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Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. We found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of SYK and ERK signaling, (i) acts as a chemotactic factor for myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Moreover, in an immune ‘hot’ tumor model, EGFL6 completely inhibited response to a-PD-L-1 therapy. Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients. Supported by Ovarian Cancer Research Alliance

Objective: We explored mechanisms of PD-L1 upregulation in ovarian cancer cells and tested in vivo the efficacy of intraperitoneal anti-PD-L1 therapy in two different ovarian cancer mouse models. Methods: We have derived several novel murine ovarian cancer cell lines from orthotopic ovarian tumors of triple transgenic mice. 129x SvJ and C57Bl/6 tumor-bearing mice were treated with 200 μg anti-PD-L1 antibody (or rat IgG as control) every two weeks for three doses. Immune gene profiling of splenocytes collected at necropsy was performed using 561 Nanostring probes. Results: Cell cycle analysis of in vitro cultured tumor cells demonstrates that PD-L1 expression increases preferentially on cells in G2/M cells. Exposure to cisplatin modifies PD-L1 levels, although the effect varies according to tumors’ baseline susceptibility. In vivo, we observed murine PD-L1 upregulation on ascites resident tumor cells, an effect reproduced ex vivo upon exposure of tumor cells to ascites fluid. PD-L1 blockade triggers increase in survival in both preclinical models with immune gene signatures pointing to cytotoxic anti-tumor immune response. Conclusion: PD-L1 expression increases in response to inflammatory stimuli from cell-free ascites and shows preference for dividing cells. PD-L1 upregulation varies according to tumor cells’ susceptibility to cisplatin. PD-L1 blockade increases systemic T cell responses and intratumoral T cell accumulation suggesting therapeutic potential in ovarian cancer.

MUC1 is a large cell surface glycoprotein overexpressed by almost all types of adenocarcinomas, including those originating in the ovaries. We recently generated a triple transgenic MKP mouse model that carry conditional Kras mutations, Pten deletion and express human MUC1 as self. Upon AdCre injection under the ovarian bursa, the MKP mice progress to endometrioid ovarian tumors. Using a freshly isolated, orthotopic ovarian tumor from a MKP mouse, we generated an ovarian cancer cell line MKP-T-2F8 (or simply 2F8) that triggers multiple MUC1 expressing intraperitoneal (IP) tumors. At baseline, the cells express low PD-L1 but expression increases after in vivo growth. Using the 2F8 transplantable tumor model, we tested in vivo the therapeutic effectiveness of anti-PD-L1 antibody (10F.9G2). MUC1.Tg or wild type mice were IP injected with 8x10E5 of 2F8 cells, followed by three doses of 200ug of anti-PD-L1 (or isotype control) antibody, administered IP every 2 weeks, starting 3 weeks post-tumor challenge. The anti-PD-L1 treated MUC1.Tg mice showed significantly increased survival (p= 0.02), with no evidence for peritoneal tumor. In contrast, wild type mice which grow 2F8 tumors and develop spontaneous anti-MUC1 antibodies in high titers, did not respond to anti-PD-L1 therapy. These results demonstrate that targeting PD-L1 is a viable therapeutic strategy in ovarian cancer; however, tumor-bearing hosts with Th2-driven immune imbalance may not benefit from PD-L1 blockade.

Endometriosis is a largely benign, chronic inflammatory disease that may predispose to some of the ovarian tumors histotypes, collectively called endometriosis-associated ovarian cancers (EAOC). Although it is widely accepted that chronic inflammation drives cancer, the immune deregulation in chronic precursors of ovarian cancer has not been systematically studied. We performed here the first comprehensive gene profile of the tissue immune microenvironment in normal endometrium, benign endometriosis and EAOC (n=133), using Nanostring and the nCounter® GX Human Immunology Kit comprising probes for 511 immune genes. We found that the complement pathway was most prominently upregulated, suggesting its involvement in the transition from chronic endometriosis to atypical (premalignant) endometriosis and to EAOC. Complement protein expression was confirmed via immunohistochemistry, which identified upregulation of complement in epithelial cells. To explore the roles of complement in early EAOC carcinogenesis, we derived several novel ovarian cell lines with conditional mutations that can be turned on and off, using the Cre-loxP system. Using this in vitro model we demonstrate that genetic instability triggers upregulation of complement genes in epithelial cells, but without an advantage on cell death. These findings reveal an early link between epithelium and innate immune environment and further support a paradigm shift on complement roles in supporting cancer growth.