M2型巨噬细胞衍生的细胞外囊泡通过MISP/IQGAP1轴调节肝细胞癌免疫治疗耐药中PD-L1表达的机制
Mechanism of M2 type macrophage-derived extracellular vesicles regulating PD-L1 expression via the MISP/IQGAP1 axis in hepatocellular carcinoma immunotherapy resistance.
作者信息
Wang Xiaobo, Ye Xuxing, Chen Yanping, Lin Junmei
机构信息
The Fourth School of Clinical Medicine, Zhejiang Chinese Medical University, 548 Binwen Road, Binjiang District, Hangzhou, 310053, China.
Department of Traditional Chinese Medicine, Jinhua Municipal Central Hospital, 351 Mingyue Street, Wucheng District, Jinhua, 321001, China.
出版信息
Int Immunopharmacol. 2023 Nov;124(Pt A):110848. doi: 10.1016/j.intimp.2023.110848. Epub 2023 Aug 24.
BACKGROUND
Hepatocellular carcinoma (HCC) is a prevailing cancer affecting human health. M2 macrophages are essential in mediating immune responses in tumors. This study investigated the action of M2 macrophages in immune escape of HCC.
METHODS
Mitotic spindle positioning (MISP), IQ motif containing GTPase activating protein 1 (IQGAP1) and programmed cell death-1 (PD-L1) levels in primary HCC/tumor-adjacent tissues were determined by Western blot, followed by correlation analysis. M2 macrophage and CD3CD8T cell percentages were estimated by flow cytometry. Hep3B and HepG2 cells were treated with M2 macrophage conditioned medium (M2-CM) and M2 macrophage-derived extracellular vesicles (M2-EVs) and/or co-cultured with CD8T cells, followed by assessment of cell viability and apoptosis. TNF-α and INF-γ levels were measured by ELISA. MISP and IQGAP1 overexpression plasmids were transfected into HCC cells to explore their role in immune escape. The interactions among MISP, IQGAP1, STAT3, and PD-L1 were analyzed by co-immunoprecipitation. The mechanism of M2-EVs in HCC immune escape was verified in nude mice.
RESULTS
MISP/IQGAP1/PD-L1 were upregulated in HCC tissues. MISP negatively-correlated with IQGAP1/PD-L1 and IQGAP1 positively-correlated with PD-L1. M2 macrophages were reduced but CD8T cells were increased in HCC tissues with high MISP expression. M2-CM or M2-EVs inhibited the killing ability of CD8T cells, increased HCC cell viability, impeded HCC cell apoptosis, induced CD8T cell apoptosis, downregulated TNF-α and INF-γ, and upregulated PD-L1. M2-EVs facilitated HCC cell immune escape by potentiating IQGAP1 nuclear translocation and activating STAT3 phosphorylation through MISP downregulation. In vivo experiments further verified the action of M2-EVs through MISP.
CONCLUSION
M2-EVs promote HCC cell immune escape by upregulating PD-L1 through the MISP/IQGAP1/STAT3 axis.
背景
肝细胞癌（HCC）是一种影响人类健康的常见癌症。M2巨噬细胞在介导肿瘤免疫反应中至关重要。本研究调查了M2巨噬细胞在HCC免疫逃逸中的作用。
方法
采用蛋白质免疫印迹法测定原发性HCC/癌旁组织中有丝分裂纺锤体定位（MISP）、含IQ模体的GTP酶激活蛋白1（IQGAP1）和程序性细胞死亡蛋白1（PD-L1）的水平，随后进行相关性分析。通过流式细胞术估计M2巨噬细胞和CD3CD8T细胞百分比。用M2巨噬细胞条件培养基（M2-CM）和M2巨噬细胞衍生的细胞外囊泡（M2-EVs）处理Hep3B和HepG2细胞和/或与CD8T细胞共培养，随后评估细胞活力和凋亡。通过酶联免疫吸附测定法测量肿瘤坏死因子-α（TNF-α）和干扰素-γ（INF-γ）水平。将MISP和IQGAP1过表达质粒转染到HCC细胞中，以探索它们在免疫逃逸中的作用。通过免疫共沉淀分析MISP、IQGAP1、信号转导和转录激活因子3（STAT3）和PD-L1之间的相互作用。在裸鼠中验证M2-EVs在HCC免疫逃逸中的机制。
结果
HCC组织中MISP/IQGAP1/PD-L1上调。MISP与IQGAP1/PD-L1呈负相关，IQGAP1与PD-L1呈正相关。在MISP表达高的HCC组织中，M2巨噬细胞减少，但CD8T细胞增加。M2-CM或M2-EVs抑制CD8T细胞的杀伤能力，增加HCC细胞活力，阻碍HCC细胞凋亡，诱导CD8T细胞凋亡，下调TNF-α和INF-γ，并上调PD-L1。M2-EVs通过增强IQGAP1核转位并通过下调MISP激活STAT3磷酸化促进HCC细胞免疫逃逸。体内实验进一步验证了M2-EVs通过MISP的作用。
结论
M2-EVs通过MISP/IQGAP1/STAT3轴上调PD-L1促进HCC细胞免疫逃逸。
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