Manganese facilitated cGAS-STING-IFNI pathway activation induced by ionizing radiation in glioma cells.

Purpose: After irradiation, double-stranded DNA leaked into the cytoplasm activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, leading to the production of type I interferon (IFNI). In this study, we sought to probe the effect of ionizing radiation on activity of cGAS-STING-IFNI pathway in normoxic or hypoxic glioma cells and explore a more effective method to activate the signaling pathway, thereby activating the anti-tumor immune response and improving the therapeutic effect of radiotherapy for glioma.

Materials and methods: Human glioma cells U251 and T98G cultured in normoxia or hypoxia (1% O₂) were irradiated with different doses of X-ray. The relative expressions of cGAS, IFN-I stimulated genes (ISGs), and three-prime repair exonuclease 1 (TREX1) were detected by qPCR. The expression levels of interferon regulatory factor 3 (IRF3) and p-IRF3 proteins were detected by Western blot. The production of cGAMP and IFN-β in the supernatant was detected by ELISA assay. U251 and T98G cell lines with stable knockdown of TREX1 were established after transfection with lentivirus vectors. EdU cell proliferation assay was used to screen suitable metal ions concentrations. The phagocytosis of DCs was observed by immunofluorescence microscope. The phenotype of DCs was detected by flow cytometry. The migration ability of DCs was detected by a transwell experiment.

Results: We found that cytosolic dsDNA, 2'3'-cGAMP, cGAS and ISGs expression, and IFN-β in cell supernatant were all increased with the doses of X-ray in the range of 0-16 Gy in normoxic glioma cells. Nevertheless, hypoxia significantly inhibited the radiation-induced dose-dependent activation of cGAS-STING-IFNI pathway. Furthermore, manganese (II) ion (Mn²⁺) significantly improved cGAS-STING-IFNI pathway activation induced by X-ray in both normoxic and hypoxic glioma cells, thereby promoting the maturation and migration of DCs.

Conclusions: The responses of cGAS-STING-IFNI pathway to ionizing radiation were mainly investigated under normoxic condition, but the experiments described here indicated that hypoxia could hinder the pathway activation. However, Mn²⁺ showed radiosensitizing effects on the pathway under either normoxic or hypoxic conditions demonstrating its potential as a radiosensitizer for glioma through activating an anti-tumor immune response.