Abstract 3738: A STING independent type-1 interferon response induced by fractionated radiotherapy coincides with altered tumor growth and clonogenicity
Authors: Goedegebuure Ruben S. , Kleibeuker Esther A. , Castricum Kitty C. , Berg Jaap van den , Derks Sarah , Verheul Henk M. , Slotman Ben J. , Harris Adrian , Thijssen Victor L. 
Published: August 2019

Abstract
Introduction To improve radiotherapy efficacy, insight in the dynamic biological responses that occur during clinical radiation schedules is vital. In the current study we explored gene expression changes during the course of fractionated radiotherapy (FrRT) that might be targeted with combination treatment strategies.
Methods Human cell lines HT29, SW480, HCT116, COLO320, RKO (colorectal carcinoma), D384 (glioblastoma) and OE19 (esophageal adenocarcinoma) as well as HT29 xenograft tumors in nude mice were irradiated up to 3 to 5 weeks with daily fractions of 2 Gy for 5 days per week. In vivo tumor growth was monitored daily and in vitro clonogenic survival was determined every other day. At the end of each treatment week tumors and cells were harvested for RNA isolation. Subsequently, gene expression analysis was performed by RNA sequencing and micro-array analysis. Altered gene expression levels were validated by qPCR. Protein expression was determined by ELISA and Western blot.
Results Tumor growth of the HT29 xenografts declined after 2 weeks of FrRT compared to non-irradiated tumors. In irradiated cell lines, clonogenic survival analyses revealed a log-linear decline in survival in the first 2 weeks, after which a steady-state-like phase was reached up to 5 treatment weeks. Gene expression analysis of both xenograft tumors as well as irradiated cell lines at this 2 week time point identified a type-1 interferon (IFN) mediated signaling pathway as the most significantly enriched biological process. The induction of a selection of IFN-stimulated genes (ISGs) was confirmed by qPCR. Comparable responses were observed in both STING positive (HT29, SW480, HCT116, D384) and negative (COLO320, RKO, OE19) cell lines. To get more insight in the dynamics of the IFN response we analyzed ISGs expression at the end of each treatment week. This revealed a peak in ISGs expression after 10 to 15 fractions, both in vivo and in vitro. The ISGs expression levels decreased with continuation of treatment but always remained above the basal level of non-irradiated cells. Single dose irradiation up to 10 Gy also induced dose-dependent ISGs expression, albeit typically 5 to 10 fold lower as compared to FrRT. Subsequent protein and mRNA expression analyses consistently revealed induction of IFN beta expression, with peak levels measured after 10 to 15 fractions. In addition, occasional induction of IFN lambda 2/3 protein and mRNA expression was observed.
Conclusion FrRT induces an intrinsic type-1 IFN response which occurs independent of STING. This response is associated with induction of IFN beta expression, which peaks after 2 to 3 weeks of treatment and coincides with a shift in in vivo tumor growth and in vitro clonogenic survival. Targeting this response during FrRT might be exploited as a novel combination treatment.
Citation Format: Ruben S. Goedegebuure, Esther A. Kleibeuker, Kitty C. Castricum, Jaap van den Berg, Sarah Derks, Henk M. Verheul, Ben J. Slotman, Adrian Harris, Victor L. Thijssen. A STING independent type-1 interferon response induced by fractionated radiotherapy coincides with altered tumor growth and clonogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3738.