[Hepatocellular carcinoma cells induce M2 macrophage polarization through upregulation of ANXA2 and ANXA5 expression]
Junying Xu1,2, Chenghao Liu2,3, Hao Huang1,2
1Department of Basic Medicine, School of Medicine, Shihezi 832000, China.
Objectives:
To explore the prognostic value of annexins A2 (ANXA2) and A5 (ANXA5) in hepatocellular carcinoma (HCC) and their impact on tumor microenvironment.
Methods:
Public datasets of HCC transcriptome and clinical data and spatial transcriptomic data were used for survival analysis, correlation analysis, immune microenvironment analysis, and enrichment analysis to explore the effects of annexin A2 (ANXA2) and ANXA5 on patient prognosis and tumor microenvironment and the potential mechanisms. Immunofluorescence assays were performed to detect ANXA2, ANXA5, and ANXA11 expressions in HCC tissues and their co-expression with CD206. THP-1 cells were treated with culture supernatant of human HCC MHCC-97H cells (TCM), and the changes in mRNA levels of IL-10, ANXA2, ANXA5, and ANXA11 and the protein levels of ANXA2, ANXA5, ANXA11, and KLF4 were examined using qRT-PCR and Western blotting. Flow cytometry was used to detect the expression of CD206 and PD-L1 in the macrophages.
Results:
ANXA2, ANXA5 and ANXA11 expressions were all significantly up-regulated in HCC tissues in correlation with poor patient prognosis. GO and GSEA revealed enrichment of IL-10 and IL-4/IL-13 signaling pathways in tumors overexpressing ANXA2, ANXA5 and ANXA11, whose high expressions were positively correlated with macrophage infiltration. Immunofluorescence staining demonstrated co-expressions of ANXA2 and ANXA5 with M2-macrophage markers in human HCC tissues. The conditioned medium from HCC cells significantly up-regulated the expressions of ANXA2, ANXA5, CD206 and PD-L1 in THP-1 cells, whereas treatment with the recombinant ANXA2 or ANXA5 protein alone increased CD206 expression only. Correlation analysis indicated that KLF4 was positively correlated with both ANXA2 and ANXA5, and inhibition of KLF4 caused significant reduction of ANXA2, ANXA5 and CD206 levels in THP-1 cells.
Conclusions:
HCC promotes M2 macrophage polarization by up-regulating ANXA2 and ANXA5 expressions, which contribute to an immunosuppressive microenvironment in HCC and potentially serve as molecular targets for HCC immunotherapy.
