S100A9 from tumor-associated macrophage enhances cancer stem cell-like properties of hepatocellular carcinoma. 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. Overall design: 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.

肿瘤相关巨噬细胞（Tumor-associated macrophages, TAMs）是肿瘤微环境（tumor microenvironment, TME）的关键组成部分，在肝细胞癌（hepatocellular carcinoma, HCC）的进展中发挥至关重要的作用。然而，TAMs与HCC细胞之间的相互作用尚未得到完全阐明。本研究通过体外使用人单核细胞系THP-1细胞诱导TAMs，以探究其在HCC进展中的功能。研究发现，S100钙结合蛋白A9（S100 calcium binding protein A9, S100A9）——一种与炎症微环境相关的分泌型蛋白——在TAMs中显著上调。肿瘤组织中S100A9的表达与HCC患者的不良预后相关。S100A9可通过Ca²+依赖的方式，经由晚期糖基化终产物特异性受体（advanced glycosylation end-product specific receptor, AGER）激活核因子-κB（nuclear factor-kappa B, NF-κB）信号通路，从而增强HepG2和MHCC-97H细胞的干细胞样特性。进一步研究发现，经S100A9处理后，HepG2和MHCC-97H细胞可通过趋化因子（C-C基序）配体2（chemokine (C-C Motif) ligand 2, CCL2）招募更多巨噬细胞，这表明TAMs与HCC细胞之间存在正反馈环路。综上，本研究结果揭示TAMs可上调分泌型蛋白S100A9的表达，增强HCC细胞的干细胞样特性，为对抗HCC提供了潜在的治疗靶点。

实验整体设计：采用佛波酯（PMA，Sigma公司，100ng/ml）处理THP-1细胞24小时，将其极化为巨噬细胞。对于THP-1来源的TAM，将经PMA处理的THP-1细胞（2×10^6个）置于6孔Transwell板的下室，同时将HepG2或MHCC-97H细胞置于上室0.4μm孔径的多孔膜上。共培养24小时后，将HepG2或MHCC-97H细胞与THP-1来源的巨噬细胞进行共培养。48小时后收集巨噬细胞进行RNA提取。随后，通过全基因组表达谱分析，对比THP-1来源的巨噬细胞，分析THP-1细胞来源的TAMs中的差异表达基因。