Ionizing Radiation Triggers The Antitumor Immunity By Inducing GSDME-mediated Pyroptosis In Tumor Cells

As one of major treatments for cancer in clinic,radiotherapy is applied for most patients with tumor at different stages of clinic treatment.The tumor cell damage and death induced by radiation has been regarded as one basic theory of radiotherapy.Due to the differences in genetic backgrounds of cancer cells and radiotherapy planning,cancer cells may go into different cell death types after radiation,then affect the radiation resistance of tumor cells and the activation of immune system.Exploring new cell death mode induced by radiation is of great significance for understanding the biological effects and providing more theoretical basis or guidance for radiotherapy.Pyroptosis is a new type of programmed cell death which characterized by the formation of plasma membrane pores and the release of intracellular contents.The occurrence of pyroptosis depends on the exposure of the N-terminal domain of the Gasdermin family proteins.In recent years,pyroptosis has attracted much attention in tumor therapy and antitumor immune response.GSDME(Gasdermin E),a member of the Gasdermin family proteins,has been confirmed to induce pyroptosis by chemotherapeutic drugs and then promote anti-tumor immune responses.However,whether radiotherapy could induce pyroptosis,the regularity of its occurrence,and the role in anti-tumor immune response remain unclear.In this study,through detecting 20 kinds of tumor cell lines in four types of cancers(lung,liver,breast,and glioma cancer),we first demonstrated that X-ray radiation could induce pyroptosis,and the different degrees of pyroptosis was correlated with the expression level of GSDME in tumor cells.Next,we examined the effect of radiation factor on pyroptosis,the result proved that pyroptosis has no radiation-type specificity because it could be induced by X,y-ray and a particle radiation.Radiation induces pyroptosis in a dose-and time-dependent manner.Furthermore,a large radiation dose fraction(1×10 Gy/time)is more effective than conventional radiation dose fractions(5×2 Gy/time)in pyroptosis induction.The mechanism study showed that radiation cleaved GSDME to induce tumor cell pyroptosis through activated the ROS/Caspase 9/Caspase 3 signaling pathway.By increasing the expression level of GSDME in tumor cells,the cell death mode was significantly changed from apoptosis to pyroptosis after radiation.In addition,western blot results reveal that radiation combined with DNA demethylation agents(decitabine,azacytidine)or some other chemotherapy agents(cisplatin,etoposide)could enhance radiation-induced pyroptosis.The results of cell viability,cloning survival assays and tumor-bearing nude mice model showed that GSDME-mediated pyroptosis did not change the radiosensitivity of tumor cells.In tumor-bearing mice(Balb/c),overexpression of GSDME could significantly promote the infiltration of CD8+T cells in the irradiated tumors,and enhance the inhibitory effect of radiation on tumor growth.The mechanism study showed that overexpressing GSDME in 4T1 cells significantly enhanced radiation-induced pyroptosis,and the result of flow cytometry indicating that GSDME-mediated pyroptosis after radiation enhances the antigen presentation of DCs through increase the expression of surface antigens such as CD40,CD83,CD86 and CD80.Moreover,the results of ELISA showed that Granzyme B,TNF-α and INF-y were significantly increased in irradiated GSDME-overexpressing tumors.Taken together,our results indicate that GSDME-mediated pyroptosis effectively promotes the infiltration of CD8+T lymphocytes by enhancing the antigen presentation of DCs,thereby boosting anti-tumor immunity after irradiation.In summary,this study systematically explored(1)the occurrence of radiation-induced pyroptosis and the regularity between dose,time,and different types of radiation;(2)the key signaling pathways and mechanisms of radiation-induced pyroptosis;(3)the role and mechanism of radiation-induced pyroptosis in activating anti-tumor immune responses.This study increases the understanding in the biological effects of radiation,explores the molecular mechanism of radiation-induced cell death,and provides theoretical and practical basis for the optimization of clinical radiotherapy.