Alternative activated (M2) macrophages promoted tumor growth and invasiveness in Hepatocellular Carcinoma

Abstract

Background and Objective Chronic inflammation is a significant risk factor for Hepatoceullar Carcinoma (HCC) development yet inadequate studies have focused on studying the roles of these immunological components for the disease. Different from classic (M1) activated macrophages, alternative activated (M2) macrophages promote anti-inflammation and growth induction representing a perfect potential tumor regulator. The aim of this study is to investigate the role of M2 macrophages in HCC progression. Methods and Results Analyzed by immunohistochemistry and quantitative PCR on 100 clinical specimens with five macrophage markers (CD14, CD68, CD163, scavenger receptor class A and mannose receptor), we first discovered that M2 macrophages were widely expressed in liver tumor comprising 10-30% of total cell population. High level M2 specific CD163 (hazard ratio=2.693; p = 0.043) and scavenger receptor A (hazard ratio=3.563; p = 0.044) indicated poor prognosis and correlated with increased tumor nodules and venous infiltration in HCC patients. Consistent findings were found in orthotopic mice injected with M2 macrophages which led to increased 32.6% of tumor volume and 50% of metastasis in compared with negative control (p<0.05). Methods and Results (cont’d) Enhanced proliferation (2±0.2-fold) and migration (3±0.4-fold) of a HCC cell line MHCC97L were observed after co-cultivated with M2 macrophages in vitro. All the results collectively demonstrated the tumor promoting phenotype of M2 macrophages and we proceeded to identify the underneath molecular mechanisms in HCC. Applying cytokine antibody array, chemokine CCL22 was discovered to be strongly secreted by M2 macrophages in response to MHCC97L during cocultivation. Chemotaxis analysis revealed that CCL22 increased MHCC97L migration capacities and excess level led to increased venous infiltration in HCC patients (p<0.05). High level of CCL22 receptor CCR4 were discovered to be selectively expressed in HCC tumors and cell lines. Further antagonist inhibition of the receptor CCR4 significantly hindered the migration of MHCC97L in response to CCL22. More importantly, addition of M2 macrophages as well as recombinant CCL22 induced the activation of SNAIL and subsequent epithelial-to mesenchylmal transition (EMT) mechanisms in MHCC97L which was responsible for the increased invasiveness of HCC by M2 macrophages. Conclusion Clinical analysis and both the in vitro and in vivo studies presented here collectively illustrated the critical roles of M2 macrophages in promoting tumor growth and migration through CCL22-CCR4 mechanism in HCC. Targeting these M2 macrophages and associated products in situ represents a novel approach for treating the disease. Key words: Macrophages; Hepatocellular Carcinoma; Epithelial mesenchymal transition; Cytokine