The Synergistic Effect And Mechanism Of Microwave Ablation In Combination With TIGIT Inhibitor Against Cancer

Objective:Immunotherapy based on immune checkpoint inhibitors has achieved great success which is involved in the cancer therapy and the combination of immunotherapy with chemotherapy,radiotherapy and local treatment has become the mainstream of comprehensive cancer treatment at present.Microwave ablation(MWA)is a minimally invasive anti-tumor therapy,which can trigger the specific T cell anti-tumor immune response.MWA in combination with immunotherapy has made important progress in some preclinical and clinical studies.T cell immunoglobulin and immunoreceptor tyrosine inhibitory motif domain(TIGIT)is a novel immune checkpoint inhibitor molecular.In this study,we used the mouse colon cancer model to detect the expression of TIGIT in spleen lymphocytes and contralateral tumor infiltrating lymphocytes(TILs)before and after MWA treatment,and to prove the synergistic effect and mechanism of MWA in combination with TIGIT inhibitor against cancer.We hope MWA in combination with immunotherapy can provide new ideas to reverse the immunosuppressive tumor microenvironment and improve the therapeutic effect.Methods:In this study,we constructed the bilateral dorsal transplanted tumor model of mice,and then detected the expression of TIGIT in spleen lymphocytes and tumor infiltrating lymphocytes before MWA.We also detected the expression of TIGIT in contralateral tumor infiltrating lymphocytes before and after MWA treatment.We analyzed single cell RNA sequencing(scRNA-seq)data of the published MC38 colon cancer model and Panc02 subcutaneous tumor model,then analyzed the expression of TIGIT before and after ablation of TILs.Subsequently,we evaluated the antitumor effect of MWA alone or in combination with TIGIT inhibitor by monitoring tumor growth and survival in mice.In our study,we chose the blockade monoclonal antibody as TIGIT inhibitor.In addition,the percentages of TILs and the effector molecular changes of CD8+T and NK cells were detected by flow cytometry.Through the depletion of CD8 and NK,it is further verified that TIGIT inhibitor in combination with MWA can reverse immunosuppression by enhancing the function of CD8+T and NK cells.Finally,we constructed the bilateral dorsal transplanted tumor model of MC38 colon cancer and divided them into MWA and MWA in combination with TIGIT inhibitor group.We used magnetic bead enrichment and flow sorting to collect tumor infiltrating CD45+immune cells.And then single cell RNA sequencing(scRNA-seq)was performed.We analyzed scRNA-seq data in our study to compare the changes of cell cluster,proportion and function of immune cell subsets between MWA and MWA in combination with TIGIT inhibitor groups.At the same time,the cellular communication between lymphocytes and myeloid cells was analyzed by CellChat software to prove the synergistic effect and mechanism of MWA in combination with TIGIT inhibitor against cancer.Results:In this study,we found that the expression of immunosuppressive receptor TIGIT was highly in tumor infiltrating CD4+T(11.80±2.27%)than of spleen(6.52±1.54%),and the statistical analysis revealed significant difference(P<0.05).The expression of TIGIT in tumor infiltrating CD8+T and spleen was 15.30±0.60%,4.29±0.26%,respectively.And the statistical analysis revealed significant difference(P<0.0001).TIGIT was highly expressed in tumor infiltrating NK than of spleen(20.60±1.54%vs.9.24±2.40%,P<0.001).TIGIT was highly expressed in tumor infiltrating Foxp3+Tregs than of spleen(37.3±6.71%vs.5.06±1.49%,P<0.001).ScRNA-seq data also showed that TIGIT was expressed in conventional T cells,NK and Tregs in MC38 colon cancer model,and the expression was the most obvious in Tregs.We constructed the bilateral dorsal transplanted tumor model of mice treated with MWA.After MWA treatment,we found that the expression of TIGIT was upregulated in the contralateral tumor infiltrating CD4+T,CD8+T and NK cells.Furthermore,the expression of TIGIT was upregulated in the T lymphocytes in the contralateral tumor after ablation by analyzed scRNA-seq data,while the expression of other immune cells was very low.MWA in combination with TIGIT inhibitor could synergistically inhibit the growth of contralateral tumors.And we found that the survival time of the combined mice was prolonged.MWA in combination with TIGIT inhibitor could increase the frequencies of CD45+TILs,CD8+TILs,CD4+TILs,and NK cells.The combination with MWA and TIGIT inhibitor could increase the production of IFN-γ+CD8+,TNF-α+CD8+and GZMB+CD8+which can promote the antitumor immune response and the production of CD107a+NK,IFNγ+NK and TNF-α+NK in distant tumors.Using scRNA-seq analysis,we found that there was no significant difference in the proportion of stem like CD8+T,interferon-induced CD8+T and cycling CD8+T cells between the mice treated with MWA and MWA in combination with TIGIT inhibitor,while MWA in combination with TIGIT inhibitor could increase the percentage of effector CD8+T cells,reduce the number of exhausted CD8+T cells.We found that pathways associated with TNFα,TGF-β,oxidative phosphorylation,and glycolysis responses were upregulated in the effector CD8+T cells in the MWA in combination with TIGIT inhibitor group,whereas NFκB-mediated TNF-α signaling,TGF-β signaling,G2/M checkpoint,IFN-y,fatty-acid metabolism,oxidative phosphorylation,and glycolysis pathways were upregulated in the exhausted CD8+T cells in the MWA in combination with TIGIT inhibitor group.We also found that CXCR3 was up-regulated in stem-like CD8+T cells,effector CD8+T cells,interferon-induced CD8+T cells,and cycling CD8+T cells.And the percentage of neutrophils,monocytes,tumor-associated macrophages 1(TAM1),TAM2,circulating macrophages and Dendritic cell(DC)was similar in MWA and MWA in combination with TIGIT inhibitor groups.The expression of chemokines CCL5,CXCL9 and CXCL16 were upregulated and the expression of CCL2 and CCL7 were decreased in the MWA in combination with TIGIT blockade group.After MWA in combination with TIGIT inhibitor,the intercellular communication of immune subsets was changed,the interaction between myeloid cells was significantly weakened,and the interaction between T cells was significantly enhanced.Furthermore,the CCL and CXCL signals in CD8+T cells in MWA in combination with inhibitor group were significantly increased.Conclusion:TIGIT which is an immune checkpoint is involved in the immunosuppression of the tumor microenvironment after MWA treatment.The combination treatment of MWA and TIGIT inhibitor can boost the antitumor immune response which is mediated by CD8+TILs,NK cells in distant tumors,reverse the exhaustion of CD8+T cells after MWA,reshape myeloid cells and change the intercellular communication of immune subsets in TME.MWA in combination with TIGIT inhibitor is a new combination of immunotherapy strategy which is of potential clinical application value.