CD24 as a therapeutic vulnerability in osimertinib-induced drug-tolerant persister cells of EGFR-mutant NSCLC

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.