| Background:Although immunotherapies have achieved encouraging success in the clinical treatment of tumors,the response rate among tumor patients remains low.One of the major reasons is the presence of immunosuppressive tumor microenvironment(ITME),which is responsible for the poor effectiveness of current tumor immunotherapy.Tumor cells establish pro-tumor ITME by recruiting various immunosuppressive cells,resulting in a low infiltration of cytotoxic T lymphocytes(CTLs)in tumor tissues and the failure of activating an effective anti-tumor immune response.In addition,the low immunogenicity and high heterogeneity of tumors allow them to evade surveillance and attack by the immune system.Therefore,how to improve the immunogenicity of tumors and remodel ITME has become a critical issue to be addressed in current tumor immunotherapy.Induction of immunogenic cell death(ICD)in tumor cells has become an effective strategy to improve ITME.In addition to releasing neoantigenic epitopes,tumor cells undergoing ICD also release damage associated molecular patterns(DAMPs)such as high mobility group protein 1(HMGB1),ATP and calreticulin(CRT),which activate adaptive immune responses and produce long-lasting antitumor immune effects.Therefore,induction of ICD in tumor cells is considered to be a very promising strategy for tumor immunotherapy.However,there are few chemotherapeutic agents that can be used as ICD inducers in clinical practice due to their severe side effects and unclear mechanism.Therefore,finding novel effective ICD inducers,elucidating their mechanism and applying them to tumor therapy are of great significance to activate antitumor immunity and produce long-term effects.Raddeanin A(RA)is a natural triterpenoid saponin isolated from the Chinese Medicinal Herb Anemone Raddeana Regel.Its role in inducing ICD has not been reported so far.In this study,taking the induction of ICD and activation of T cells as the starting point,we evaluated the in vitro and in vivo antitumor activity of RA,and conducted an in-depth study on the mechanism of its induction of ICD.Methods:Screening for compounds that induce tumor ICD while activating T cells from a library of natural active compounds was based on HMGB1-Gluc reporter gene assay and LacZ reporter gene assay.The protein levels of interleukin-2(IL-2),interferon gamma(IFN-y)and tumor necrosis factor alpha(TNF-α)secreted by B3Z/OT-I T cells after coculturing with B16 tumor cells and bone marrow-derived dendritic cells(BMDCs)were measured by enzyme linked immunosorbent assay(ELISA).The changes in levels of interferon bata(IFN-β),TNF-α and C-X-C motif chemokine ligand 10(CXCL10)in B16/MC38 tumor cells were also determined by ELISA.The expression of CRT on the surface of B16/MC38 tumor cells treated with RA,and levels of CD69,IFN-y and granzyme B(GZMB)on B3Z/OT-I T cells and CD40,CD80,CD86,MHC-Ⅰ,MHC-Ⅱand MHC-Ⅰ SIINFEKL on the surface of BMDCs were detected by flow cytometry.RNA sequencing was used to screen for differentially expressed genes in tumor cells treated with DMSO or RA.qPCR detected mRNA changes of Cxcl10,Isg15,Ifnb1,Irf9,Ifit1,Ccl5 and Tnf in tumor cells treated with RA.Western Blot was carried out to detect TBK1,IRF3 and P65 proteins and their phosphorylation levels in tumor cells treated with RA.The potential binding proteins of RA in tumor cells were screened by biotin-streptavidin binding system and LC-MS/MS,and the direct targets of RA were validated in combination with siRNA gene silencing assays.Cellular thermal shift assay(CETSA)was performed to verify the direct interaction of RA with TAR DNA binding protein-43(TDP-43).Surface plasmon resonance analysis experiments were performed to determine the dissociation constants of TDP-43 with RA or oleanolic acid(OA).Prediction of the reciprocal amino acid sites of TDP-43 and RA was realized by molecular docking model.The nucleus,cytoplasm and mitochondria in B16 and MC38 cells were extracted and separated by subcellular hierarchical separation assay,and the distribution of TDP-43 in tumor cells treated with RA was detected by Western Blot.Flow cytometry was used to detect the apoptosis of tumor cells treated with RA and the changes in ROS levels in mitochondria and mitochondrial membrane potential.Co-localization of cGAS and mitochondrial DNA(mtDNA)was observed by immunofluorescence experiments and laser confocalization.Genes such as Tdp43 were silenced by siRNA.Knockout of Tdp43 in tumor cells was carried out with CRISPR-Cas9 gene editing technology.The in vivo antitumor activity of RA was evaluated by B16 and MC38 transplantation tumor models in mice,and changes in body weight,tumor volume and tumor weight were measured.ELISA was performed to detect the changes of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in mice serum.Multicolor flow cytometry detects the changes in the ratios of T cells and dendritic cells(DCs)and their activity in the tumor microenvironment(TME),as well as the infiltration of immunosuppressive cell populations such as myeloid-derived suppressor cells(MDSCs)and regulatory T cells(Tregs).Results:Screening of ICD-inducing compounds from a library of natural active compounds by HMGB1-Gluc reporter gene and LacZ reporter gene assays revealed that RA significantly induced HMGB1 release from tumor cells and LacZ activity in co-cultured B3Z T cells.Experiments in a mouse bilateral tumor model showed that RA induced immunogenicity and immune memory in vivo,suggesting that RA is an effective ICD inducer.Further validation by ELISA and flow cytometry revealed that RA induced ATP release from tumor cells and promoted CRT exposure on the surface of tumor cell;meanwhile,RA promoted co-cultured B3Z/OT-I T cells to express CD69 and secrete IL-2,IFN-y.TNF-α,and GZMB,which confirms that RA enhances the immunogenicity of tumors.Flow cytometry further showed that RA significantly increased the expression of CD40,CD80,CD86,MHC-Ⅰ,MHC-Ⅱ and MHC-Ⅰ SIINFEKL on the surface of BMDCs co-incubated with tumor cells,indicating that RA promotes the maturation of DCs and enhances their function of antigen presentation.The results of MC38 and B16 mouse transplantation tumor models showed that RA had good anti-tumor activity in vivo and significantly inhibited the growth of transplanted tumors,while promoting the infiltration and activation of CD8+T cells and DCs in TME.However,RA showed no significant tumor suppressive effect in nude mice or when used together with CD8 neutralizing antibody,confirming that the anti-tumor activity of RA was dependent on T cells.Moreover,the tumor suppressive effect of RA was also reversed when DCs were eliminated from mice using cytochrome C,suggesting that the antitumor effect of RA also requires DCs to mediate.Targets draping results revealed that RA could interact directly with the TDP-43 protein in tumor cells,and the binding sites are Ala228 and His256.Further mechanistic studies revealed that RA binding to TDP-43 protein in tumor cells promotes its enrichment in mitochondria,leading to a decrease in mitochondrial membrane potential,and an increase in ROS levels and mtDNA release,activating the cGAS-STING-type I IFN signaling pathway,which induces ICD and activates DCs and CD8+T cells in TME.The mouse MC38 transplantation experiment further confirmed that RA in combination with PD-1 monoclonal antibody synergistically increased tumor-infiltrating CD8+T cells and DCs and reduced the accumulation of Tregs and M-MDSCs,thereby enhancing the in vivo tumor immunotherapeutic effects of PD-1 monoclonal antibody.Conclusion:This study found that RA induced ICD through the mtDNA-cGAS/STING pathway,promoted DCs-dependent activation of CD8+T cells and reshaped the tumor immune microenvironment;meanwhile,it revealed the important role of TDP-43 in tumor immunotherapy.Moreover,the combination of RA with immunotherapy such as PD-1 monoclonal antibody has great potential and broad application prospects,which has far-reaching implications for the research of natural active product RA. |
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