MC38结肠癌小鼠肿瘤浸润免疫细胞的动态变化

doi:10.12300/j.issn.1674-5817.2020.157

摘要/Abstract

摘要： 目的观察荷瘤小鼠肿瘤浸润免疫细胞在肿瘤发展过程中的动态变化。方法将小鼠结肠癌MC38细胞移植到24只C57BL/6J小鼠皮下,荷瘤后每周一次记录小鼠体质量及肿瘤大小的变化,每周一次采用流式细胞术检测肿瘤浸润免疫细胞水平,包括CD45⁺细胞、CD3⁺ T细胞、CD8⁺ T细胞、CD4⁺ T细胞、自然杀伤（natural killer,NK）细胞、树突状细胞、巨噬细胞、M1型巨噬细胞、M2型巨噬细胞、髓源抑制细胞（myeloid-derived suppressor cells,MDSC）、单核样髓源抑制细胞（monocytic-MDSC,M-MDSC）、粒样髓源抑制细胞（granulocytic-MDSC,M-MDSC）,共检测4周。结果随着小鼠结肠癌肿瘤的增长,CD45⁺细胞水平总体表现出下降趋势,CD8⁺ T细胞水平稳步下降至不可逆的水平,而CD4⁺ T细胞比例在荷瘤早期迅速增长,单位体积中浸润细胞数增长了近8倍,肿瘤浸润的T细胞几乎为CD4⁺表型。以NK细胞为代表的非特异性免疫应答水平持续下降,而树突状细胞在肿瘤内持续累积,并通过激活精氨酸酶1（Arginase-1,ARG1）和诱导型一氧化氮合成酶（inducible nitric oxide synthetase,iNOS）,进一步加深免疫抑制作用。巨噬细胞浸润水平整体无显著变化,但Ⅰ型巨噬细胞浸润细胞数的下滑速度显著高于Ⅱ型。单位体积肿瘤内MDSC数未出现下降趋势,在肿瘤内持续累积;其中G-MDSC浸润水平呈稳降趋势,M-MDSC浸润水平持续上升,且占比远高于G-MDSC。结论小鼠移植MC38结肠癌细胞后,肿瘤组织内免疫细胞浸润水平整体下降,促肿瘤免疫逐渐占据主导地位,从而促进结肠癌的发展。肿瘤内产生了Th1/Th2漂移现象,特异性和非特异性免疫应答水平均显著降低,浸润的单核细胞和粒细胞主要是Ly6C⁺表型。肿瘤相关树突状细胞和巨噬细胞发挥了重要的免疫抑制作用,CD4⁺ T细胞和M-MDSC可能是发挥促肿瘤免疫效应的关键点。

关键词: 结肠癌, 肿瘤浸润免疫细胞, 免疫微环境, 小鼠

Abstract: Objective To observe the dynamic changes of tumor-infiltrating immune cells in tumor-bearing mice during tumor development. Methods Mouse colon cancer MC38 cells were transplanted subcutaneously into 24 C57BL/6J mice, and the changes in body mass and tumor size of mice were recorded once a week after tumor-bearing, The levels of tumor-infiltrating immune cells, including CD45⁺ T cells, CD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, natural killer (NK) cells, dendritic cells, macrophages, M1-type macrophages, M2-type macrophages, myeloid-derived suppressor cells (MDSCs), mononuclear-like MDSCs (M-MDSCs), and granulocytic MDSCs (G-MDSCs) were detected by flow cytometry once a week for 4 weeks. Results As the colon cancer tumor of the mouse grows, total CD45⁺ T cellular level decreased, and CD8⁺ T cellular level steadily decreased to an irreversible level, but CD4⁺ T cells proportion rapidly increased at the early stage of the tumor-bearing, quantity of infiltrating cells in the unit volume increased by 8 times, and T cells of tumor-infiltration almost were CD4⁺ phenotype. The nonspecific immune response level represented by NK cells continued to decline, but the dendritic cells continued to accumulate in the tumor. The immunosuppressive action was further enhanced through the activation of arginase-1 (ARG1) and inducible nitric oxide synthetase (iNOS). Macrophage infiltration levels showed no significant change, but the gliding speed for the quantity of infiltrating type-Ⅰ macrophages was obviously higher than that of type-Ⅱ macrophages. The proportion of MDSCs per unit volume showed no decreasing trend, and the cells continued to accumulate in the tumor. G-MDSC infiltration level showed a steady decrease, while M-MDSC infiltration level showed an increasing trend, the proportion of M-MDSCs were higher than that of G-MDSCs. Conclusion After transplanting MC38 colon cancer cells in mice, the level of immune cell infiltration in the tumor tissue declined, tumor immunology gradually became dominat, thus promoting the development of colon cancer. The Th1/Th2 drift occurred in the tumor. Specific and nonspecific immune response levels were significantly decreased on an average, and the infiltrating monocytes and granulocytes were mainly Ly6C⁺. Tumor-associated dendritic cells and macrophages play an important immunosuppressive role. CD4⁺T cells and M-MDSCs might be the key for promoting tumor immunity.

Key words: Colon cancer, Tumor-infiltrating immune cells, Tumor immune microenvironment, Mice

中图分类号:

Q95-33
R-332

张波扬, 陈焕鹏, 余文兰, 刘忠华, 黄朝峰. MC38结肠癌小鼠肿瘤浸润免疫细胞的动态变化[J]. 实验动物与比较医学, 2021, 41(5): 409-417.

ZHANG Boyang, CHEN Huanpeng, YU Wenlan, LIU Zhonghua, HUANG Chaofeng. Dynamic Changes of Tumor-infiltrating Immune Cells in Mice with MC38 Colon Cancer[J]. Laboratory Animal and Comparative Medicine, 2021, 41(5): 409-417.

图/表 5

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