对于大多数携带EGFR突变的非小细胞肺癌（NSCLC）患者，使用EGFR抑制剂（EGFRi）进行治疗能够引起临床反应。尽管在治疗初期能够减小肿瘤大小，但仍然存在残留疾病导致疾病复发。阐明对于敏感细胞和耐受细胞之间的生物学差异以及揭示耐受细胞在治疗过程中的进化规律，有助于寻找提高EGFRi疗效的策略。在本研究中，我们采用了一种表达条形码系统与单细胞RNA测序相结合的方法，对耐药细胞的起源和克隆进化进行了高分辨率追踪。该平台可以纵向地分析多个克隆中基因表达和药物敏感性在EGFRi治疗中的变化。耐药细胞在基线时具有更高的关键生存通路（例如YAP和EMT）的表达，并且在接受EGFRi治疗后也可以不同程度地改变基因表达，与敏感细胞相比。此外，针对共同下游组分（MAPK）或正交因子（化疗）的药物联合治疗比单独使用EGFRi具有更高的疗效，这很可能归因于对肿瘤中存在的多种EGFRi耐受机制进行更广泛的靶向。总体而言，该方法有助于全面研究治疗应答中的克隆进化，从而为开发改进的诊断方法和针对耐药细胞的治疗策略提供信息。

For a majority of non-small cell lung cancer (NSCLC) patients with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the pre-existing biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug tolerant cells at a high resolution by using an expressed barcoding system coupled with single cell RNA-sequencing. This platform enabled longitudinally profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared to sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is likely attributable to broader targeting of the multiple EGFRi tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug tolerant cells.