上海口腔医学

上海口腔医学 ›› 2024, Vol. 33 ›› Issue (6): 586-593.doi: 10.19439/j.sjos.2024.06.004

• 论著 • 上一篇    下一篇

C3H/He鼠头颈鳞癌转移淋巴结内CD8+ T细胞的关键基因、通路和免疫检查点表达分析

周迪*, 韩楠男*, 李华盛, 严明, 阮敏   

  1. 上海交通大学医学院附属第九人民医院 口腔颌面-头颈肿瘤科, 上海交通大学口腔医学院, 国家口腔医学中心, 国家口腔疾病临床医学研究中心, 上海市口腔医学重点实验室, 上海市口腔医学研究所, 上海 200011
  • 收稿日期:2024-02-01 修回日期:2024-04-16 出版日期:2024-12-25 发布日期:2025-01-07
  • 通讯作者: 阮敏,E-mail: doctorruanmin@sjtu.edu.cn
  • 作者简介:周迪（1997-）,女,硕士研究生,E-mail: castaliazhou@163.com;韩楠男（1993-）,女,博士研究生,E-mail: hannannan111@126.com。*并列第一作者

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

ZHOU Di, HAN Nan-nan, LI Hua-sheng, YAN Ming, RUAN Min   

  1. Department of Oromaxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology. Shanghai 200011, China
  • Received:2024-02-01 Revised:2024-04-16 Online:2024-12-25 Published:2025-01-07

PDF (PC)

10

摘要： 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小鼠腘窝淋巴结转移模型,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Pdcd1、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。

关键词: 头颈鳞癌, 淋巴结转移, C3H/He小鼠, CD8+ T细胞, PD-1

Abstract: 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 HNSCC, 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 differentially expressed genes. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed sequentially. Flow cytometry and multiplex immunohistochemical were used to detect the expression of four immune checkpoints of CD8+ T cells in metastatic lymph nodes of tumor-bearing mice. SPSS 26.0 software package was used for statistical 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 immune 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 targeting CD8+ T cells may become a new strategy for the prevention and cure for lymph node metastasis of HNSCC.

Key words: Head and neck squamous cell carcinoma, Lymph node metastasis, C3H/He mice, CD8+ T cell, PD-1

中图分类号: