| Objective:Radiotherapy（RT）is one of the three main methods of traditional treatment of tumors.About 70%of cancer patients need RT in the course of disease.Compared with chemotherapy,RT has the advantages of relatively less systemic side effects,which is an effective treatment measure in the treatment of unresectable tumors and the prevention of local recurrence after surgery.It is well known that RT can cause tumor cell death by damaging DNA.However,some studies have shown that the immune system can promote tumor cell death in the radiation field.At present,people gradually realize the importance of host immune system to affect the curative effect of RT.Existing studies have found that immune cells in radiation fields play a crucial role in promoting tumor cell death.RT has been proved to play a beneficial regulatory role in immune response,such as DNA damage and apoptosis,triggering the death of immunogenic cells,enhancing antigen presentation,activating cytotoxic T cells and changing the characteristics of key immune cells in tumor microenvironment.Therefore,the effective combination of RT and immunotherapy has become an important means in the current research on comprehensive treatment of cancers.In recent years,cancer immunotherapy has made important breakthroughs,especially the blocking therapy of immune checkpoints,including T cell metastasis,dendritic cell vaccine,polypeptide vaccine,oncolytic virus,cytokine therapy,agonist monoclonal antibody,antagonistic monoclonal antibody and small molecule drugs.Immune checkpoint blockade therapy can effectively inhibit tumor growth and greatly change the prognosis of cancer patients.Up to now,immune checkpoint inhibitors targeting programmed cell death protein-1（PD-1）,programmed cell death protein ligand-1（PD-L1）and cytotoxic T lymphocyte-related protein-4 have become the focus of many basic research and clinical trials,and have achieved certain results in some cancers.However,while some patients get clinical response,most patients have little benefit or no response to the blockade of immune checkpoints,and some effective patients have tumor progression after transient tumor control.Therefore,researchers are exploring multi-modal treatment to improve tumor control by combining immune checkpoint blockade therapy with traditional cancer therapy.More and more preclinical studies and clinical observations have shown that RT may have a strong driving force for enhancing the efficacy of immune checkpoint blockers,because RT can effectively activate anti-tumor immune responses and overcome tumor resistance.Existing data showed that immunotherapy combined with RT can better control local and systemic tumors.However,whether internal or acquired,treatment resistance is still widespread.Therefore,it is necessary to develop new RT combined with immune checkpoint inhibitors.T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain（TIGIT）（also known as WUCAM,Vstm3,VSIG9）,which is a member of the poliovirus receptor（PVR,also known as CD155,Necl5 and Tage4）/Nectin family,the family is a subset of immunoglobulin superfamily.The expression of TIGIT was strictly limited to lymphocytes,with the highest expression in regulatory and effective CD4+T cells,auxiliary CD4+T cells,effective CD8+T cells and natural killer cells.Through the screening of secretory proteins,it was found that CD155 was a high-affinity homologous receptor of TIGIT.Due to the extensive expression of CD155 and TIGIT in tumor and immune cells,CD155/TIGIT pathway may play an important role in the whole cancer immune cycle.Blocking CD155/TIGIT pathway has achieved good results in a variety of preclinical tumor models.At the same time,clinical trials on blocking CD155/TIGIT have been carried out and some achievements have been made,especially when combined blocking PD1/PD-L1 pathway.As far as we know,whether blocking the CD155/TIGIT pathway improves the control of RT on tumors remains unknown.Therefore,this study first evaluated the expression of CD155/TIGIT in clinical samples by immunohistochemistry,analyzed the influence of its expression on the prognosis of patients receiving RT,and evaluated the value of this signaling pathway in the prognosis of tumor patients.Secondly,the effect of RT on the expression of CD155/TIGIT was explored through experimental studies to evaluate whether RT mediated radiation resistance by up-regulating CD155/TIGIT pathway and inhibit the activity of immune cells.Finally,the tumor-bearing mice model was constructed to evaluate whether blocking