A Novel Strategy For Tumor Cell Vaccine That Inductive Expression Of GSDMD N-fragment Causes The Pyroptosis

[Back ground]The low immunogenicity and high heterogeneity of tumor and the formation of immunosuppressive microenvironment and physical barrier during the development of tumor are the main means for tumor to evade immune surveillance.Pyroptosis is a new form of immunogenic cell death(ICD),which releases a large number of inflammatory cytokines and danger signal molecules to activate the immune system,and plays an important role in the regulation of anti-infection immune response and anti-tumor immune response.GSDMD is a pore-forming protein,studies have shown that ectopic expression of the N-terminal active domain of GSDMD(GSDMD-NT)can cause lytic and inflammatory pyroptosis.In addition,the pyroptosis effector molecμle GSDMB/GSDME can interact with toxic lymphocytes and induce pyroptosis by granular enzyme cleavage and activation,inducing pyroptosis in less than 15%of tumor cells is sufficient to elicit a robust immune response to eliminate the entire tumor.Different tumor cells have different induction conditions for ICD,different tumors have different responses to different inducers,and often require large doses with potential toxic side effects.Based on the unique mechanism of pyroptosis,overexpression of GSDMD-NT in tumor cells through tetracycline induction system can achieve universal and efficient pyroptosis induction,and GSDMD-NT can only play a hole effect from the cell inner membrane,without damaging the surrounding normal tissues and causing the side effect of pyroptosis.In response to highly heterogeneous tumors,pyrolysis can provide personalized,abundant tumor-associated antigens,while releasing a large number of inflammatory cytokines to assist in breaking down tumor immunosuppression mechanisms.Therefore,the induction of tumor cell immunogenic pyroptosis is of great significance for overcoming the mechanism of tumor immunosuppression and immune escape,reshaping the monitoring and killing of tumor by the immune system,and achieving significant clinical therapeutic effects.[Objective]This study based on pyroptosis clear mechanism and regμlatory role in the antitumor immunity,aims to explore safe and effective tumor cells pyroptosis induction strategy,reveals the characteristics and effect mechanism of immune and immune intervention strategy on the basis of the optimization,in order to enhance clinical therapeutic potential of tumor cell vaccine.[Methods]The gene expression system mediated by the recombinant lentiviral vector was used to screen the tumor cell lines that induce stable expression of GSDMD-NT.Cell proliferation was detected by CCK-8 in vitro,pyroptosis morphology was observed by microscope,pyroptosis ratio was detected by flow cytometry with double staining AnnexinV and 7-AAD molecμle,and the expression and localization of GSDMD-NT were detected by immunofluorescence.The immunogenicity of pyroptosis was investigated in vitro,Real-time qPCR was used to detect the expression of corresponding molecμlar transcription levels,Western blot was used to detect the expression of corresponding molecμlar protein levels and ELISA was used to detect the release of inflammatory cytokines.Transwell chamber was used to evaluate the influence of pyroptosis tumor cells on the migration and recruitment of BMDCs,and flow cytometry was used to detect the maturation of BMDCs.In vivo induction of GSDMD-NT to induce tumor cell pyroptosis in mice to conduct immune intervention on tumors,Flow cytometry was used to detect the activation of immune effector cells and the infiltration of immunosuppressor cells in spleen and tumors of mice.[Results](1)The tetracycline induced expression of GSDMD-NT lentiviral plasmid was successfully constructed,and the normal operation of the plasmid was verified by plasmid enzyme digestion and 293FT transient transfection protein expression detection;(2)Three mouse tumor cell lines(TC-1,4T1,CT26)with stable expression of GSDMD-NT induced by tetracycline were successfully screened,transcriptional and protein expression of GSDMD-NT increased after induction of tetracycline;(3)Pyrolysis of tumor cells was successfully induced in vitro,GSDMD-NT was applied to the cell membrane,the typical pyrolysis morphology of cell swelling and enlargement,membrane rupture,nuclear pyknosis and DNA rupture were observed under the microscope;(4)CCK-8 cell proliferation assay verified that the uninduced expression of GSDMD-NT gene after modification did not affect cell proliferation,but significantly inhibited cell proliferation when induced expression;(5)During the occurrence of tumor cell pyrotosis,the release of inflammatory cytokines IL-18,IL-1β,IL-33,IL-6 was increased by ELISA detection,the expression of HMGB1 was increased by Western blot detection,and the release of ATP,LDH,HMGB1 in the supernatant of cell pyrotosis was increased,the expressions of Hsp70/90,CXCL9 and H-2Kb were up-regμlated and the expressions of PD-L1 were down-regμlated by Real time qPCR;(6)The pyroptosis tumor cells coμld promote the migration and maturation of BMDCs,and the expressions of CD80,CD86,MHC I and MHC II molecules were increased;(7)The tumor model of TC-1 mouse subcutaneous transplantation which was modified by GSDMD-NT gene could completely eliminate the tumor of 20～800mm3 by inducing pyroptosis in vivo,and produced immune protection for at least 30 days against the wild-type TC-1 tumor which was re-inoculated;(8)In vivo induced pyroptosis in 4T1 breast cancer model in situ and CT26 subcutaneous graft tumor model by GSDMD-NT gene modified coμld significantly inhibit tumor growth,but could not remove the whole tumor;(9)Different doses GSDMD-NT gene modified tumor cells after subcutaneous injection of TC-1 directly induce pyroptosis,prove that gene modified tumor growth can be artificially controlled and not into the tumor,its immune protection effect and the cell dose into positive correlation;(10)We would intratumoral injection of GSDMD-NT gene modified TC-1 tumor cell vaccine to induce pyroptosis in vivo,which could significantly inhibit the growth of established TC-1 tumor,flow cytometry showed that the number of anti-tumor CTLs、NKs effector cells in spleen and tumor increased,while the number of MDSCs inhibitory cells decreased;(11)GSDMD-NT gene modified 4T1 breast cancer in situ tumor model and CT26 colorectal cancer subcutaneous graft tumor model induced cell apoptosis in vivo at different tumor sizes,significantly inhibited tumor growth at 6-40mm3 size,and showed no significant antitumor effect at 40-200mm3 size.Flow cytometry analysis showed that the new type of pyroptosis tumor cell vaccine significantly inhibited the growth of 4T1 breast tumors in situ by stimulating the number of activated systemic anti-tumor CTLs effector cells and reducing the number of MDSCs inhibitory cells.[Conclusion]The stable expression of induced pyroptosis core effector molecule GSDMD-NT is a highly effective and universal way to induce immunogenic death of tumor cells.The pyroptosis induction of tumor cells in mice provides a broad spectrum of individualized tumor antigens and sufficient expression of ICD characters,thus providing a strong immune-stimulating signal to stimulate a strong anti-tumor immune response.Among them,high immunogenic TC-1 tumor containing HPV-E6/E7 antigen has the effect of complete tumor elimination,but for 4T1/CT26 tumor with strong immunosuppression,the anti-tumor effect does not reach the same effect.In conclusion,this study proposes.a new strategy of tumor cell vaccine based on cell immunogenicity induced death,aiming at the challenge of tumor heterogeneity,which provides a useful discussion and important reference for the research of new tumor immune intervention therapy and tumor vaccine.