current management treatments. On the other hand, OC is a devastating disease since the majority of patients, when first diagnosed, are at advanced clinical stages (III/IV). Furthermore, relapse occurs in more than 70% of patients and the 5-year survival rate is lower than 40%. Thus, it is important the development of novel therapeutic approaches. In this context, remarkable progresses have been made in immunotherapy. In fact, several preclinical and clinical studies are ongoing with immunotherapeutic strategies combined or not with classic treatments. To date, the efficacy is not satisfactory, thus a detailed understanding of the biology and interactions between cancer and the immune system is essential for the recognition and development of potential new immunotherapeutic strategies and targets as well as the optimization of existing immunotherapies. Therefore, the aim of this thesis was to characterize the molecular profile, frequency, phenotype and function of immune cells in EC and OC patients. In particular, considering that the presence of activated tissue-resident gamma delta (γδ) T cells, recognized for their favorable response in various human malignancies, has been associated with a beneficial response to immunotherapies, and that their role in EC progression has not been elucidated yet, we performed a characterization of γδ T cells in EC. Flow cytometry analysis reveals that Vδ1 is the primary subset of γδ T cells infiltrating EC lesions in both early and advanced stages. Upon in vitro stimulation, these cells maintain their functional activity by producing cytokines, such as interferon (IFN)-γ and tumor necrosis factor (TNF)-α. However, within the tumor, they exhibit increased expression of PD-1, compared to their blood-matched circulating counterparts. Single-cell RNA-sequencing (scRNA-seq) analysis confirmed the upregulation of PD-1 expression in γδ T cells, exhibiting a progressive increase from peritumor to tumor specimens, in contrast to healthy tissue. Crucially, tumor-infiltrating Vδ1 T cells expressing PD-1 retain their immune competence, which can be enhanced by PD-1 blockade. Indeed, their cytotoxic response against autologous tumor cell targets significantly increases in the presence of the specific blocking anti-PD-1 monoclonal antibody compared to controls. Moreover, scRNA-seq analysis associated the presence and the specific activation of Vδ1 T cells with the response to anti-PD-1 therapy. These data not only emphasize the specific anti-tumor response of Vδ1 T cells in EC but also indicate the feasibility of targeting γδ T cells to enhance current immunotherapeutic strategies. As few studies have been conducted on the early stages (stage I) of OC due to the difficulty in diagnosing this disease, we performed a deep characterization through scRNA-seq of stage I high-grade serous ovarian cancer (HGSOC) tumor microenvironment (TME), the most common subtype of OC, to assess the impact of the TME in OC progression and to identify potential immunotherapeutic targets. Within the HGSOC lesions we detected high enrichment of CD4 regulatory T cells (Tregs) that displayed a spectrum of states linked to their naïve, effector, proliferating and destabilized transcriptomic profiles. The presence of FOXP3+ Tregs associates with a permissive, immune-suppressive TME, characterized by the prevalence of CD4 Th2 cells, exhaustion of cytotoxic CD8 T cells, and pro-tumoral states of natural killer (NK) cells and tumor-associated macrophages (TAMs). Cell-to-cell communication analysis predicted multiple molecular mechanisms underlying Treg inhibition of T and myeloid immune responses, along with the establishment of reciprocal interactions between Tregs and tumor cells, promoting tumor progression. Trajectory analysis revealed two differentiation paths for Tregs, both leading to immunosuppressive Treg profiles. While the FOXP3high profile converges with proliferating Tregs, the second path of cytotoxic FOXP3+ Tregs converged with FOXP3-KLRB1+ ex-Tregs, distinguished for their anti-tumoral CXCL13+IFNG+ transcriptomic profile. These findings highlight the pivotal role of Tregs in the early establishment of the immunosuppressive TME conducive to HGSOC, also highlighting the potential of manipulating Treg cell fate and their de-stabilization as a promising target for therapeutic interventions.