Tumor-infiltrating macrophages (Mϕs), known as tumor-associated macrophages (TAMs), play a crucial role in the tumor microenvironment. Immunohistochemistry (IHC) revealed that intratumoral CD68-positive Mϕs are associated with poor prognosis and clinicopathological factors in patients with hepatocellular carcinoma (HCC). Subsequently, an indirect co-culture system involving HCC cells and peripheral blood-derived Mϕs was developed. Complementary DNA (cDNA) microarray analysis revealed that C-C motif chemokine ligand 2 (CCL2) was highly expressed in HCC cells co-cultured with Mϕs. CCL2 neutralization suppressed proliferation, migration, and phosphorylation of extracellular signal-regulated kinase (Erk) in HCC cells and Mϕs enhanced through co-culture. In contrast, recombinant human CCL2 (rhCCL2) addition facilitated these malignant phenotypes and increased Erk phosphorylation levels in HCC cells and Mϕs. The primary CCL2 receptor, C-C motif chemokine receptor 2 (CCR2) was expressed in HCC cells and Mϕs and was upregulated in co-cultured HCC cells. CCR2 inhibition suppressed malignant phenotypes and reduced phosphorylated levels of Erk enhanced by rhCCL2. Additionally, the inhibition of Erk signal suppressed rhCCL2-enhanced malignant phenotypes. Moreover, serum CCL2 levels were higher in patients with HCC than those in healthy donors. Based on IHC, CCL2-positive cases with high CCR2 expression and phosphorylated Erk-positive cases exhibited poor survival outcomes. Therefore, CCL2 upregulation through interactions between HCC cells and Mϕs contributed to HCC progression, making the CCL2/CCR2/Erk signal a potential target for HCC treatment.
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