Abstract 6545: Targeting NOS2 and the tumor microenvironment in ovarian cancer: Switching the immune system from “cold” to “hot”
Authors: Gianmarco Melone, Ivonne Uzair, Mailin Li, Maria Chervo, Karina Ortega, Jenying Deng, Wei Qian, Jianying Zhou, Liliana Guzman, Kristina Zambo, Harika Nagandla, Hong Zhao, Christoforos Thomas, Fotis Nikolo, Keith Chan, Lewis Francis, Jenny Chang, Aireana Phillips, Xen Ping Hoi
Journal: Cancer Research (2025)

Abstract Purpose of the study: The goal of this study is to understand how targeting inducible Nitric Oxide Synthase 2 (NOS2) may alter the ovarian cancer tumor microenvironment. NOS2 is part of many proinflammatory pathways and plays a key role in immune response regulation and cancer cell proliferation. By combining pan-NOS inhibitor L-NMMA with conventional Cisplatin administration, we want to convert the immune environment from “cold” or non-responsive to “hot” or responsive to chemotherapy. Experimental procedures: We established an in vivo late-stage syngeneic ovarian cancer model by intraperitoneal (IP) injection of ID8 cells in C57BL/6 mice. We administered Cisplatin (2 mg/kg, IP), L-NMMA (4 mg/Kg at day 1, 200 mg/Kg starting from day 2, IP), and Cisplatin + L-NMMA combination at the above-mentioned doses starting from week five (ascites formation phase). At the endpoint (11 weeks), ascites was analyzed through flow cytometry to describe the immune population in different treatment regimens. We also developed a NOS2 knockout (KO) human ovarian cancer cell line (OVCAR8) through CRISPR double nickase system. The NOS2 KO and the reduction of NOS2 downstream proteins like ZEB1 and Vimentin were validated through western blot. Sensitivity to Cisplatin for the OVCAR8 KO cell line was tested through RealTime-Glo™ MT Cell Viability Assay. A p-value of &amp;lt;0.05, as measured using the One-way Anova test, was considered significant. Results: In comparison to single agents, the combination treatment into ascites showed a significative increase of CD62L- CD44+ markers in CD4+ T cells (35.67% in Cisplatin treatment vs. 59.72% in Combination treatment, p &amp;lt; 0.05), increased infiltration of T cells (30.74% in Cisplatin treatment vs. 43.57% in Combination treatment, p &amp;lt; 0.05), NK (0.3408% In Cisplatin treatment vs 3.270% in Combination treatment, p &amp;lt; 0.05) and NK/NKT cells (2.677% in Cisplatin treatment vs. 7.090% in Combination treatment, p &amp;lt; 0.05). A reduction of the ascites formation in the combination treatment group (6.83 cm vs. 6.619 cm abdomen diameter) was also observed. The development of an in vitro OVCAR8 NOS2 KO model showed that the depletion of NOS2 can reduce Vimentin and ZEB1 expression together with a reduced KO cell proliferation after treatment with Cisplatin, compared to OVCAR8 parental cell line (90.73% residual proliferation in parental cell line vs 38.17% residual proliferation in the KO clone after 72 h administration of 4 µM Cisplatin). Conclusions: Our results showed the potential of the combination therapy to increase the infiltration of NK and NK/NKT cells and T lymphocytes into ascites together with the increase of effector markers in CD4+ T cells. The development of a second in vivo model based on the OVCAR8 NOS2 KO cell line will provide a better understanding of the role of NOS2 in the metastasis formation process and the impact of NOS2 on patients' survival. Citation Format: Gianmarco Melone, Ivonne Uzair, Mailin Li, Maria Chervo, Karina Ortega, Jenying Deng, Wei Qian, Jianying Zhou, Liliana Guzman, Kristina Zambo, Harika Nagandla, Hong Zhao, Christoforos Thomas, Fotis Nikolo, Keith Chan, Lewis Francis, Jenny Chang, Aireana Phillips, Xen Ping Hoi. Targeting NOS2 and the tumor microenvironment in ovarian cancer: Switching the immune system from "cold" to "hot" [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6545.