中国临床药学杂志

目的 揭示癌症相关成纤维细胞(CAFs)通过分泌CXCL12降低CCL5的表达，进而影响食管鳞癌(ESCC)中CD8~+T细胞增殖的可能分子机制。方法 从TCGA数据库获取ESCC患者的癌组织转录组数据，利用R语言中“CIBERSORT”“MCPcounter”“xCell”分析细胞比例、丰度和富集情况，依据CD8~+T细胞评分中位数将样本划分为高、低表达2组。基于GEO数据库中GSE160269数据集的ESCC单细胞RNA测序(scRNA-seq)数据，采用“Seurat”包识别CD8~+T细胞特征基因；利用“limma”包对GSE164158数据集的bulk RNA-seq数据进行差异表达分析。收集10例ESCC患者的癌及癌旁组织进行全转录组测序，免疫组化验证CCL5表达。自3例患者新鲜组织分离CAFs与成纤维细胞(NFs)，从健康人外周血单个核细胞(PBMC)中提取CD8~+T细胞，建立共培养体系。通过免疫荧光、RT-qPCR、ELISA和流式细胞术，评估细胞表型、CCL5表达及CD8~+T细胞功能。结果 基于TCGA-ESCC数据集分析发现，趋化因子(CXCL9、CXCL10、CXCL11、CCL4L2、CCL5)与ESCC-CD8~+T细胞数量和增殖活性相关。scRNA-seq分析发现CCL5可能是ESCC-CD8~+T细胞的marker基因。通过GSE164158 bulk RNA-seq数据集差异分析发现CCL5和CD8A在人ESCC样本中呈低表达。体外细胞实验结果表明：CAFs通过分泌CXCL12抑制CCL5表达并降低CD8~+T细胞数量。结论 ESCC相关CAFs通过分泌CXCL12降低CCL5表达，抑制CD8~+T细胞增殖，从而对ESCC肿瘤微环境产生影响。

AIM To determine how cancer-associated fibroblasts(CAFs) in esophageal squamous cell carcinoma(ESCC) inhibit CD8⁺ T cell proliferation by downregulating CCL5 expression via CXCL12 secretion. METHODS The transcriptome data from the patient's cancer tissue were downloaded from the TCGA database, and the R language "CIBERSORT", "MCPcounter" and "xCell" packages were used to analyze cell type proportion, cell abundance, and cell enrichment. According to the median of the calculated results, the samples were divided into high and low CD8⁺ T groups. The ESCC scRNA-seq data of CD8⁺ T cells in the GSE160269 data set of the GEO database were analyzed by the "Seurat" package of the R language, and the differential genes of ESCC bulk RNA-seq data in the GSE164158 data set of the GEO database were analyzed by the "limma" package of the R language. Ten cases of ESCC patient cancer tissues and their adjacent normal tissues for whole transcriptome sequencing were collected, and the CCL5 expression levels in clinical tissues were detected using immunohistochemistry. CAFs cells and normal fibroblasts(NFs) from fresh cancer tissues and adjacent normal tissues of 3 ESCC patients were isolated, and CD8⁺ T cells were obtained from human peripheral blood mononuclear cells(PBMC) to establish a co-culture system. ESCC cell lines were selected to verify the mechanism in vitro. The cell phenotype, CCL5 expression and CD8~+T cell function were evaluated by immunofluorescence, RT-qPCR, ELISA and flow cytometry. RESULTS Chemokines(CXCL9, CXCL10, CXCL11, CCL4L2, CCL5) were correlated with the number and proliferation activity of ESCC-CD8~+T cells based on the TCGA-ESCC dataset. Single-cell RNAsequencing(scRNA-seq) analysis revealed that CCL5 might be a marker gene for ESCC-CD8⁺ T cells. The differential analysis of the GSE164158 bulk RNA-seq dataset showed that CCL5 and CD8A were lowly expressed in human ESCC samples. In vitro cell experiments showed that CAFs inhibited the expression of CCL5 and reduced the number of CD8⁺ T cells by secreting CXCL12. CONCLUSION CAFs inhibit CD8⁺ T cell proliferation by reducing CCL5 expression through CXCL12 secretion, thereby affecting the tumor microenvironment of ESCC.