ZEB2 upregulation modulates the polarization of TAMs toward the immunosuppressive state in EGFR-TKI-resistant NSCLC

<b>Aim:</b> Immune checkpoint inhibitors (ICIs) have revolutionized the treatment approach for NSCLC. However, the effectiveness of ICI therapy in patients with EGFR-driven NSCLC, particularly those resistant to EGFR-TKI, has been disappointing. The immunosuppressive tumor microenvironment (TME) following EGFR-TKI therapy has been proved to significantly affected the effectiveness of ICIs. Therefore, studying the mechanism behind the development of a suppressive TME and exploring potential interventions is crucial for research on EGFR-TKI-resistant NSCLC. <b>Methods:</b> ZEB2 levels were quantified in human NSCLC cell lines and in tumor specimens from NSCLC patients by quantitative RT-PCR (qRT-PCR), WB, and immunohistochemical staining. To examine how ZEB2 affected macrophage polarization, M1/M2 marker profiles were measured with qRT-PCR and flow cytometry. Changes in cytokine production triggered by altered ZEB2 expression were determined with qRT-PCR, ELISA, and Meso Scale Discovery electrochemiluminescence assays. The direct binding of ZEB2 to cytokine-gene promoters was tested using a dual-luciferase reporter system. Upstream regulatory pathways were investigated by correlating LUAD transcriptomic data from TCGA with ZEB2 expression and validating key findings via western blotting. Finally, cell-derived xenograft (CDX) models were generated by subcutaneously implanting pre-treated PC9 or HCC827 cells into BALB/c nude mice to verify the impact of EGFR-TKI resistance and ZEB2 on tumor-associated macrophage (TAM) polarization <i>in vivo</i>. <b>Results:</b> It was elucidated that EGFR-TKI resistance upregulated the M2 polarization biomarkers, Arg-1 (PC9-GR: <i>P</i> < 0.01; HCC827-GR: <i>P</i> < 0.05) and IL4 (PC9-GR: <i>P</i> < 0.01; HCC827-GR: <i>P</i> < 0.01), while downregulated the M1 polarization biomarkers, TNF-α (PC9-GR: <i>P</i> < 0.01; HCC827-GR: <i>P</i> < 0.01), IL1β (PC9-GR: <i>P</i> < 0.01; HCC827-GR: <i>P</i> < 0.01), and IL6(PC9-GR: <i>P</i> < 0.001; HCC827-GR: <i>P</i> < 0.001) in NSCLC cell lines. Meanwhile, CD206⁺ TAMs (PC9-GR: <i>P</i> < 0.05; HCC827-GR: <i>P</i> < 0.01) were increased and CD86⁺ TAMs (PC9-GR: <i>P</i> < 0.05; HCC827-GR: <i>P</i> < 0.05) were decreased in both EGFR-TKI-resistant mice models. Apart from the formation of suppressive TME, ZEB2 was found to be upregulated in PC9-GR (qRT-PCR: <i>P</i> < 0.0001; WB: <i>P</i> < 0.05) and HCC827-GR (qRT-PCR: <i>P</i> < 0.0001; WB: <i>P</i> < 0.05) cells. The same trend was also noticed in clinical samples, with ZEB2 upregulated after gefitinib resistance in NSCLC patients (<i>P</i> < 0.0001). Based on these findings, ZEB2 knockdown was proved to downregulate Arg-1 (PC9-GR: <i>P</i> < 0.01; HCC827-GR: <i>P</i> < 0.05) and IL4 (PC9-GR: <i>P</i> < 0.01; HCC827-GR: <i>P</i> < 0.001), while upregulate the TNF-α (PC9-GR: <i>P</i> < 0.0001; HCC827-GR: <i>P</i> < 0.0001), IL1β (HCC827-GR: <i>P</i> < 0.001), and IL6 (PC9-GR: <i>P</i> < 0.01; HCC827-GR: <i>P</i> < 0.001), indicating its role in M1/M2 polarization in both EGFR-TKI-resistant NSCLC cell lines. The downregulation of CD206⁺ TAMs (PC9-GR: <i>P</i> < 0.05; HCC827-GR: <i>P</i> < 0.01) and the upregulation of CD86⁺ TAMs (PC9-GR: <i>P</i> < 0.001; HCC827-GR: <i>P</i> < 0.05) also demonstrated the reversion of suppressive TME after ZEB2 knockout in EGFR-TKI-resistant mice models. Additionally, after the intervention of MK2206, which was an Akt inhibitor, ZEB2 expression was suppressed at both low (PC9-GR: <i>P</i> < 0.001; HCC827-GR: <i>P</i> < 0.001) and high concentrations (PC9-GR: <i>P</i> < 0.001; HCC827-GR: <i>P</i> < 0.0001). Finally, the mechanism underlying ZEB2's regulation on TAM polarization was proved to be associated with cytokine secretion. According to the results of ELISA, apart from its inducement on TGF-β1 secretion (PC9-GR: <i>P</i> < 0.0001; HCC827-GR: <i>P</i> < 0.0001), ZEB2 could directly bind to the promoter region of CSF-1 to elevate its secretion (PC9-GR: <i>P</i> < 0.0001; HCC827-GR: <i>P</i> < 0.0001). <b>Conclusion:</b> In EGFR-TKI-resistant NSCLC, activation of the PI3K-Akt cascade drove a marked rise in ZEB2 expression. The elevated ZEB2 increased CSF-1 and TGF-β1 release, steering macrophages toward an M2 phenotype while impeding M1 polarization. Accordingly, suppressing ZEB2 had the potential to reshape the TME and enhance the effectiveness of ICIs once EGFR-TKI resistance had emerged.