Mechanism of M2 type macrophage-derived extracellular vesicles regulating PD-L1 expression via the MISP/IQGAP1 axis in hepatocellular carcinoma immunotherapy resistance

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 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.

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 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.

Conclusion: M2-EVs promote HCC cell immune escape by upregulating PD-L1 through the MISP/IQGAP1/STAT3 axis.

CD8(+)T cells; Hepatocellular carcinoma; IQGAP1; Immune escape; M2 macrophages; MISP; PD-L1; STAT3.