CCL2/CCR2/Erk signal induced by interaction between cancer cells and macrophages contributes to hepatocellular carcinoma progression

Macrophages infiltrating into tumors are known as tumor-associated macrophages (TAMs) and play a central role in tumor progression. In immunohistochemistry, intra-tumoral CD68-positive macrophages correlated with poor prognosis and clinicopathological factors of patients with hepatocellular carcinoma (HCC). Next, we established an indirect co-culture assay between HCC cells and peripheral blood-derived macrophages. cDNA microarray analysis revealed that C-C motif chemokine ligand 2 (CCL2) was highly expressed in HCC cells co-cultured with macrophages. CCL2 bound to CCR2, known as a main receptor of CCL2, and promoted the proliferation and migration of HCC cells and macrophages. The Erk pathway as a downstream signal of the CCL2/CCR2 axis contributed to controlling these phenotypes of HCC cells and macrophages. In the ELISA using serum samples, the serum CCL2 levels of patients with HCC were significantly elevated compared with healthy volunteer donors. The survival outcomes of patients with intra-tumoral CCL2 expression Positive based on immunohistochemistry tended to be worse, without statistical significance. Consequently, the CCL2 upregulated through the interactions between HCC cells and macrophages contributed to HCC progression and the CCL2/CCR2/Erk signal might be an effective candidate for the treatment target of HCC and macrophages. Peripheral blood CD14-positive monocytes (PBMos) were selectively collected from healthy volunteer donors using autoMACS Pro Separator. Thereafter, PBMos were incubated with 10 ng/mL recombinant human M-CSF and 1 ng/mL recombinant human GM-CSF for six days to induce macrophage (M0). For macrophage polarization to TAM-like macrophage, macrophages were washed and incubated with 50% HCC-conditioned medium for 24 hours.