PO.TB09.01 · 肿瘤生物学
EGFR抑制剂耐药肿瘤上皮细胞簇的时空分析
Spatio-temporal analysis of EGFR inhibitor resistant tumor epithelial clusters
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
[背景]耐药持存（DTP）细胞是一种可逆的、对药物不敏感的肿瘤亚群，与微小残留病灶（MRD）和复发相关。然而，其出现机制和治疗脆弱性仍不明确。我们此前利用遗传条形码在体内追踪克隆动力学，并显示特定克隆的扩增有助于EGFR抑制剂的耐药。在此，我们分析了接受EGFR抑制剂治疗的非小细胞肺癌，以（1）鉴定治疗耐药的肿瘤细胞群体，（2）阐明DTP细胞出现的空间机制。
[方法]分析了PIT3试验（UMIN000026197）中新辅助EGFR抑制剂治疗前后的肿瘤样本。进行了单细胞（sc）RNA测序（n = 8）和Visium HD空间转录组学（n = 10）。空间数据集使用HueTracer（https://github.com/MANO-B/HueTracer）进行处理，从而能够提取单细胞水平的表达谱并与scRNA-seq进行整合。
[结果]整合的scRNA-seq分析（8份标本，16,419个细胞）鉴定出多个肿瘤上皮细胞簇，包括一个在应答不佳者中富集的EGFR耐药亚簇。该亚簇显示出更高比例的G1期细胞、EGFR降低以及EMT相关基因升高，提示治疗耐药表型。在应答者中也检测到具有类似转录特征的稀有细胞，表明存在存活的DTP样细胞。
空间转录组学分析（20份标本，2,415,876个细胞）鉴定出肿瘤簇和治疗应答区域，后者以伴有坏死和促纤维增生的岛状残留病灶为特征。少量存活细胞被指定为DTP候选细胞，并与其他肿瘤上皮细胞进行比较。在五个病例中，对差异表达基因的荟萃分析揭示了炎症相关和可塑性相关基因的一致上调，包括ILR-X、TF-X、LAMB3和TXLNB（FDR < 0.05）。GSEA显示TNF-alpha/NFκB信号、干扰素应答、EMT和组织重塑通路的富集，表明DTP候选细胞中存在共有的炎症增强和可塑性增强状态。
[结论]单细胞和空间分析表明，EGFR抑制剂治疗后持存的DTP样细胞表现出共有的分子表型，其特征为炎症加剧和上皮可塑性增强。这些特征可能是能够驱动治疗后复发的微小残留病灶的基础，支持将DTP细胞作为潜在治疗靶点的概念。
查看英文原文 English abstract
[Background]Drug-tolerant persister (DTP) cells are a reversible drug-insensitive tumor subpopulation implicated in minimal residual disease (MRD) and recurrence. However, their emergence mechanisms and therapeutic vulnerabilities remain unclear. We previously tracked clonal dynamics in vivo using genetic barcodes and showed that expansion of specific clones contributes to the resistance of EGFR inhibitors. Here, we analyzed non-small cell lung cancer treated with EGFR inhibitors to (1) identify treatment-resistant tumor cell populations and (2) elucidate spatial mechanisms underlying DTP cell emergence.
[Methods]Tumor samples from the PIT3 trial (UMIN000026197) were analyzed before and after the neoadjuvant EGFR inhibitor therapy. Single-cell (sc) RNA-seq (n = 8) and Visium HD spatial transcriptomics (n = 10) were performed. Spatial datasets were processed using HueTracer (https://github.com/MANO-B/HueTracer), enabling extraction of single-cell-level expression profiles and integration with scRNA-seq.
[Results]Integrated scRNA-seq analysis (8 specimens, 16,419 cells) identified multiple tumor epithelial clusters, including an EGFR-resistant subcluster enriched in poor responders. This subcluster showed a higher proportion of G1-phase cells, reduced EGFR, and elevated EMT-related genes, suggesting treatment-resistant phenotypes. Rare cells with similar transcriptional features were also detected in responders, indicating the presence of surviving DTP-like cells.
Spatial transcriptomic analysis (20 specimens, 2,415,876 cells) identified tumor clusters and treatment-responsive regions characterized by island-like residual lesions with necrosis and desmoplasia. Small numbers of surviving cells were designated as DTP candidates and compared with other tumor epithelial cells. Across five cases, meta-analysis of differentially expressed genes revealed consistent upregulation of inflammation- and plasticity-related genes, including ILR-X , TF-X , LAMB3 , and TXLNB (FDR < 0.05). GSEA showed enrichment of TNF-alpha/NFκB signaling, interferon response, EMT, and tissue remodeling pathways, indicating a shared inflammatory and plasticity-enhanced state in DTP candidates.
[Conclusion]Single-cell and spatial analyses demonstrate that DTP-like cells persisting after EGFR inhibitor therapy exhibit a shared molecular phenotype characterized by heightened inflammation and epithelial plasticity. These features may underlie microscopic residual disease capable of driving post-treatment recurrence, supporting the concept of DTP cells as a potential therapeutic target.
利益披露 Disclosure
S. Watanabe, None..
Y. Suehara, None..
M. Ikegami, None..
S. Park, None..
Y. Kimura, None..
T. Ueno, None..
T. Hayashi, None..
K. Takamochi, None..
S. Kohsaka, None.