Molecular Mechanism Of Immunogenic Death Induced By Trifluoprazine In Non-small Cell Lung Cancer Cells

Lung cancer is one of the most common tumors in China,with high morbidity and mortality.In recent years,immunotherapy,represented by PD-1/PD=L1 immune checkpoint blocking therapy(ICB),has become a new type of therapy with great anticancer potential.However,due to the low immunogenicity of some tumors,immunotherapy often does not have the maximum effect.Converting cold tumors into hot tumors by inducing immunogenic cell death(ICDs)with chemotherapeutic drugs is one of the ways to enhance immunotherapy,showing great potential to cooperate with ICBs.Trifluoperazine(TFP)is clinically used in the treatment of schizophrenia.Recently,it has been reported that TFP can induce autophagy,apoptosis and cell cycle arrest of tumor cells.These studies indicate that TFP has good anticancer potential,but the anti-tumor mechanism of TFP needs more research.In this paper,non-small cell lung cancer as the research object,through cell experiments and animal experiments,to study the mechanism of TFP induced ICD.SRB cell survival test showed that TFP had a significant inhibitory effect on non-small cell lung cancer.Western blot and flow cytometry confirmed that TFP could induce apoptosis of NSCLC cells.After the knockdown of the pyroptotic protein GSDME and treatment with TFP,the rate of AnnexinV-FITC double positive cells decreased significantly by flow cytometry.After GSDME knockout,lytic cell death assay was performed to observe the changes of plasma membrane integrity caused by TFP treatment,and the number of red fluorescent highlights decreased,which further indicated that TFP caused pyroptosis.Previous laboratory studies have shown that TFP can cause ER stress,and the combination of ER stress inhibitor 4-PBA and TFP showed that GSDME cleavage was significantly reduced,suggesting that TFP may cause caustic death through ER stress,thus inducing ICD and improving the immunogenicity of tumors.Pyroptosis can induce severe ICDs.Immunofluorescence detection of CRT translocation and ATP release showed that TFP treatment induced ICD.Knockdown of GSDME reduced the translocation of CRT to the plasma membrane and ATP release,indicating that TFP caused immunogenic cell death of tumor cells through pyroptosis.In vivo experiments also further confirmed that TFP treatment can inhibit the growth of subcutaneous transplanted tumors in LLC1 tumor-bearing mice,and increase the infiltration of CD8+T cells in tumor tissues,suggesting that TFP treatment may improve the immunogenicity of tumor cells.In summary,this study shows that TFP can induce pyroptosis through endoplasmic reticulum stress,leading to ICD,thereby improving the infiltration of immune cells in tumor tissues,which provides a possibility for the application of TFP in the field of anti-tumor immunotherapy and provides a new idea for improving the efficacy of immunotherapy.