PO.ET03.05 · 实验与分子治疗
CD24作为EGFR突变型NSCLC奥希替尼诱导的耐药持留细胞的治疗易感性靶点
CD24 as a therapeutic vulnerability in osimertinib-induced drug-tolerant persister cells of EGFR-mutant NSCLC
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
耐药持留（DTP）细胞在治疗压力下存活并驱动残留病灶，限制了靶向治疗的长期疗效。第三代表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI）奥希替尼显著改善了EGFR突变型非小细胞肺癌（NSCLC）患者的结局。然而，耐药不可避免地会出现。这种耐药在很大程度上由DTP细胞的持留和适应性演化所驱动。尽管如此，奥希替尼诱导的DTP细胞形成和维持的分子机制仍知之甚少。为更好地界定这些机制，我们对接受新辅助奥希替尼治疗的EGFR突变型NSCLC患者的切除肿瘤标本进行了单细胞RNA测序（scRNA-seq）。分析包括配对的治疗前活检样本和治疗后手术标本，并按病理反应分层（主要病理缓解[MPR]与非MPR）。关键发现通过TCGA/GTEx、公共scRNA-seq数据集、临床残留肿瘤的IHC以及CRISPR/Cas9编辑细胞系和长期药物处理模型中的功能实验加以验证。与MPR肿瘤相比，非MPR肿瘤中治疗前上皮细胞表现出显著更高的CD24表达和CD24⁺细胞比例，且这些水平在治疗后保持稳定，提示存在一种预先存在的CD24⁺状态，与较低的初始反应相关。相反，MPR肿瘤在治疗后表现出CD24表达和CD24⁺上皮亚群的强劲增加，与公共EGFR-TKI DTP数据集一致。对MPR上皮细胞的重新聚类揭示了七个亚群，其中C1、C3和C5富集治疗后细胞并被归类为DTP样。CD24表达在C1和C6中达到峰值，C6包含比例均衡的治疗前/后细胞。拟时序分析确定了三条转录轨迹，将CD24高表达亚群C1和C6置于富集治疗后细胞的终末状态，且CD24表达朝该终点逐渐升高。值得注意的是，C6内的治疗前细胞全部为CD24高表达，提示对预先存在的CD24⁺亚群的选择性保留。CD24高表达亚群富集铁死亡、ROS信号和自噬这些经典DTP程序。体外DTP模型也表明，奥希替尼处理后CD24显著上调，而CD24缺失降低了基础增殖、增强了奥希替尼的抗增殖效应，并延迟了撤药后的再生长。总之，这些结果将CD24确认为预先存在和治疗诱导的上皮状态的标志物，这些状态维持DTP的存活和再生长，凸显CD24作为在EGFR突变型NSCLC中实现对奥希替尼更持久反应的潜在治疗易感性靶点。
查看英文原文 English abstract
Drug-tolerant persister (DTP) cells survive therapeutic pressure and drive residual disease, limiting the long-term efficacy of targeted therapies. The third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib has markedly improved outcomes for patients with EGFR-mutant non-small cell lung cancer (NSCLC). However, resistance inevitably emerges. This resistance is largely driven by the persistence and adaptive evolution of DTP cells. Nevertheless, the molecular mechanisms underlying the formation and maintenance of osimertinib-induced DTP cells remain poorly understood. To better define these mechanisms, we performed single-cell RNA sequencing (scRNA-seq) on resected tumor specimens from patients with EGFR-mutant NSCLC treated with neoadjuvant osimertinib. Analyses included paired pre-treatment biopsy samples and post-treatment surgical specimens, stratified by pathological response (major pathological response [MPR] vs. non-MPR). Key findings were validated using TCGA/GTEx, public scRNA-seq datasets, IHC of clinical residual tumors, and functional assays in CRISPR/Cas9-edited cell lines and long-term drug-treatment models. Pre-treatment epithelial cells in non-MPR tumors exhibited markedly higher CD24 expression and CD24⁺ fractions compared with MPR tumors, and these levels remained stable post-treatment, indicating a pre-existing CD24⁺ state associated with reduced initial response. In contrast, MPR tumors displayed a robust post-treatment increase in CD24 expression and CD24⁺ epithelial subsets, consistent with public EGFR-TKI DTP datasets. Re-clustering of MPR epithelial cells revealed seven subclusters, among which C1, C3, and C5 were enriched for post-treatment cells and classified as DTP-like. CD24 expression peaked in C1 and C6, with C6 containing balanced pre/post cells. Pseudotime analysis identified three transcriptional trajectories, placing CD24-high clusters C1 and C6 at terminal states enriched for post-treatment cells, with CD24 expression progressively increasing toward this endpoint. Notably, pre-treatment cells within C6 were exclusively CD24-high, suggesting selective preservation of a pre-existing CD24⁺ subset. CD24-high clusters were enriched for ferroptosis, ROS signaling, and autophagy-canonical DTP programs. In vitro DTP models also demonstrated significant upregulation of CD24 following osimertinib treatment, while CD24 depletion reduced basal proliferation, enhanced the antiproliferative effect of osimertinib, and delayed regrowth after drug withdrawal. Together, these results identify CD24 as a marker of both pre-existing and therapy-induced epithelial states that sustain DTP survival and regrowth, highlighting CD24 as a potential therapeutic vulnerability for achieving more durable responses to osimertinib in EGFR-mutant NSCLC.
利益披露 Disclosure
J. Ko, None..
J. Moon, None..
M. Yun, None.