Nitric oxide inhibition improves cytotoxic immune response in a murine invasive bladder cancer model

BELÉN AMATO, ; DENISE BELGOROSKY, ; YANINA LANGLE ; ANA MARIA EIJAN

MODALIDAD VIRTUAL

Congreso; Reunion Conjunta de Sociedades de Biociencias SAIC SAI SAFE; 2020

SAIC SAI SAFE

Bladder cancer (BC) is a common malignancy of male urinary tract. According to the invasion degree, tumors are classified as non-muscle invasive (NMI) and invasive (MI). The constitutive expression of the inducible isoform of nitric oxide synthase (iNOS), which produces high levels of nitric oxide (NO), is a poor prognostic marker in human BC, associated with invasion and early recurrence. We previously showed that its pharmacological inhibition with L-NAME in NMI tumor-bearing mice (TBM) reversed tumor immunosuppression and improved the response to gold standard BCG treatment.Objectives: To evaluate the variations on cytotoxic (CD8+ and NK cells) and immunosuppressive (Treg) immune populations on iNOS-expressing MI murine BC model under L-NAME treatment and to analyze its combination with BCG.Methods: Ex vivo: Normal splenocytes were stimulated in vitro with MB49-I cells in presence of L-NAME (2mM) +/- BCG (2mg/ml). In vivo: Splenic and tumor leucocytes of normal mice and MI MB49-I TBM treated with 0.5 g/L L-NAME in drinking water were analyzed. The percentage of CD8+, NK cells and Treg were quantified by flow cytometry.Results: The ex vivo stimulation of splenocytes with MB49-I cells, was able to increase CD8+ (p≤0,01) and reduce Treg (p≤0,05), respect to unstimulated splenocytes. The combination of L-NAME with BCG increased NK cells (p≤0,01) and reduced Treg (p≤0,05), inducing the highest relation CD8+/Treg (p≤0,001). MB49-I TBM presented a decrease in CD8+ and NK cells (p≤0,001) and an increase in Treg (p≤0,05) compared to normal mice. L-NAME treatment was able to decrease Treg and increase CD8+ (p≤0,001).Conclusion: NO inhibition with L-NAME reverses an immunosuppressive state in MB49-I TBM. Its combination with BCG improves the relation CD8+/Treg, suggesting an increased anti-tumor immunity. Inhibitors of NO production may be potential immunomodulators for patients with iNOS positive BC.