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Development of a combination therapy to destabilize ovarian cancer and its tumor microenvironment through inducible Nitric Oxide Synthase (iNOS) inhibition. / GIANMARCO MELONE

Swansea University Author: GIANMARCO MELONE

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DOI (Published version): 10.23889/SUThesis.72010

Abstract

Ovarian cancer represents a significant challenge in oncology, largely because reliable strategies for early detection are still lacking. In its initial stages, the disease is frequently asymptomatic or associated with vague and nonspecific symptoms that often resemble different gynecological or abdomi...

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Published: Swansea 2026
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
Supervisor: Francis, L., and Chang, J.
URI: https://cronfa.swan.ac.uk/Record/cronfa72010
Abstract: Ovarian cancer represents a significant challenge in oncology, largely because reliable strategies for early detection are still lacking. In its initial stages, the disease is frequently asymptomatic or associated with vague and nonspecific symptoms that often resemble different gynecological or abdominal disorders. Consequently, most patients receive a diagnosis only when the disease has already progressed to an advanced stage, typically after extensive peritoneal dissemination has occurred. This late diagnosis severely limits therapeutic options and is associated with poor clinical outcomes. The management of ovarian cancer is further complicated by the high rate of relapse and by the immunosuppressive characteristics of the tumour microenvironment. Among these factors, nitric oxide (NO) plays a particularly relevant role. In many cancers, including ovarian cancer, NO is predominantly produced by inducible nitric oxide synthase (iNOS), and elevated NO levels have been associated with enhanced tumour progression as well as suppression of antitumour immune responses. Given the challenges posed by late-stage diagnosis, the presence of an immunologically “cold” tumour microenvironment, and the aggressive dissemination of ovarian cancer cells, this study explores an alternative therapeutic strategy to complement conventional chemotherapy. Specifically,the work investigates the potential of the iNOS inhibitor L-NMMA as a means of modulating nitric oxide production, both as a single agent and in combination with Cisplatin. The experimental approach integrates in vitro and in vivo models to evaluate whether targeting NO signalling can simultaneously impair tumour cell behaviour and disrupt protumoural interactions within the tumour microenvironment. The main objective of this research is therefore to develop a therapeutic strategy capable not only of directly affecting ovarian cancer cells but also of altering the tumour microenvironment by limiting excessive nitric oxide production. This goal was addressed through the identification of Vimentin, a marker of epithelial–mesenchymal transition (EMT), as a molecular target affected by L-NMMA treatment. In OVCAR8 ovarian cancer cells, L-NMMA administration resulted in a reduction of Vimentin expression exceeding 50%. To further investigate the role of nitric oxide in tumour biology, OVCAR8 NOS2 knockout cell lines were specifically developed and used for both in vitro and in vivo studies. In addition, the combined treatment with Cisplatin and L-NMMA was associated in a syngeneic in vivo model with enhanced immune activation, characterized by increased markers of CD4⁺ T-cell activation and a reduction in CD4⁺ naïve T-cell markers, together with an overall increase in T cells and natural killer (NK) cells. This combination therapy demonstrated the capacity to modulate the immune response while simultaneously reducing total tumour cell number, percentages of tumour cell numbers and tumour area. Complementary evidence was obtained using a xenograft model based on NSG mice implanted with OVCAR8 NOS2 knockout cells. In this system, reduced peritoneal dissemination was observed, suggesting diminished migratory capacity associated with decreased Vimentin expression and impaired nitric oxide production. Furthermore, mice treated with Cisplatin displayed prolonged survival and improved control of tumour progression when compared with models using NOS2 parental OVCAR8 cell lines. Overall, the results presented in this work highlight the potential of targeting nitric oxide signalling as a strategy to enhance the efficacy of standard chemotherapy in ovarian cancer. By integrating experimental approaches across two research institutions, this project expands current understanding of the role of nitric oxide in ovarian cancer biology and provides evidence supporting the development of therapeutic interventions aimed at modulating the tumour microenvironment. In conclusion, the findings presented in this study, may contribute to the design of more effective treatment strategies capable of improving clinical outcomes for patients affected by ovarian cancer.
Keywords: Ovarian cancer, iNOS, L-NMMA, tumour microenvironment, Vimentin
College: Faculty of Medicine, Health and Life Sciences
Funders: EPSRC doctoral training grant

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