The radiosensitizing effect of immunoadjuvant OM-174 requires cooperation between immune and tumor cells through interferon-gamma and inducible nitric oxide synthase

Purpose: To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-γ (IFN-γ) and thereby radiosensitize tumor cells. Methods and Materials: Splenocytes from BALB/c mice were stimulated by OM-174 at plasma-achievable concentrations (0.03-3 μg/mL), and afterward analyzed for the expression and secretion of IFN-γ by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Stimulated splenocytes were used as a source of IFN-γ to radiosensitize hypoxic EMT-6 tumor cells through the cytokine-inducible isoform of nitric oxide synthase (iNOS). Results: OM-174 activated the production of IFN-γ at high levels that reached 70 ng/mL in normoxia (21% oxygen) and 27 ng/mL in tumor-relevant hypoxia (1% oxygen). This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of the radiosensitizing molecule nitric oxide, as confirmed by accumulation of its oxidative metabolite nitrite, Western blot analysis, and reverse transcriptase-polymerase chain reaction. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-γ. The same mechanism of radiosensitization through the IFN-γ secretion pathway was identified for IL-12 + IL-18, which are known to mediate IFN-γ responses. Hypoxia displayed a dual effect on the immune-tumor cell interaction, by downregulating the expression of the IFN-γ gene while upregulating iNOS at transcriptional level. Conclusion: Immunoadjuvant OM-174 is an efficient radiosensitizer of tumor cells through activation of the IFN-γ secretion pathway in immune cells. This finding indicates a rationale for combining immunostimulatory and radiosensitizing strategies and extends the potential therapeutic applications of OM-174