Study On The Correlation Between Immune Cell Subsets In TME And Peripheral Blood And The Efficacy Of Immunotherapy In Esophageal Cancer

Background:Esophageal cancer is one of the most common malignant tumors in the digestive tract,its incidence is only second to gastric cancer and colorectal cancer.Esophageal squamous cell carcinoma is the most common histological subtype of esophageal carcinoma in China,and its onset is often occult.Besides,the traditional treatment effect is poor,and the overall prognosis is not good.In recent years,immunocheckpoint inhibitors acting on tumor microenvironment(TME)have made major breakthroughs in the treatment of esophageal squamous cell carcinoma,but only a fraction of patients benefit from them.Tumor microenvironment is a highly complex ecosystem composed of multiple cell types,which plays an important role in tumor biology,including proliferation,migration,and metastasis.Moreover,the heterogeneity of immune cell composition in tumor microenvironment is also closely related to immunotherapy efficacy,and single cell sequencing technology can provide a method to fully study immune cells in the complex tumor microenvironment at the single cell level.This study is based on single-cell sequencing data and flow cytometry analysis of peripheral blood immune cells of patients with esophageal cancer who have received immunotherapy,in order to identify the subsets of immune cells related to the efficacy of immunotherapy.Methods:1.Download the GSE145370 dataset from Gene Expression Omnibus(GEO).The dataset stores single-cell RNA sequencing data from cancer tissues and adjacent tissues of 7 patients with esophageal squamous cell carcinoma(ESCC)who underwent surgical resection.2.R software kit Seurat(v3)was applied for data quality control,dimension reduction,clustering,and visualization of cell subsets.3.Annotated and identified cell subsets according to known marker genes.4.Seurat was used to detect the difference in the expression of CD4 and CD8 in tumor tissues and adjacent tissues.The expression differences of CD134,CD137,CD44,CD62L and FOXP3 on CD4+T cells in tumor and paracancer tissues were detected.The expression differences of CD39,TIM3,PD1,CD44,CD62L,CD40L,CD137 and CD 103 on CD8+ T cells were detected.The expression difference of CD 103 and TIM3 in DCs was detected.5.Twenty patients with advanced esophageal squamous cell carcinoma who received anti-PD-1 antibody combined with chemotherapy as first-line treatment in the First Affiliated Hospital of Soochow University were selected and peripheral blood samples were collected before the first treatment and the third treatment.6.Through the preparation of cell suspension,antibody staining and flow cytometry analysis,the software FlowJo was used to analyze and sort out the data of flow cytometry,and the levels of peripheral blood immune cell subsets(CD39+CD8+T cells,PD1+CD8+T cells,TIM3+CD8+T cells,CD40L+CD8+T cells,CD44+CD62L+CD8+T cells,CD8+CD137+T cells,CD134+CD4+T cells,and TIM3+dendritic cells)were obtained before and after immunotherapy.7.Statistical methods such as survival analysis and Kruskal Wallis test were used to analyze whether various immune cell subsets were related to the efficacy of immunotherapy.Results:1.The expression level of CD8 in T cells in cancer or adjacent tissues is higher than that of CD4.The expression levels of CD 8 and CD4 in cancer tissues were higher than those in adjacent tissues.2.The number of CD8+T cells,CD39+CD8+T cells,TIM3+CD8+ T cells,PD1+CD8+T cells,CD137+CD8+T cells,and CD103+CD8+T cells in tumor tissue was significantly higher than that in adjacent tumor tissue.However,CD40L+CD8+T cells,CD44+CD8+T cells,and CD62L+CD8+T cells in adjacent tissue were significantly higher than those in tumor tissue.3.The expression levels of CD134,CD137,and FOXP3 on CD4+T cells in tumor tissue were significantly higher than those in adjacent tissues;The expression of CD44 was higher in the adjacent tissues;There was no significant difference in CD62L between the two.4.In tumor tissue,the expression level of TIM3 in DC cells is significantly higher than that of CD 103.In dendritic cells(DCs)of tumor tissue,the expression levels of TIM3 and CD103 are higher than those in adjacent tissues.5.After immunotherapy,the median change value of TIM3+CD8+T lymphocytes in peripheral blood of patients with esophageal cancer was 3.35%.Compared with patients with change value<3.35%,patients with change value>3.35%had shorter PFS The changes of TIM3+CD8+ T cells in patients with CR or PR were significantly lower than those in patients with PD.However,there was no statistically significant difference in change values between CR+PR and SD,or between SD and PD.CD39+CD8+T cells.PD1+CD8+ T cells,CD44+CD62+CD8+T cells,CD137+CD8+ T cells,CD40L+CD8+T cells had no correlation with PFS and efficacy.6.After immunotherapy in patients with esophageal cancer,the changes in the level of CD134+CD4+T cells in peripheral blood were not related to PFS or efficacy.7.After immunotherapy,the median change in peripheral blood TIM3+DCs levels in patients with esophageal cancer was 6.4%.Compared with patients with change value<6.4%,patients with change value>6.4%had a shorter trend in PFS,with no statistically significant difference.However,there were differences in the changes in TIM3+DCs among different therapeutic groups(P=0.039).Among them,the change value of TIM3+DCs in CR or PR patients was significantly lower than that in PD patients(P=0.042).There was no significant difference between CR+PR and SD.SD and PD(P>0.05).Conclusion:The CD8 T cell subpopulations that express CD39,TIM3,PD1,CD 137.and CD103,the CD4 T cell subpopulations that express CD134,CD137,and FOXP3,and the DC cell subpopulations that express TIM3,and CD 103 in cancer tissue are significantly higher than those in adjacent tissue,indicating that immune cells in TME of esophageal cancer are specifically activated by tumor antigens,constituting a unique immune microenvironment for cancer tissue.Different individuals with different levels of immune cell subsets within TME have different immune monitoring effects,which can affect the occurrence and development of tumors,and may also affect the effectiveness of immunotherapy.TIM3 is an immunosuppressive molecule and a surface marker of depleted T cells.Our peripheral blood T cell subpopulation assay found that patients with significantly increased TIM3+CD8+T cell subpopulations after immunotherapy for esophageal cancer have short PFS.It is suggested that changes in peripheral blood TIM3+CD8+T cell subsets may be an effective indicator for judging the efficacy of immunotherapy for esophageal cancer,which can be used to guide clinical immunotherapy,and the method is relatively convenient.