Radiation fractionation effect on IDO1 through NF-kappa B/COX2 axis in non-small cell lung cancer

e20613 Background: Lung cancer is the leading cancer mortality worldwide. Radiotherapy(RT) plays an important role in lung cancer treatment. Different does fractionation of RT has different effects on the prognosis of lung cancer. For example, stereotactic body radiotherapy can significantly improve the prognosis compared with conventionally Fractionated Radiotherapy. In non-small cell lung cancer, we have previously shown that Indoleamine 2,3-dioxygenase 1 (IDO1) metabolites and radiation induced changes had significant effects on long-term survival in patients treated with either Stereotactic body Radiation Therapy of 10Gy fraction size or conventional fractionated radiotherapy of 2Gy fraction size. However, the underlining molecular mechanism is unknown. In this study, we aimed to elaborate the molecular mechanism of such clinical differential effects in the laboratory. Methods: This study applied two dose/fractionation regimens with a similar BED10: 10Gyx1 and 2Gyx8. 10Gyx1 and 2Gyx8 were tested in cell lines of A549, H1299, Calu-1, TC-1 of human and murine NSCLC as well as normal rat cardiomyocyte cell line H9c2, and in vivo (TC-1 implanted C57Bl/6 mice). The mRNA and protein levels of IDO1, p-NF-kB, COX2 from cells and tumor tissues were determined by using qPCR, western blot and immunohistochemistry. Survival outcome was tested by colony formation assay in vitro and tumor growth curve in vivo. Results: In vitro, 10Gyx1 and 2Gyx8 induced IDO1 expression in all tested cell lines and the level of IDO1 expression induced by 10Gyx1 was lower than 2Gyx8. Both 10Gyx1 and 2Gyx8 increased p-NF-kB and COX2 expression and the level induced by 10Gyx1 was lower than 2Gyx8. In tumor bearing mice, two dose/fractionation regimens also induced intratumor IDO1 expression and increased the levels of p-NF-kB and COX2. Interestingly, the increased levels of IDO1, p-NF-kB, COX2 after 10Gyx1 were also significantly lower than 2Gyx8 in vivo. IDO inhibitor synergized with RT and enhanced cell killing efficacy of RT. Conclusions: The findings of this study suggest different does fractionation of RT induced up-regulation of IDO1 through NF-kB/COX2 axis in NSCLC and non-cancerous cell lines, what’s more, hypofractionated radiotherapy 10Gyx1 had less upregulation that of conventionally fractionated radiotherapy 2Gy x8 on IDO1 immune inhibitory pathway. The finding of hypofractionated radiotherapy less immune suppression differ from the traditional belief of more immune stimulation. Our current study provides a rationale in using IDO1 as a biomarker to design RT dose fractionation and/or immunotherapy in combination and possibilities for clinical trials to design IDO1 inhibitors in combination with radiotherapy.