The online version of this article (https://doi.org/10.1186/s12957-018-1312-y) contains supplementary material, which is available to authorized users.
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.
THP-1 cells were induced to differentiate into M2-like macrophages through treatments with IL-4, IL-13, 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.
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 M2-CM. Furthermore, the TLR4/(signal transducer and activator of transcription 3 (STAT3) signaling pathway contributed to the effects of M2-CM on HCC cells.
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.
Additional file 1: Figure S1. Cells treated with LPS in the absence of M2-CM did not exhibit increased migration. (A) Cell migration in the control and LPS only groups was determined using the transwell assay (× 100). (B) The distance traveled by migrating HCC cells in the control and LPS only groups was measured using a wound-healing assay (× 50). (C) Analysis of the data from the transwell migration assay. (TIFF 16142 kb)12957_2018_1312_MOESM1_ESM.tif
Additional file 2: Figure S2. Levels of the p-ERK, p-Ikkα/β, total STAT3, ERK, Ikkα, and Ikkβ proteins were detected using western blotting. (A) Levels of the p-STAT3 and STAT3 proteins in the control, LPS, and M2-CM + LPS groups. (B) Levels of the p-ERK and ERK proteins in the control, M2-CM + LPS, and M2-CM groups. (C) Levels of the p-Ikkα/β, Ikkα, and Ikkβ proteins in the control, M2-CM + LPS, and M2-CM groups. (TIFF 601 kb)12957_2018_1312_MOESM2_ESM.tif
Additional file 3: Figure S3. Language edit certification. (JPEG 147 kb)12957_2018_1312_MOESM3_ESM.jpg
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