Investigating the role of ZEB2 upregulation in TAM polarization and immune modulation in EGFR-TKI resistant NSCLC.

Abstract

e20669 Background: Immune checkpoint inhibitors (ICIs) pushed therapeutic strategy of NSCLC into a new era. Nevertheless, the clinical benefit of ICI therapy in EGFR-driven NSCLC patients, especially patients resistant to EGFR-TKI was unsatisfactory. Immunosuppressive tumor microenvironment (TME) after EGFR TKI treatment proved huge influence on the efficacy of ICIs. Hence, investigation of the mechanism underlying the formation of suppressive TME and exploration of its potential intervention was the urgent requirement for EGFR-TKI resistant NSCLC research. Methods: ZEB2 expression in human NSCLC cell lines and NSCLC patients’ samples were assessed utilizing qRT-PCR, western blotting, and immunohistochemistry (IHC). The effects of EGFR-TKI resistance and ZEB2 on TAMs polarization were investigated via qRT-PCR analysis of M1/M2-like phenotypic biomarkers and flow cytometry. The modulation of ZEB2 on cytokines’ secretion was evaluated through qRT-PCR, ELISA and MSD electrochemiluminescence. The direct influence of ZEB2 on promoter regions of cytokines was explored using dual luciferase reporter assay. The regulation of signaling pathway on ZEB2 was analyzed by correlative analysis using LUAD data from TCGA database, the results were then consolidated using western blotting. CDX mouse models, established by injecting PC9 and HCC827 cells after intervention subcutaneously into the BALB/c nude mice, were used to further confirm the influence of EGFR-TKI resistance and ZEB2 on TAMs polarization. Results: It was elucidated that EGFR-TKI resistance induced M2 polarization and inhibited M1 polarization of TAMs, which was critical for the formation of immunosuppressive TME. ZEB2 was found to be upregulated in EGFR-TKI resistant NSCLC in vitro, in vivo and in silicon. Based on these results, ZEB2 upregulation could induce M2 polarization and impede M1 polarization of TAMs, demonstrating its essential role in the influence of EGFR-TKI resistant NSCLC on TAMs polarization. Besides, ZEB2 overexpression was dependent on PI3K-Akt signaling pathway which was upregulated after EGFR-TKI resistance in NSCLC. Finally, the mechanism underlying ZEB2’s regulation on TAMs polarization was proved to be associated with cytokines’ secretion. Apart from its inducement on TGF-β1 secretion, ZEB2 could directly bind to the promoter region of CSF-1 to elevate its secretion. Conclusions: ZEB2, which was upregulated in EGFR-TKI resistant NSCLC on a PI3K-Akt signaling pathway dependent manner, could induce M2 polarization and hinder M1 polarization via elevating the secretion of CSF-1 and TGF-β1. Devoting to improving the efficacy of ICI therapy, ZEB2 could become a potential target for intervention to modulate TME after the development of EGFR-TKI resistance.