this signaling pathway by anti-TIGIT antibody combined with RT can produce synergistic anti-tumor effect,and to explore the interaction mechanism between RT and anti-TIGIT treatment,so as to provide theoretical and experimental basis for the development of new combined treatment models.Methods:1.The clinical pathological data and pathological sections of 114 patients with primary small cell carcinoma of the esophagus（PSCCE）were collected.The expression of CD155/TIGIT in PSCCE tumor and adjacent tissues was evaluated by immunohistochemical staining.The correlation between CD155/TIGIT expression and clinicopathological factors was evaluated.The effect of CD155 and TIGIT expression on prognosis were evaluated by univariate and multivariate analysis.On the basis of multivariate analysis,the Nomogram was constructed.The prediction performance of Nomogram is evaluated by consistency index（C-index）and calibration diagram.The clinical utility of the nomogram was assessed via decision curve analysis（DCA）.2.The expression of CD155 in tumor cells and its influence on clinical prognosis were analyzed through the public databases（Kaplan-Meier plotter database,Gene Expression Profiling Interactive Analysis and EMBL-EBI expression profile database）.The clinicopathological data of 118 patients with esophageal squamous cell carcinoma（ESCC）undergoing radical chemoradiotherapy and 49 patients undergoing neoadjuvant chemoradiotherapy（n CRT）were collected.The expression of CD155 was evaluated by immunohistochemical staining.The effect of CD155 expression in tumor biopsy samples before treatment on the prognosis of patients receiving RT was analyzed.The difference of CD155 expression in tumor samples of ESCC patients before and after RT was compared.3.Western blot（WB）was used to analyze the expression of CD155 in human lung cancer cell lines,human ESCC cell lines and ESCC radioresistant cell lines,and to evaluate the effect of RT on the expression of CD155.The potential mechanism of RT regulating CD155 expression was verified by small interfering RNA silencing target gene,WB analysis and dual luciferase assay.CD155 knockout mouse colon cancer cell line（MC38）was constructed by CRISPR/Cas9 technology.In vitro,the effects of CD155 on tumor radiosensitivity were evaluated by cell counting kit-8（CCK-8）,clone formation assay,apoptosis assay and other experimental methods.Through the establishment of nude mouse tumor model,we found that the tumor growth was further inhibited by CD155 knockout combined with RT compared with RT alone.In addition,the tumor-bearing mouse model with normal immune function confirmed that tumor growth was more significantly inhibited in CD155 knockout combined with RT group than RT alone.4.The tumor tissue samples of 23 ESCC patients receiving n CRT before and after RT were subjected to multiple immunofluorescence staining.Firstly,the difference in the number of CD8+T cells,CD4+T cells and NK cells infiltrated by tumor tissue before and after RT was evaluated.At the same time,the changes of TIGIT expression on CD8+T cells,CD4+T cells and NK cells after RT were compared to evaluate the effect of TIGIT expression level on the clinical pathological remission of ESCC patients after receiving n CRT.MC38,LLC and B16 F10 tumor-bearing mice models were established.The effects of anti-TIGIT treatment on RT were evaluated by comparing tumor growth curve,survival curve and distant tumor control.The potential value of CD103+dendritic cells（DCs）in the combination of radiotherapy and anti-TIGIT treatment was evaluated by establishing the wild type and Batf3 defect(Batf3-/-)tumor-bearing mice model.Fms-related tyrosine kinase 3 ligand（Flt3L）was used to further confirm the role of CD103+DCs in RT combined with anti-TIGIT treatment.Results:1.Through immunohistochemical scoring,we found that CD155 and TIGIT were often overexpressed in PSCCE tumor tissues.The expression of CD155 was positively correlated with TIGIT（P<0.001）,and CD155 was significantly correlated with tumor size,T stage,distant metastasis,TNM stage and Ki-67 score（P<0.05）.The expression of TIGIT was significantly correlated with tumor size,T stage,distant metastasis and TNM stage（P<0.05）.Patients with high expression of CD155 and TIGIT had significantly shorter overall survival（OS）and progression free survival（PFS）than those with low expression.Multivariate regression analysis showed that CD155expression and treatment strategy were independent prognostic factors for PSCCE patients.OS was calibrated well（C-index=0.724）in