fig6

Figure 6. ZEB2 promotes M2 polarization and impedes M1 polarization of TAMs by elevating the secretion of CSF-1 and TGF-β1. (A) Heatmap demonstrating the relationships between 17 most highly upregulated DEGs after EGFR-TKI resistance in HCC827 and HCC4006 cells and cytokines that were proved to be associated with macrophage polarization; (B) A GSEA was conducted with the ZEB2-related genes in the TCGA LUAD dataset using the LinkedOmics online platform. The results of the KEGG pathway enrichment analysis showed that the chemokine signaling pathway and leukocyte transendothelial migration were among the upregulated pathways; (C) qRT-PCR was conducted to determine the alterations of cytokines associated with macrophage polarization after EGFR-TKI resistance in PC9 and HCC827 cells. Data represent results from three independent experiments; (D) Results of ELISA and MSD electrochemiluminescence of PC9 and HCC827 cells before and after EGFR-TKI resistance. The concentrations of TGF-β1 and CSF-1 were detected using ELISA, while the concentrations of CCL8, IL4, and CXCL9 were detected using MSD electrochemiluminescence. Data represent results from three independent experiments; (E) qRT-PCR analysis revealed that ZEB2 knockdown could reverse the alterations of CSF-1, TGF-β1, CCL8, IL4, and CXCL9 in PC9-GR and HCC827-GR cells. Data represent results from three independent experiments; (F) Results of ELISA and MSD electrochemiluminescence of PC9-GR and HCC827-GR cells with or without ZEB2 knockdown. The concentrations of TGF-β1 and CSF-1 were detected using ELISA, while the concentrations of CCL8, IL4, and CXCL9 were detected using MSD electrochemiluminescence. Data represent results from three independent experiments. ^*P < 0.05, ^**P < 0.005, ^***P < 0.0005, ^****P < 0.0001.