Alternatively activated macrophages promoted tumor growth and metastasis in hepatocellular carcinoma

Abstract

Background and Aim Hepatocellular Carcinoma (HCC) is the fifth most frequent malignancy worldwide with high mortality and recurrence rate. Chronic inflammation is a dominant risk factor for the malignancy and the roles of tumor associated immunological infiltrates during the disease development and progression remain unclear. The significance of alternative activated macrophages (M2) with pro-tumor phenotypes has been demonstrated in many cancers except HCC. M2 macrophages are associated with tumor angiogenesis, invasion and growth which represent a potential target to be investigated. In this study, we intend to investigate the role of M2 macrophage on HCC.

Materials and Methods M2 macrophages in 100 clinical specimens collected from HCC patients were detected by immunohistochemical (IHC) staining and quantitative PCR using common macrophage markers CD14 and CD68, and M2 specific markers CD163, Scavenger receptor class A (SA) and Mannose receptor (MR). The protein and transcript expression levels were further correlated with clinical pathological parameters. Phenotypic and functional characteristics of M1 and M2 macrophages derived from THP-1 cell line were validated and their roles in promoting HCC growth and invasion were studied in vitro co-culture system and in vivo orthotopic mice model. Secretory profiles of M2 macrophages after cocultivated with HCC cells were analyzed by cytokines antibody array screening. C-C motif chemokine 22 (CCL22) was identified to be upregulated in M2 macrophages in response to HCC cells. The functional roles of the chemokine in HCC was further studied by chemotaxis and migration assay. Underlying molecular mechanisms induced by CCL22 in HCC cells were determined.

Results In clinical analysis, we first discovered that macrophages were widely expressed in liver tumor comprising 10-30% of total cell population. Noteworthy, the density of a M2 macrophage marker CD163 was found to had a significant prognostic impact as an independent factor associated disease free survival (HR: 3.79; 95% CI 1.3-10.4; p<0.01). Strong expression of the CD163+ population was also correlated with poor relapse free survival, multiple tumor nodules and increased venous infiltration. In vivo studies revealed that the tumor volume injected with M2 macrophages increased 3.26-fold (1.27cm3±0.36) compared to the control group (0.39cm3±0.05) (P=0.032). In contrast, mice injected with M1 macrophages had a significant reduction of tumor volume by 2.79-fold (0.14cm3±0.02) (P=0.044). Increasing rate of lung metastases (57%) was also observed in M2 treated group compared to the control (25%) (P<0.05). In vitro, M2 macrophages increased the number of HCC cells (MHCC97L) and migration events by 1.3-fold and 3.2-fold after cocultivation compared to negative control (P<0.05). In cytokine antibody array study, M2 macrophages derived CCL22 was discovered to be strongly induced by MHCC97L. Chemotaxis analysis confirmed that CCL22 increased MHCC97L migration capacities and excess level led to increased venous infiltration in HCC patients (p<0.05). Upon addition of CCL22, the epithelial mesenchymal transition through SNAIL activation in MHCC97L was observed.

Conclusion Clinical analysis and both the in vitro and in vivo studies presented here collectively illustrated the significances 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.