the verification step after Nomogram model was constructed,and DCA proved that the model had satisfactory clinical efficacy.In conclusion,our results show that CD155 and TIGIT are highly expressed in PSCCE patients,and are associated with shorter OS and PFS,which can be used as biomarkers for predicting PSCCE patients survival and RT efficacy.2.Kaplan-Meier plotter database analysis showed that patients with high expression of CD155 often had poor OS and PFS in lung cancer patients receiving RT.Through the analysis of ESCC patients receiving radical CRT,we found that the OS rate of patients with low expression of CD155 was significantly higher than that of patients with high expression of CD155,and PFS also obtained similar results.Through the immunohistochemical score of CD155 expression,we found that in ESCC patients receiving n CRT,the high expression of CD155 was correlated with tumor regression grading（TRG）,indicating poor response to RT.We found that CD155 was often overexpressed in tumor tissues and cell lines through gene expression profile interaction analysis data set and EMBL-EBI expression profile database analysis（96.9%）.WB analysis of common lung cancer and ESCC cell lines showed that CD155 protein levels in five lung cancer（5/7）and four ESCC（4/6）cell lines were significantly high,which further confirmed that CD155 was often highly expressed in tumor cells.3.RT significantly increased the expression of CD155 in lung cancer cell lines H1299 and SPCA-1.The expression of CD155 in radioresistant ESCC cell lines（KYSE150R and KYSE 30R）was significantly higher than that in wild-type cell lines.RT increased the expression of CD155 in tumor tissues of patients receiving n CRT.q PCR and WB analysis showed that the protein levels of CD155 in LLC and MC38 cells were slightly up-regulated after RT,but the m RNA levels were significantly increased.In mechanism,RT can increase Rel B entry into the nucleus and promote CD155 m RNA transcription.Dual luciferase assay confirmed that Rel B binds to CD155 promoter region.In vitro experiments confirmed that CD155 knockout could increase cell apoptosis,decrease cell survival rate and decrease clone formation ability after RT.The establishment of nude mouse tumor model further confirmed that CD155 could affect the sensitivity to RT by regulating the activity of tumor cells.In addition,the tumor-bearing mice model with normal immune function confirmed that CD155 could regulate radiosensitivity by affecting CD8+T cells in tumor microenvironment.4.Through multiple immunofluorescence staining of tumor tissues,we found that RT could significantly increase the number of CD8+T cells and NK cells infiltrated by tumor.In addition,TIGIT expression was significantly up-regulated in CD8+T cells,CD4+T cells and NK cells after RT.In the tumor-bearing mouse model,the TIGIT expression of lymphocytes in tumor and tumor drainage lymph nodes was also significantly increased.Through the study of three tumor-bearing mice models,it was found that anti-TIGIT treatment could increase the control of RT on primary tumors and distant tumors,prolong the survival time of tumor-bearing mice,and establish the specific immune memory effect.In mechanism,the synergistic anti-tumor effect of anti-TIGIT combined with RT is mainly through increasing the infiltration of CD8+T cells in tumors and the secretion of TNF-αand IFN-γin CD8+T cells.In the Batf3-/-mouse model,it was found that the therapeutic effect of RT combined with anti-TIGIT was offset,indicating that CD103+DCs were needed to promote anti-tumor effect in RT combined with anti-TIGIT treatment.In addition,Flt3L treatment enhanced the tumor response to RT combined with anti-TIGIT treatment.Conclusion:1.CD155 and TIGIT are highly expressed in PSCCE patients and are related to short OS and PFS.At the same time,the high expression of CD155 indicates poor tumor pathological complete response rate,which supports the role of CD155 and TIGIT as prognostic markers for cancer patients.2.RT can mediate tumor immunosuppression and reduce radiosensitivity by up-regulating the expression of CD155/TIGIT.3.Blocking the CD155/TIGIT signaling pathway can increase the sensitivity of RT and improve the efficacy of RT by affecting the tumor cells themselves and the activity of tumor infiltrating CD8+T lymphocytes.4.The efficacy of RT combined with anti-TIGIT depends on CD103+DCs,suggesting that targeting CD155/TIGIT pathway and increasing CD103+DCs tissue infiltration can improve the clinical application potential of RT. |
