The TGF-β signaling activated by cancer-associated macrophages promotes cancer stemness and Gefitinib resistance in EGFR-mutant non-small-cell lung cancer

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) remains an inevitable challenge in the management of EGFR-mutant non-small cell lung cancer (NSCLC). Although genetic alterations have been extensively characterized, the contribution of tumor microenvironment remodeling and cancer stemness to EGFR-TKIs resistance remains incompletely defined. Here, we investigated the role of transforming growth factor-β (TGF-β) signaling in mediating Gefitinib resistance through tumor–macrophage interactions. Bioinformatic analyses were performed to identify signaling pathways associated with Gefitinib resistance. Gefitinib-sensitive and resistant NSCLC cell lines were used to evaluate stemness features and drug response by Western blotting, sphere formation, colony formation, and cell viability assays. A macrophage co-culture system was established to assess tumor-associated macrophages (TAMs) polarization and its impact on resistance. Xenograft models were used to validate in vivo effects. The TGF-β inhibitor Disitertide was applied to determine therapeutic potential. TGF-β/SMAD3 signaling was significantly activated in Gefitinib-resistant cells and correlated with poor prognosis. Resistant cells exhibited enhanced stem-like properties, including increased sphere formation and upregulation of stemness-associated markers. TGF-β1 stimulation promoted stemness and reduced Gefitinib sensitivity in sensitive cells, whereas pharmacological inhibition of SMAD3 attenuated these effects and partially restored drug responsiveness. Mechanistically, resistant tumor cells induced macrophage polarization toward a TAMs phenotype via TGF-β1 secretion. TAMs, in turn, reinforced stemness and resistance in tumor cells, establishing a TGF-β–driven positive feedback loop. In vivo, TAMs accelerated tumor growth, while TGF-β inhibition suppressed macrophage polarization and enhanced Gefitinib sensitivity. Our findings demonstrate that TGF-β signaling integrates cancer stemness and macrophage polarization to sustain acquired Gefitinib resistance in EGFR-mutant NSCLC. Targeting the TGF-β axis may represent a promising combinatorial strategy to overcome EGFR-TKIs resistance.