Radiation Induced Upregulation of DNA Sensing Pathways is Cell-Type Dependent and Can Mediate the Off-Target Effects

SIMPLE SUMMARY: Radiotherapy was shown to act as in situ vaccination, which can mediate systemic response to cancer in conjunction with immune checkpoint inhibitors. We aimed to investigate the DNA sensing pathways as a fundamental mechanism of immunostimulation by DNA after tumor irradiation. Key distinct DNA sensing pathways were identified in two different cell types abundant in the microenvironment of solid tumors, tumor cells and macrophages. We evaluated dose- and time-dependent expression of DNA sensors and cytokines and assayed for radiation-induced bystander effects. Understanding the timing of the activation of these pathways and subsequent cytokine expression induced by different irradiation doses could improve the treatment schemes for combining immune checkpoint inhibitors or other immunotherapies with the radiation therapy in order to improve the therapy of cancer. Therefore, this paper could complement and provide basis for current clinical studies investigating radio-immunotherapy schedules. ABSTRACT: Irradiation of tumors generates danger signals and inflammatory cytokines that promote the off-target bystander and abscopal effects, evident especially when radiotherapy is administered in combination with the immune checkpoint inhibitors (ICI). The underlying mechanisms are not fully understood; however, cGAS-STING pathway was recognized as the main mediator. In our study, we demonstrate by immunofluorescent staining that tumor cells as well as macrophages, cell types abundant in the tumor microenvironmeent (TME) accumulate DNA in their cytosol soon after irradiation. This accumulation activated several distinct DNA sensing pathways, most prominently activated DNA sensors being DDX60, DAI, and p204 in tumor cells and DDX60, DAI, p204, and RIG-I in macrophages as determined by PCR and immunofluorescence imaging studies. This was accompanied by increased expression of cytokines evaluated by flow cytometry, TNFα, and IFNβ in tumor cells and IL1β and IFNβ in macrophages, which can alter the TME and mediate off-target effects (bystander or abscopal effects). These results give insight into the mechanisms involved in the stimulation of antitumor immunity by radiation.