TGF-β1 secreted by M2 phenotype macrophages enhances the stemness and migration of glioma cells via the SMAD2/3 signalling pathway

Abstract

The positive correlation between the number of M2 phenotype TAMs (M2-TAMs) and tumour development suggests a supportive role of M2-TAMs in glioma progression. In the present study, the molecular link between glioma cells and M2-TAMs was investigated and it was demonstrated that transforming growth factor-β1 (TGF-β1) secreted by M2-TAMs is key in facilitating the stemness and migration of glioma cells. Cluster of differentiation (CD )133 and CD 44, markers for the M2 phenotype, were assessed by western blotting. A sphere formation assay and trans-well assay were applied to test the stemness and migration abilities of glioma cells following co-cultured with M2-TAMs. Stemness markers CD 133 and CD 44, epithelial-mesenchymal transition-associated markers and mothers against decapentaplegic homolog (SMAD)2/3 and sex determining region Y-box 4/2 (SOX4/2) levels were also evaluated by western blotting. A xenograft tumor mouse model was used to demonstrate the tumor forming ability of glioma cells. The results showed that the U251 glioma cells co-cultured with M2-TAMs exhibited high level of sphere formation, stemness and migration ability. Recombinant TGF-β1 protein treatment was able to achieve the same effects on U251 cells, whereas a TGF-β pathway inhibitor reversed the stemness and migration abilities of the glioma cells induced by M2-TAMs. It was also demonstrated that TGF-β1 secreted by M2-TAMs upregulated the phosphorylation of SMAD2/3 and the expression of SOX4/2 in glioma cells. In a mouse xenograft model, solid tumours formed by U251 cells co-cultured with M2-TAMs or pre-treated with TGF-β1 were larger in size and had a higher growth rate. Taken together, results of the present study demonstrated that M2-TAMs promoted the stemness and migration abilities of glioma cells by secreting TGF-β1, which activated the SMAD2/3 pathway and induced the expression of SOX4 and SOX2. These results highlight the mechanism by which M2-TAMs and glioma interact and demonstrate potential therapeutic strategies for glioma treatment.