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

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. 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. 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. 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.

电离辐射后，渗漏进入细胞质的双链DNA（double-stranded DNA）会激活环GMP-AMP合酶（cyclic GMP-AMP synthase, cGAS）-干扰素基因刺激因子（stimulator of interferon genes, STING）通路，进而诱导I型干扰素（type I interferon, IFN-I）的产生。本研究旨在探究电离辐射对常氧或低氧胶质瘤细胞中cGAS-STING-IFN-I通路活性的影响，并探索更为有效的该信号通路激活方法，从而激活抗肿瘤免疫应答，提升胶质瘤放射治疗的疗效。本研究对常氧或低氧（1% O₂）培养的人胶质瘤细胞U251和T98G施加不同剂量的X射线照射。通过实时定量聚合酶链式反应（qPCR）检测cGAS、I型干扰素刺激基因（IFN-I stimulated genes, ISGs）及3'端修复核酸外切酶1（three-prime repair exonuclease 1, TREX1）的相对表达量；采用蛋白质免疫印迹（Western blot）检测干扰素调节因子3（interferon regulatory factor 3, IRF3）及其磷酸化形式p-IRF3的蛋白表达水平；通过酶联免疫吸附测定（ELISA）检测细胞上清液中cGAMP和干扰素β（IFN-β）的含量。通过慢病毒载体转染，构建稳定敲低TREX1的U251和T98G细胞系。采用EdU细胞增殖实验筛选适宜的金属离子浓度。通过免疫荧光显微镜观察树突状细胞（Dendritic Cells, DCs）的吞噬功能，采用流式细胞术（flow cytometry）检测DCs的表型，利用Transwell小室实验检测DCs的迁移能力。研究结果显示，在常氧胶质瘤细胞中，胞浆双链DNA、2′3′-cGAMP、cGAS及ISGs的表达水平，以及细胞上清液中的IFN-β含量，均随0~16 Gy范围内的X射线剂量升高呈剂量依赖性增加。但低氧可显著抑制辐射诱导的cGAS-STING-IFN-I通路剂量依赖性激活。此外，二价锰离子（manganese (II) ion, Mn²⁺）可显著增强常氧及低氧胶质瘤细胞中X射线诱导的cGAS-STING-IFN-I通路激活，进而促进DCs的成熟与迁移。以往针对cGAS-STING-IFN-I通路电离辐射响应的研究多聚焦于常氧条件，而本研究实验结果表明，低氧会阻碍该通路的激活。然而，无论在常氧还是低氧条件下，Mn²⁺均对该通路具有放射增敏效应，提示其可通过激活抗肿瘤免疫应答，成为胶质瘤放射治疗的潜在增敏剂。