Liu Yunhuan, Yu Yong, Hu Congli, Jiang Minlin, Zhao Chao, Li Xuefei, Cheng Lei, Zhou Caicun
Department of oncology, Shanghai Pulmonary Hospital Affiliated to Tongji University, Shanghai 200433, China.
Authors contributed equally.
Cancer Drug Resist. 2025 May 29;8:25. doi: 10.20517/cdr.2024.206. eCollection 2025.
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. 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 . It was elucidated that EGFR-TKI resistance upregulated the M2 polarization biomarkers, Arg-1 (PC9-GR: < 0.01; HCC827-GR: < 0.05) and IL4 (PC9-GR: < 0.01; HCC827-GR: < 0.01), while downregulated the M1 polarization biomarkers, TNF-α (PC9-GR: < 0.01; HCC827-GR: < 0.01), IL1β (PC9-GR: < 0.01; HCC827-GR: < 0.01), and IL6(PC9-GR: < 0.001; HCC827-GR: < 0.001) in NSCLC cell lines. Meanwhile, CD206 TAMs (PC9-GR: < 0.05; HCC827-GR: < 0.01) were increased and CD86 TAMs (PC9-GR: < 0.05; HCC827-GR: < 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: < 0.0001; WB: < 0.05) and HCC827-GR (qRT-PCR: < 0.0001; WB: < 0.05) cells. The same trend was also noticed in clinical samples, with ZEB2 upregulated after gefitinib resistance in NSCLC patients ( < 0.0001). Based on these findings, ZEB2 knockdown was proved to downregulate Arg-1 (PC9-GR: < 0.01; HCC827-GR: < 0.05) and IL4 (PC9-GR: < 0.01; HCC827-GR: < 0.001), while upregulate the TNF-α (PC9-GR: < 0.0001; HCC827-GR: < 0.0001), IL1β (HCC827-GR: < 0.001), and IL6 (PC9-GR: < 0.01; HCC827-GR: < 0.001), indicating its role in M1/M2 polarization in both EGFR-TKI-resistant NSCLC cell lines. The downregulation of CD206 TAMs (PC9-GR: < 0.05; HCC827-GR: < 0.01) and the upregulation of CD86 TAMs (PC9-GR: < 0.001; HCC827-GR: < 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: < 0.001; HCC827-GR: < 0.001) and high concentrations (PC9-GR: < 0.001; HCC827-GR: < 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: < 0.0001; HCC827-GR: < 0.0001), ZEB2 could directly bind to the promoter region of CSF-1 to elevate its secretion (PC9-GR: < 0.0001; HCC827-GR: < 0.0001). 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.
免疫检查点抑制剂（ICI）彻底改变了非小细胞肺癌（NSCLC）的治疗方法。然而，ICI疗法在表皮生长因子受体（EGFR）驱动的NSCLC患者中，尤其是对EGFR酪氨酸激酶抑制剂（TKI）耐药的患者中的有效性一直令人失望。EGFR-TKI治疗后的免疫抑制肿瘤微环境（TME）已被证明会显著影响ICI的有效性。因此，研究抑制性TME形成背后的机制并探索潜在的干预措施对于EGFR-TKI耐药NSCLC的研究至关重要。通过定量逆转录聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法（WB）和免疫组织化学染色对人NSCLC细胞系和NSCLC患者的肿瘤标本中的锌指蛋白E盒结合因子2（ZEB2）水平进行定量。为了研究ZEB2如何影响巨噬细胞极化，用qRT-PCR和流式细胞术检测M1/M2标志物谱。用qRT-PCR、酶联免疫吸附测定（ELISA）和微尺度发现电化学发光测定法确定由ZEB2表达改变引发的细胞因子产生的变化。使用双荧光素酶报告系统测试ZEB2与细胞因子基因启动子的直接结合。通过将来自癌症基因组图谱（TCGA）的肺腺癌（LUAD）转录组数据与ZEB2表达相关联，并通过蛋白质免疫印迹法验证关键发现，研究上游调控途径。最后，通过将预处理的PC9或HCC827细胞皮下植入BALB/c裸鼠中建立细胞源异种移植（CDX）模型，以验证EGFR-TKI耐药性和ZEB2对肿瘤相关巨噬细胞（TAM）极化的影响。结果表明，在NSCLC细胞系中，EGFR-TKI耐药上调了M2极化生物标志物精氨酸酶-1（PC9-GR：<0.01；HCC827-GR：<0.05）和白细胞介素4（IL4，PC9-GR：<0.01；HCC827-GR：<0.01），而下调了M1极化生物标志物肿瘤坏死因子-α（TNF-α，PC9-GR：<0.01；HCC827-GR：<0.01）、白细胞介素1β（IL1β，PC9-GR：<0.01；HCC827-GR：<0.01）和白细胞介素6（IL6，PC9-GR：<0.001；HCC827-GR：<0.001）。同时，在两种EGFR-TKI耐药小鼠模型中，CD206阳性TAM（PC9-GR：<0.05；HCC827-GR：<0.01）增加，而CD86阳性TAM（PC9-GR：<0.05；HCC827-GR：<0.05）减少。除了形成抑制性TME外，还发现ZEB2在PC9-GR（qRT-PCR：<0.0001；WB：<0.05）和HCC827-GR（qRT-PCR：<0.0001；WB：<0.05）细胞中上调。在临床样本中也观察到相同的趋势，NSCLC患者吉非替尼耐药后ZEB2上调（<0.0001）。基于这些发现，证明ZEB2基因敲低可下调精氨酸酶-1（PC9-GR：<0.01；HCC827-GR：<0.05）和白细胞介素4（PC9-GR：<0.01；HCC827-GR：<0.001），同时上调肿瘤坏死因子-α（PC9-GR：<0.0001；HCC8-GR：<0.0001）、白细胞介素1β（HCC827-GR：<0.001）和白细胞介素6（PC9-GR：<0.01；HCC827-GR：<0.001），表明其在EGFR-TKI耐药NSCLC细胞系的M1/M2极化中的作用。在EGFR-TKI耐药小鼠模型中，ZEB2基因敲除后，CD206阳性TAM下调（PC9-GR：<0.05；HCC827-GR：<0.01），CD86阳性TAM上调（PC9-GR：<0.001；HCC827-GR：<0.05），这也证明了抑制性TME的逆转。此外，在使用Akt抑制剂MK2206干预后，ZEB2表达在低浓度（PC9-GR：<0.001；HCC827-GR：<0.001）和高浓度（PC9-GR：<0.001；HCC827-GR：<0.0001）下均受到抑制。最后，证明ZEB2调节TAM极化的机制与细胞因子分泌有关。根据ELISA结果，除了诱导转化生长因子-β1（TGF-β1）分泌（PC9-GR：<0.0001；HCC827-GR：<0.0001）外，ZEB2可直接结合集落刺激因子-1（CSF-1）的启动子区域以增加其分泌（PC9-GR：<0.0001；HCC827-GR：<0.0001）。 在EGFR-TKI耐药的NSCLC中，磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-Akt）级联的激活导致ZEB2表达显著升高。升高的ZEB2增加了CSF-1和TGF-β1的释放，使巨噬细胞转向M2表型，同时阻碍M1极化。因此，一旦出现EGFR-TKI耐药，抑制ZEB2有可能重塑TME并提高ICI的有效性。
