M2-polarized tumor-associated macrophages facilitated migration and epithelial-mesenchymal transition of HCC cells via the TLR4/STAT3 signaling pathway

Yao et al. World Journal of Surgical Oncology (2018) 16:9 DOI 10.1186/s12957-018-1312-y RESEARCH Open Access M2-polarized tumor-associated macrophages facilitated migration and epithelial-mesenchymal transition of HCC cells via the TLR4/STAT3 signaling pathway Rong-Rong Yao1,2†, Jing-Huan Li1,2†, Rui Zhang1,2, Rong-Xin Chen1,2 and Yan-Hong Wang1,2* Abstract Background: M2-polarized macrophages are tumor-associated-macrophages (TAMs), which are important contents of tumor-infiltrating immune cells. Toll-like receptor 4 (TLR4) is a molecular biomarker of tumor aggressiveness and poor prognosis. Toll-like receptors (TLRs) have important roles in the immune system and M2-polarized macrophages. However, the effects of TLR4 on M2-polarized macrophages in hepatocellular carcinoma (HCC) are unknown. Here, TLR4 expressed on HCC cells mediates the pro-tumor effects and mechanisms of M2-polarized macrophages. Methods: THP-1 cells were induced to differentiate into M2-like macrophages through treatments with IL-4, IL13, and phorbol myristate acetate (PMA). We used the HCC cell lines SMMC-7721 and MHCC97-H cultured in conditioned medium from M2-like macrophages (M2-CM) to investigate the migration potential of HCC cells and epithelial-mesenchymal transition (EMT)-associated molecular genetics. Signaling pathways that mediated M2-CM-promoted HCC migration were detected using western blotting. Results: HCC cells cultured with M2-CM displayed a fibroblast-like morphology, an increased metastatic capability, and expression of EMT markers. TLR4 expression was markedly increased in M2-CM-treated HCC cells. TLR4 overexpression promoted HCC cell migration, and a TLR4-neutralizing antibody markedly inhibited HCC EMT in cells cultured with M2CM. Furthermore, the TLR4/(signal transducer and activator of transcription 3 (STAT3) signaling pathway contributed to the effects of M2-CM on HCC cells. Conclusions: Taken together, M2-polarized macrophages facilitated the migration and EMT of HCC cells via the TLR4/ STAT3 signaling pathway, suggesting that TLR4 may be a novel therapeutic target. These results improve our understanding of M2-polarized macrophages. Keywords: Hepatocellular carcinoma, Epithelial-mesenchymal transition, Tumor-associated macrophages, Toll-like receptor 4 Background Hepatocellular carcinoma (HCC) is the primary liver cancer, which causing cancer-related death is the third of globally [1], in spite of recent advances in HCC diagnosis and treatment. The 5-year survival rate of HCC patients is still very low due to recurrence and metastasis, emphasizing the need * Correspondence: † Equal contributors 1 Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Shanghai 200032, China 2 Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China to understand the molecular mechanisms of HCC recurrence and metastasis and to suggest novel therapeutic targets. The important role of the immune microenvironment in HCC pathogenesis has been widely explored in recent years. M2-polarized macrophages are generally considered tumor-associated macrophages (TAMs) that sustain tumor progression and one of the primary tumorinfiltrating immune cells [2]. M2-polarized macrophages further tumor cell growth, invasion, and metastasis by secreting several cytokines [3–5] and stimulating specific © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yao et al. World Journal of Surgical Oncology (2018) 16:9 signaling pathways, such as the transforming growth factor β1 (TGF-β1) [6] and the IL-10 signaling pathways [7]. Furthermore, according to several clinical studies, an increased number of TAMs in the tumor site frequently correlates with a poorer prognosis [8, 9]. However, the exact mechanisms by which M2-polarized macrophages modulate the migration potential of HCC are not completely defined. Toll-like receptors (TLRs) are important receptors in the immune system and help to provide an advantageous microenvironment for cancer cells. But, the rigid roles of TLRs in cancer biology are still barely understood. TLR4 is overexpressed in macrophages and cancer cells, including colon cancer cells, lung cancer cells, and melanoma cells [10]. TLR4 is a cancer stem cell marker in HCC [11], and TLR4 expression increases tumor-initiating activity and chemoresistance during HCC development [12]. Recent research revealed an association between TLR4 expression and tumor aggressiveness and a poor prognosis for patients with HCC [13], although the mechanisms by which TLR4 promotes cancer progression are still unknown. Therefore, we initially hypothesized that M2-polarized macrophages induce epithelial-mesenchymal transition (EMT) and accelerate the invasion and migration of HCC cells via a TLR4-dependent signaling pathway. Methods Cell lines and culture conditions HCC cell lines (SMMC-7721 and MHCC97-H) were cultured in DMEM (GNM12800) medium, and THP-1 cells were maintained in RPMI 1640 (GNM31800) medium. Both of the mediums are supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 50 U/ml streptomycin sulfate (Beyotime, China), and 50 U/ml penicillin (Beyotime, China). MHCC97-H cells were obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University. The Shanghai Cell Bank of the Chinese Academy of Sciences provided THP-1 and SMMC-7721 cells. All cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. PMA treatment for differentiating THP-1 cells into M2polarized macrophages THP-1 cell could be induced macrophage differentiation by phorbol myristate acetate (PMA, Sigma).THP-1 cells were cultured in RPMI 1640 complete medium with PMA treatment (320 nM/106 cells) for 6 h, and then addition 20 ng/ml IL-13 and 20 ng/ml IL-4 incubated for 18 h to generate M2-polarized macrophages [14, 15]. CD68 expression in THP-1 cells and in induced M2-polarized macrophages was analyzed using a FACS Calibur flow cytometer. The differences in the relative expression levels of IL-10, TGF-β1, IL-12, and IL-23 between M2-polarized macrophages and macrophages were detected by quantitative real-time PCR (qRT-PCR). Page 2 of 9 Collection of M2-CM and treatment of HCC cell lines Macrophages and M2-polarized macrophages were washed three times with serum-free RPMI 1640 after treatment and cultured in RPMI 1640 supplemented with 10% FBS for 24 h. After culturing for 24 h, the fresh medium is replaced. The (...truncated)