S100A9 from tumor-associated macrophage enhances cancer stem cell-like properties of hepatocellular carcinoma

Tumor-associated macrophages (TAMs) are crucial components of the tumor microenvironment (TME). They play vital roles in hepatocellular carcinoma (HCC) progression. However, the interactions between TAMs and HCC cells have not been fully characterized. In this study, TAMs were induced using human monocytic cell line THP-1 cells in vitro to investigate their functions in HCC progression. S100 calcium binding protein A9 (S100A9), an inflammatory microenvironment-related secreted protein, was identified to be significantly upregulated in TAMs. S100A9 expression in tumor tissues was associated with poor survival of HCC patients. It could enhance the stem cell-like properties of HepG2 and MHCC-97H cells by activating nuclear factor-kappa B (NF-κB) signaling pathway through advanced glycosylation end-product specific receptor (AGER) in a Ca2+-dependent manner. Furthermore, we found that, after treatment with S100A9, HepG2 and MHCC-97H cells recruited more macrophages via chemokine (C-C Motif) ligand 2 (CCL2), which suggests a positive feedback between TAMs and HCC cells. Taken together, our findings reveal that TAMs could upregulate secreted protein S100A9 and enhance the stem cell-like properties of HCC cells, and provide a potential therapeutic target for combating HCC. THP-1 cells were polarized into macrophages using PMA (Sigma, 100ng/ml) treatment for 24 hours. As for THP-1 derived TAM, THP-1 cells (2×106 cells) treated with PMA were placed on the lower chamber of a 6-well transwell plate. Meanwhile, HepG2 or MHCC-97H cells were placed on the 0.4μm porous membrane of upper chamber. After 24 hours, we co-cultured HepG2 or MHCC-97H cells with THP-1 derived macrophages. Then 48 hours later, macrophages were collected for RNA extraction. Next, we analyzed the differentially expressed genes in THP-1 cells-derived TAMs using whole genome expression profile analysis compared with THP-1 derived macrophages.