肝细胞癌（HCC）是一种影响人类健康的常见癌症。M2巨噬细胞在肿瘤中介导免疫应答中起着关键作用。本研究调查了M2巨噬细胞在HCC免疫逃避中的作用。通过Western blot确定了主导HCC/肿瘤相邻组织中有丝分裂纺锤体定位（MISP）、IQ基因家族GTP酶激活蛋白1（IQGAP1）和程序性细胞死亡-1（PD-L1）的水平，然后进行相关性分析。通过流式细胞术估计了M2巨噬细胞和CD3+CD8+T细胞的百分比。将Hep3B和HepG2细胞分别经M2巨噬细胞调制培养基（M2-CM）和M2巨噬细胞源性细胞外囊泡（M2-EVs）处理，或与CD8+T细胞共培养，然后评估细胞存活和凋亡。通过ELISA测量了TNF-α和INF-γ的水平。通过转染HCC细胞进行MISP和IQGAP1过表达质粒，以探索它们在免疫逃避中的作用。通过共免疫沉淀分析了MISP、IQGAP1、STAT3和PD-L1之间的相互作用。在裸鼠体内验证了M2-EVs在HCC免疫逃避中的作用。MISP/IQGAP1/PD-L1在HCC组织中上调。MISP与IQGAP1/PD-L1呈负相关，IQGAP1与PD-L1呈正相关。高表达MISP的HCC组织中，M2巨噬细胞减少而CD8+T细胞增加。M2-CM或M2-EVs抑制了CD8+T细胞的杀伤能力，增加了HCC细胞的存活能力，阻碍了HCC细胞的凋亡，诱导CD8+T细胞凋亡，降低了TNF-α和INF-γ的水平，上调了PD-L1。M2-EVs通过加强IQGAP1核转位并通过MISP下调激活STAT3磷酸化，促进HCC细胞免疫逃避的机制在裸鼠实验证实。M2-EVs通过MISP上调通过MISP/IQGAP1/STAT3轴上调PD-L1促进HCC细胞免疫逃避。 版权所有 © 2023 Elsevier B.V.。保留所有权利。

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.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 CD3+CD8+T 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 CD8+T 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.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 CD8+T cells were increased in HCC tissues with high MISP expression. M2-CM or M2-EVs inhibited the killing ability of CD8+T cells, increased HCC cell viability, impeded HCC cell apoptosis, induced CD8+T 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.M2-EVs promote HCC cell immune escape by upregulating PD-L1 through the MISP/IQGAP1/STAT3 axis.Copyright © 2023 Elsevier B.V. All rights reserved.