Analysis of hub genes,pathways and immune checkpoints of CD8+T cells in metastatic lymph nodes of head and neck squamous cell carcinoma in C3H/He mice
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目的:探讨C3H/He鼠头颈鳞癌(head and neck squamous cell carcinoma,HNSCC)转移淋巴结内CD8+T细胞的关键基因、通路和抑制性免疫检查点的表达.方法:应用头颈鳞癌SCC-7细胞系,构建免疫健全C3H/He小鼠胭窝淋巴结转移模型,利用免疫荧光染色技术确定淋巴结转移状态.利用流式细胞术分别分选出正常和转移淋巴结内的CD8+T细胞,进行转录组测序分析,筛选出差异表达基因,并进行GO功能富集分析和KEGG通路富集分析.采用流式细胞术和多重免疫荧光组织化学技术,检测荷瘤小鼠转移淋巴结内CD8+T细胞的4个免疫检查点的表达水平.采用SPSS 26.0软件包对数据进行统计学分析.结果:足垫注射SCC-7细胞4周后,C3H/He小鼠出现明显的腘窝引流淋巴结转移.对正常淋巴结和转移淋巴结内的CD8+T细胞进行转录组测序分析,筛选出差异表达基因912个,包括3个表达上调的抑制性免疫检查点相关基因Pdcdl、Lag3和Tigit.KEGG网络分析筛选出8个上调的肿瘤相关信号通路,包括Toll样受体信号通路、NF Kappa-B信号通路、TNF信号通路、MAPK信号通路、IL-17信号通路、NOD样受体信号通路、肿瘤中PD-L1和PD-1免疫检查点通路和p53信号通路.流式细胞术显示,小鼠转移淋巴结内CD8+T细胞出现PD-1和TIM-3蛋白高表达,多重免疫荧光进一步在头颈鳞癌患者的肿瘤转移淋巴结中确认CD8+T细胞高表达PD-1.结论:小鼠肿瘤转移淋巴结内CD8+T细胞的肿瘤相关信号通路显著激活,小鼠和头颈鳞癌患者转移淋巴结内CD8+T的PD-1表达水平显著增高,靶向CD8+T细胞的免疫治疗有可能成为头颈鳞癌淋巴结转移防治的新策略.
PURPOSE:To explore the expression of hub genes,pathways and inhibitory immune checkpoints of CD8+T cells in metastatic lymph nodes of head and neck squamous cell carcinoma(HNSCC)in C3H/He mice.METHODS:A popliteal lymph node metastasis model of immunocompetent C3H/He mice was constructed with SCC-7 cell line of HN-SCC,and the lymph node metastasis status was determined by immunofluorescence.CD8+T cells in normal and metastatic lymph nodes were sorted by flow cytometry,and transcriptome sequencing analysis was performed to screen out differen-tially expressed genes.GO functional enrichment analysis and KEGG pathway enrichment analysis were performed se-quentially.Flow cytometry and multiplex immunohistochemical were used to detect the expression of four immune check-points of CD8+T cells in metastatic lymph nodes of tumor-bearing mice.SPSS 26.0 software package was used for statisti-cal analysis.RESULTS:After 4 weeks of foot pad injection of SCC-7 cells,C3H/He mice displayed obvious metastasis in popliteal drainage lymph node.A total of 912 differentially expressed genes were screened out by transcriptome sequencing analysis of CD8+T cells in normal and metastatic lymph nodes,including three inhibitory immune checkpoint-related genes which were upregulated(Pdcd1,Lag3 and Tigit).Eight tumor-related signaling pathways were screened out by KEGG network analysis,including Toll-like receptor signaling pathway,NF Kappa-B signaling pathway,TNF signaling pathway,MAPK signaling pathway,IL-17 signaling pathway,NOD-like receptor signaling pathway,PD-L1 and PD-1 im-mune checkpoint pathway in tumors and p53 signaling pathway.Flow cytometry showed high expression level of PD-1 and TIM-3 in CD8+T cells in mouse metastatic lymph nodes.It was further confirmed by multiplex immunohistochemical that PD-1 was highly expressed in CD8+T cells in metastatic lymph nodes of patients with HNSCC.CONCLUSIONS:Tumor-related signaling pathway of CD8+T cells in mouse metastatic lymph nodes is significantly activated.The PD-1 expression level of CD8+T in metastatic lymph nodes of mice and patients with HNSCC is markedly increased.Immunotherapy target-ing CD8+T cells may become a new strategy for the prevention and cure for lymph node metastasis of HNSCC.
Head and neck squamous cell carcinomaLymph node metastasisC3H/He miceCD8+T cellPD-1
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