iNOS regulates the therapeutic response of pancreatic cancer cells to radiation therapy

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to radiation therapy (RT), chemotherapy, or a combination of these modalities, and surgery remains the only curative intervention for localized disease. Although cancer-associated fibroblasts (CAFs) are abundant in PDAC tumors, the effects of RT on CAFs and the response of PDAC cells to RT are unknown. Using patient samples and orthotopic PDAC biological models we showed that RT increased inducible nitric oxide synthase (iNOS) in the tumor tissues. Mechanistic in vitro studies showed that, although undetectable in RT-activated tumor cells, iNOS expression and nitric oxide (NO) secretion were significantly increased in CAFs secretome following RT. Culture of PDAC cells with conditioned media from RT-activated CAFs increased iNOS/NO signaling in tumor cells through nuclear factor kappa B (NFĸB), which in turn elevated the release of inflammatory cytokines by the tumor cells. Increased NO after RT in PDAC contributed to an acidic microenvironment that was detectable using the radiolabeled pH (low) insertion peptide (pHLIP ® ). In murine orthotopic PDAC models pancreatic tumor growth was delayed when iNOS inhibition was combined with RT. These data show the important role that iNOS/NO signaling plays in the effectiveness of RT to treat PDAC tumors. (low) insertion peptide (pHLIP ® ). Importantly, we demonstrate that this phenomenon can be mitigated with iNOS inhibition in vitro and in vivo, providing a therapeutic option for overcoming this pro-tumorigenic phenotype. human pancreatic cancer cell lines cultured with CM of RT-activated human CAFs demonstrated that an in vitro RT dose of 5 Gy increases NO 2– secretion by cancer cells ( Supplementary Fig. 7 ). These results show that RT-activated CAFs produce high levels of iNOS and NO secretion, which are further induced by PDAC cells and that RT enhances iNOS/NO paracrine signaling in PDACs. IL1R1, IL6Rα, Interferon regulatory factor 7 (IRF7), and signal transducer and activator of transcription 1 (STAT1) ( Supplementary Fig. 9 ). These results suggest that RT-activated fibroblasts induce increased secretion of NO and iNOS expression in the tumor cells through NFĸB, which in turn leads to an elevated release of inflammatory cytokines by the tumor cells. an with In sum, our indicate that in iNOS/NO signaling in RT-activated fibroblasts upregulates PDAC glycolysis. The mechanisms of iNOS/NO signaling and their actions on PDAC response to RT remain to be completely understood. Here, we demonstrate that RT enhances iNOS expression and subsequent NO secretion in PDAC. Mechanistic studies demonstrated that the CM of irradiated CAFs drives the production of iNOS in PDACs. Cultures of PDAC cells with CM of RT-activated CAFs demonstrate that NO secretion and iNOS expression by RT-CAFs are further induced by pancreatic tumor cells through NFĸB, which in turn leads to the release of inflammatory cytokines by the tumor cells. In addition, we found that NO increases after RT in PDAC, results in acidification of the microenvironment, which can be detected by molecular imaging using pHLIP ® . These data support that NO has a pro-tumor function in irradiated PDACs by promoting a glycolytic phenotype as detected in vivo by 18 F-FDG PET imaging, which leads to extracellular acidosis. iNOS pharmacological inhibition or knockdown improved RT efficacy and decreased PDAC tumor growth, suggesting an integral role for NO and RT-mediated iNOS activity in PDAC therapy. Until our study, a role for RT-mediated iNOS/NO signaling in PDAC remained unexplored. activated CAFs contribute to PDAC pathology and that targeting iNOS/NO signaling may be a valid therapeutic approach for improving RT outcomes in PDAC.