Introduction: Sustained oxidative stress in castration-resistant prostate cancer (CRPC) cells potentiates the overall tumor microenvironment (TME). Targeting the TME using colony-stimulating factor 1 receptor (CSF1R) inhibition is a promising therapy for CRPC. However, the therapeutic response to sustained CSF1R inhibition (CSF1Ri) is limited as a monotherapy. We hypothesized that one of the underlying causes for the reduced efficacy of CSF1Ri and increased oxidation in CRPC is the upregulation and uncoupling of endothelial nitric oxide synthase (NOS3). Methods: Patient biopsies belonging to different Gleason grades were used to determine the levels of NOS3, CSF1, and GSNOR activity. A CSF1-receptor inhibitor, GW2580, was tested in-vitro and in-vivo, singularly and in combination with S-nitrosoglutathione (GSNO), an active NO donor. In-vivo dosage of 40mg/kg/day was used for GW2580, and 10mg/kg/day for GSNO was considered. Markers were checked for their expression in tumor grafts and human specimens using immunohistochemistry. Moreover, we evaluated the percentage of different immune cells using immune phenotyping in tumor cells exposed to different treatment conditions. Results: We show that in high-grade PCa human specimens, NOS3 abundance positively correlates with CSF1-CSF1R signaling and remains uncoupled. The uncoupling diminishes NOS3 generation of sufficient nitric oxide (NO) required for S-nitrosylation of CSF1R at specific cysteine sites (Cys 224, Cys 278, and Cys 830). Exogenous S-nitrosothiol administration (with S-nitrosoglutathione (GSNO)) induces S-nitrosylation of CSF1R and rescues the excess oxidation in tumor regions, in turn suppressing the tumor-promoting cytokines, which are ineffectively suppressed by CSF1R blockade. Together these results suggest that the NO administration could act as an effective combinatorial partner with the CSF1R blockade against CRPC. In this context, we further show that exogenous NO treatment with GSNOR successfully augments the anti-tumor ability of CSF1Ri to reduce the overall tumor burden effectively, decreases the intratumoral percentage of anti-inflammatory macrophages, myeloid-derived progenitor cells and increases the percentage of pro-inflammatory macrophages, cytotoxic T lymphocytes, and effector T cells, respectively. Conclusions: Together, these findings support the concept that the NO-CSF1Ri combination can act as a therapeutic agent that restores control over TME, which in turn could improve the outcomes of PCa patients. SOURCE OF Funding: AUA Research Scholar Award to HA American Cancer Society Grant to RR
