M2-polarized tumor-associated macrophages promoted epithelial-mesenchymal transition in pancreatic cancer cells, partially through TLR4/IL-10 signaling pathway

摘要

M2-polarized tumor-associated macrophages (TAMs) are key regulators of the link between inflammation and cancer. A negative correlation between infiltration intensity of M2-polarized TAMs and prognosis of pancreatic cancer has been reported. Epithelial-mesenchymal transition (EMT) is an important biological process in the progression of primary tumors toward metastasis. Inflammation-induced EMT has been previously shown, therefore, we hypothesized M2-polarized TAMs could induce EMT in pancreatic cancer. Toll-like receptor 4 (TLR4) signaling has an active role in tumor progression during chronic inflammation and the receptor is primarily expressed on macrophages. Activation of TLR4 on M2-polarized TAMs stimulates an increase in the cytokine interleukin-10 (IL-10); consequently, another aim was to investigate the potential role of TLR4/IL-10 signaling in the EMT of pancreatic cancer. Treatment with IL-4 (20 ng/ml) for 24 h successfully induced the polarization of macrophage cell line RAW 264.7 to M2 phenotype, IL-10 high, IL-12 low, and IL-23 low, and high expression of CD204 and CD206. A coculture system allowed investigation of the roles of M2-polarized TAMs and TLR4/IL-10 signaling in the EMT of Panc-1 and BxPC-3 pancreatic cancer cell lines. Our results showed that coculture with M2-polarized TAMs increased fibroblastic morphology, upregulated mesenchymal markers vimentin and snail at the mRNA and protein levels, and increased proliferation, migration, and metalloproteinase (MMP)2 and MMP9 proteolytic activity in pancreatic cancer cells. Simultaneously, coculture with M2-polarized TAMs decreased the expression of the epithelial marker E-cadherin. Coculture with pancreatic cancer cells increased TLR4 mRNA and protein expression in M2-polarized TAMs. Application of TLR4 siRNA and neutralizing antibodies against TLR4 and IL-10 markedly inhibited E-cadherin reduction and the upregulation of snail and vimentin. Furthermore, activation of TLR4 signaling by lipopolysaccharide profoundly increased the EMT of pancreatic cancer cells. In conclusion, M2-polarized TAMs promoted EMT in pancreatic cancer cells partially through TLR4/IL-10 signaling, suggesting novel therapeutic strategies and enhancing our understanding of M2-polarized TAMs.