Activated splenocytes radiosensitize hypoxic tumor cells through paracrine secretion of IFN-gamma.

5177 Purpose: Activated immune cells are known to elicit anti-tumoral effects through the pro-Th1 cytokine IFN-gamma, resulting in the appearance of cytotoxic T/NK cells. In this study, we examined whether secretion of IFN-gamma may enhance tumor cell response to radiation and the possible role of nitric oxide (NO) released by the cytokine-inducible enzyme iNOS. Methods: Spleen cells from Balb/c mice were exposed to the selected Th1-type immunostimulators lipid A, poly(I:C) or IL-12/IL-18 for 24 h, and the conditioned medium (CM) was analyzed for the secretion of IFN-gamma by ELISA. EMT-6 mammary carcinoma cells were exposed to CM and their hypoxic cell radiosensitivity was estimated by clonogenic survival. The transcriptional activation of iNOS was examined by RT-PCR, the protein level by Western blotting and the enzymatic activity by Griess assay. Results: Non-stimulated splenocytes secreted low levels of IFN-gamma, which did not affect the hypoxic radioresponse of EMT-6 tumor cells. Upon stimulation, the production of IFN-gamma increased in a cell density dependent manner up to 10 million splenocytes per ml. The maximal rates of IFN-γ secretion induced by lipid A, poly(I:C) and IL-12/18 were 10, 80 and 110 ng/ml respectively. This caused the activation of iNOS-mediated NO production in EMT-6 tumor cells and enhanced their hypoxic radioresponse by more than 2-times. Radiosensitization was abrogated by neutralizing anti-IFN-gamma antibodies and by the metabolic iNOS inhibitor aminoguanidine. The efficient activation of iNOS and radiosensitization of EMT-6 tumor cells could be also achieved by combining the immunostimulators with purified IFN-gamma. Conclusions: This study demonstrates that activated splenocytes may enhance the radioresponse of hypoxic tumor cells through paracrine secretion of IFN-gamma. The mechanism of radiosensitization was associated with the release of NO through the cytokine-inducible iNOS pathway.