Mechanisms for immune escape in epithelial ovarian cancer

Sammanfattning: Tumors develop mechanisms to subvert the immune system, constituting immune escape. Epithelial ovarian cancer (EOC), the deadliest of all gynecological malignancies, uses a variety of mechanisms to undermine immune surveillance, aiding its establishment and metastatic spreading. Despite progress in oncoimmunology, a lot remains unknown about the cancer-immune system interplay. The aim of this thesis was to study tumor-mediated mechanisms for immune escape in EOC patients, focusing on the role of cytokines and EOC- derived exosomes. Cytokines are key molecules regulating immune effector functions in health and disease. We used real-time RT-qPCR and a set of primers and probes for 12 cytokines, discriminating between different immune responses and compared the cytokine mRNA expression profiles locally in the TME and systemically in peripheral blood immune cells of EOC patients, to women with benign ovarian conditions and women with normal ovaries. The cytokine mRNA expression was in general most prominent in EOC patients, confirming the immunogenicity of EOC. We found significant dominance of inflammatory and immunosuppressive/ regulatory cytokines, known to promote tumor progression by priming and activating T regulatory cell-mediated immune suppression. In contrast, IFN-γ, crucially important for evoking a cytotoxic anti-tumor response, was not upregulated. Instead, a systemic increase of IL-4 prevailed, deviating the immune defense towards humoral immunity. With regard to our cytokine study, we performed comparative analyses of cytokine mRNA versus protein expression in the EOC cell lines OVCAR-3 and SKOV-3. We found that cytokine mRNA signals were universally detected, and in some instances translated into proteins, but the protein expression levels depended on the material analyzed and the method used. Due to the high sensitivity of real-time RT-qPCR, we suggest that cytokine mRNA expression profiles can be used for some instances, such as in studies of mechanistic pathways and in comparisons between patient groups, but cannot replace expression at the protein level. Exosomes are nanometer-sized vesicles of endosomal origin, released by virtually all cells, participating in normal and pathological processes. Like many tumors, EOC is a great exosome producer. We isolated exosomes from EOC ascitic fluid and supernatant from tumor explant cultures to study their effect on the NK cell receptors NKG2D and DNAM-1, involved in tumor killing. We found that EOC exosomes constitutively expressed NKG2D ligands on their surface while DNAM-1 ligand expression was rare and not associated with the exosomal membrane. Consistently, the major cytotoxic pathway of NKG2D-mediated killing was dysregulated by EOC exosomes while the accessory DNAM-1- mediated pathway remained unchanged. Our results provide a mechanistic explanation to the previously made observation that in EOC patients, tumor killing is only dependent on the accessory DNAM-1 pathway. Following these iii iv results, we studied NKG2D-mediated cytotoxicity in vivo in EOC patients before and after surgery. We found that the serum exosomes isolated from EOC patients were able to downregulate the NKG2D receptor and suppress NKG2D-mediated cytotoxicity in NK cells from healthy donors, in a similar way as exosomes from EOC ascites. We also found that surgery of the primary EOC tumor has a beneficial effect on the patients’ anti-tumor cytotoxic immune response. One mechanistic explanation could be a decrease in circulating NKG2D ligand- expressing exosomes, thus improving the cytotoxic NK cell function. In conclusion, our results contribute to the understanding of the mechanisms responsible for tumor immune escape in general, and in EOC patients in particular, and might be useful in developing novel antitumor therapies. Our studies highlight the prevailing immunosuppression in the local TME and the immunosuppressive role of EOC exosomes. Furthermore, they support the notion that cancer surgery is also a way of removing exosome-producing cells and reducing the serum concentration of immunosuppressive exosomes, thus boosting the patients’ cytotoxic anti-tumor response.