Evaluation of PD-1/PD-L1 and CTLA-4 immune checkpoints and angiogenic activity in ovarian cancer after oncotherad immunotherapy associated to erythropoietin.

person Bianca Ribeiro de Souza Laboratory of Urogenital Carcinogenesis and Immunotherapy (LCURGIM), Department of Structural and Functional Biology, Institute of Biology-University of Campinas (UNICAMP), Campinas, Brazil info_outline Bianca Ribeiro de Souza, Gabriela Oliveira, Giovana Leme, Felippe Augusto Tossini Cabral, Juliane Lima Baggio de Paula, Daniel Henrique da Silva Santos, Claudia Ronca Felizzola, Nelson Duran, Wagner José Fávaro, Ianny Brum Reis

Authors person Bianca Ribeiro de Souza Laboratory of Urogenital Carcinogenesis and Immunotherapy (LCURGIM), Department of Structural and Functional Biology, Institute of Biology-University of Campinas (UNICAMP), Campinas, Brazil info_outline Bianca Ribeiro de Souza, Gabriela Oliveira, Giovana Leme, Felippe Augusto Tossini Cabral, Juliane Lima Baggio de Paula, Daniel Henrique da Silva Santos, Claudia Ronca Felizzola, Nelson Duran, Wagner José Fávaro, Ianny Brum Reis Organizations Laboratory of Urogenital Carcinogenesis and Immunotherapy (LCURGIM), Department of Structural and Functional Biology, Institute of Biology-University of Campinas (UNICAMP), Campinas, Brazil, Laboratory of Urogenital Carcinogenesis and Immunotherapy (LCURGIM), Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil, Laboratory of Urogenital Carcinogenesis and Immunotherapy, Institute of Biology - University of Campinas (UNICAMP), Campinas, Brazil, Laboratory of Urogenital Carcinogenesis and Immunotherapy (LCURGIM), Institute of Biology-University of Campinas (UNICAMP), Campinas, Brazil, Nanomedicine Research Unit (Nanomed), Federal University of ABC (UFABC), Santo André, Brazil, Laboratory of Urogenital Carcinogenesis and Immunotherapy (LCURGIM), Department of Structural and Functional Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil Abstract Disclosures Research Funding Other Background: Ovarian cancer (OC) ranks fifth among cancer deaths in women and remains the deadliest of all gynecological cancers. OncoTherad is a nanostructured complex developed by University of Campinas/Brazil, which exhibits immunomodulatory and antitumor properties. Erythropoietin (EPO) can exert non-hematopoietic functions and the association of immunotherapy and EPO is a reality in anemic patients due to cancer or chemotherapy who may use immunotherapies. We evaluated the effects of OncoTherad and EPO, alone or in combination, on PD-1/PDL-1 and CTLA-4 checkpoints, pro-angiogenic (VEGF) and anti-angiogenic (Endostatin) markers, and microvessel density (CD31-labeled capillaries) in chemically induced ovarian cancer in rats. Methods: Thirty-five Fischer rats were distributed into groups: Control (Sham surgery); Cancer (7,12-dimethylbenzoanthracene – DMBA injection in the ovarian bursa, 1.25 mg/kg); OncoTherad (20mg/kg); EPO (8.4 µg/kg); and OncoTherad+EPO (same doses of the isolated treatments). Intraperitoneal doses were administered twice a week for 4 weeks. Immunohistochemistry was analyzed as total immunoreactivity and intensity of immunoreaction. Results: The Cancer group showed an increase (p < 0.05) in microvascular density and histopathological lesions, predominantly high-grade dysplasias with non-metastatic phenotype, which was related to the little involvement of PD-1/PD-L1 and CTLA-4 checkpoints in this carcinogenesis stage. The treatments altered the frequency of ovarian lesions, especially OncoTherad promoted a significant reduction of cystic lesions, while in the EPO group there was marked hyperplasia of the interstitial tissue and the associated treatment led to intermediate results. OncoTherad immunotherapy was effective in reducing angiogenesis, promoting a decrease (p < 0.05) in VEGF immunostaining and microvessel count, and an increase (p < 0.05) in endostatin immunoreactivity. EPO treatment decreased (p < 0.05) endostatin levels in comparison with OncoTherad group and led to a weaker (p < 0.05) immunoreaction intensity of PD-1/PD-L1 and CTLA-4 compared to the Cancer group. These effects might be related to the modification of the tumor microenvironment modulated by EPO with a decrease in the population of immune cells with immunosuppressive phenotype as well as histopathological alterations including reduction of folliculogenesis and luteogenesis. Conclusions: OncoTherad was able to prevent histopathological lesions and reduce the angiogenesis involved in the ovarian carcinogenesis. EPO treatment decreased endostatin, which possibly had effects on angiogenic activity. However, EPO also modulated the OC microenvironment by reducing PD-1/PD-L1 and CTLA-4. The association of OncoTherad+EPO might have triggered a compensatory effect between the isolated treatments.