Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment

Background: Bacillus Calmette-Guerin (BCG) is the standard treatment for patients with high-risk non-muscle invasive bladder cancer (BC). Despite its success, about 30-50% of patients are refractory. It was reported that inducible nitric oxide synthase (iNOS) tumor expression is presented in 50% of human BC, associated with bad prognosis and BCG failure. Objective: to evaluate in human bladder tumors the association between iNOS expression and the tumor microenvironment focusing on the immunosuppressive protein S100A9. Also, investigate in a preclinical murine MB49-BC model the tumor immunoresponse induced by BCG in combination with the nitric oxide production inhibitor L-NAME. Results: In human bladder tumors, we detected a positive association between iNOS and S100A9 tumor expression, suggesting a relationship between both immunomodulatory proteins. We also found a positive correlation between iNOS tumor expression and the presence of S100A9(+) tumor-infiltrating cells, suggesting an immunosuppressive tumor microenvironment induced by the nitric oxide production. Using the subcutaneous murine BC model, we show that similarly to the human pathology, MB49 tumors constitutively expressed iNOS and S100A9 protein. MB49 tumor-bearing mice presented an immunosuppressive systemic profile characterized by fewer cytotoxic cells (CD8(+) and NK) and higher suppressor cells (Treg and myeloid-derived suppressor cells -MDSC-) compared to normal mice. BCG treatment reduced tumor growth, increasing local CD8(+) -infiltrating cells and induced a systemic increase in CD8(+) and a reduction in Treg. BCG combined with L-NAME, significantly reduced tumor growth compared to BCG alone, diminishing iNOS and S100A9 tumor expression and increasing CD8(+)-infiltrating cells in tumor microenvironment. This local response was accompanied by the systemic increase in CD8(+) and NK cells, and the reduction in Treg and MDSC, even more than BCG alone. Similar results were obtained using the orthotopic BC model, where an increase in specific cytotoxicity against MB49 tumor cells was detected. Conclusion: The present study provides preclinical information where NO inhibition in iNOS-expressing bladder tumors could contribute to improve BCG antitumor immune response. The association between iNOS and S100A9 in human BC supports the hypothesis that iNOS expression is a negative prognostic factor and a promising therapeutic target.