Introduction: Prostate cancer (PCa) is the most common non-skin cancer among American men, and growing evidence suggests that targeting the tumor microenvironment (TME) could be essential in combating the progression of metastasis and resistance development in cancer. The colony-stimulating factor 1 (CSF1)/colony-stimulating factor 1 receptor (CSF1R) axis is critical for potentiating the tumor microenvironment (TME) by keeping a check on macrophage polarization and therefore CSF1R inhibition is a major target against CRPC. We demonstrated that increased nitric oxide (NO) reduces tumor burden in murine models for CRPC through its action on TME and specifically reducing ligands (CSF1) which binds to CSF1R. Therefore we hypothesized that increased Nitric oxide augments the action of CSF-1R inhibition against tumor associated macrophages in castration resistant prostate cancer.
Methods: The castrated SCID mice were grafted with 22RV1 cells and treated with CSF1R inhibitor (GS2580) (40mg/kg/day IP) and/or GSNO (10mg/kg/day IP) for 15 days, post treatment animals were survived for additional 2 weeks before sacrificing them and extracting tumors, organs and blood for further analysis. RNA library was generated from tumors for RNA sequencing analysis. Tumor RNA and proteins were studied for the markers that are important for prostate cancer progression using qPCR, western blots. Cytokine antibody array was performed using tumor proteins to evaluate cytokines altered upon different treatment conditions. Immunostaining was performed to check the macrophage expression in the tumor grafts. GraphPad Prism (GraphPad Software) was used for statistical analysis. All data were presented as the means ± SEM. The statistical significance between two groups was estimated by unpaired two-tailed t test.
Results: Invitro results showed that, in the presence of increased NO levels, CSF1Ri effects on inhibiting the expression of AR, ARV7, PSA, TMRPSS2, pERK (terminal markers of CRPC) etc in 22RV1 cells are significantly induced at RNA and protein levels. In leu of these findings, in-vivo results demonstrated that, although the CSF1R inhibitor monotherapy was able to decrease the tumor burden significantly, in over 50% of the mice, the expression of markers like AR, pERK, p-GSK, and VEGF was increased. On the contrary, the most significant reduction of tumor burden was observed in mice which received GSNO+CSF1Ri combination compared to CSF1Ri or GSNO monotherapy. Moreover, the expression of markers- AR, ARV7, PSA, TMRPSS2, p-GSK, p-ERK, and p90RSK was found to be least in these tumor suggesting that increased Nitric oxide could augment the action of CSF-1R inhibition against CRPC reduction. Furthermore, cytokine antibody array, RNA sequencing and immunostaining showed that the presence of increased NO levels could enhance the capability of CSF1Ri in suppressing signature of markers that are critical for PCa progression and metastasis (Interferon Alpha, Interferon Gamma, Myc targets) and cytokines (like CXCL5, FGF4, IGFBP-3, MCP-4, IL-6, and TNFalpha) that are potentially TME promoting.
Conclusions: Our findings suggest that Increased NO levels positively affects the CSF1Ri induced changes with respect to a) suppressing tumor burden, b) suppressing cytokines that are potentially TME promoting, c) suppressing signature of markers that are critical for PCa progression and metastasis. Identifying the mechanism of how combination of increasing nitric oxide with CSF1R inhibition affects tumor can have therapeutic potential for treating AR-v7 subtype of CRPC. Source of
Funding: American cancer society, Clinician Scientist Development Grant
