Single cell lineage tracing reveals clonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer

We investigate non-genomic mechanisms determining cellular response to EGFR inhibitors in triple negative breast cancer (TNBC). We integrate methods for cellular barcoding and single-cell transcriptomics to enable cell lineage tracing and explore the subclonal evolution of adaptation in an established preclinical model of TNBC in response to incremental concentrations of Afatinib, a second generation EGFR-TKI that irreversibly inhibits both EGFR and HER2. Retrospective lineage tracing data analysis uncovered a pre-existing subpopulation of rare Afatinib-tolerant cells displaying distinct biological features, such as elevated mRNA levels of the IGFBP2 gene. Furthermore, we investigated temporal coordination of transcriptional programs in drug resistant clones with high replication fitness by reordering cells along a pseudotime trajectory. Interestingly, it revealed the activation of biological processes, such as fatty acid metabolism, which have previously been linked to EGFR-TKIs resistance mechanisms. We integrated commercially available platform for cellular barcoding and single-cell transcriptomics to enable cell lineage tracing

本研究针对三阴性乳腺癌（triple negative breast cancer, TNBC）中调控细胞对表皮生长因子受体（EGFR）抑制剂应答的非基因组机制展开系统探究。我们整合细胞条形码技术（cellular barcoding）与单细胞转录组学（single-cell transcriptomics）方法，以实现细胞谱系追踪（cell lineage tracing），并在已构建的三阴性乳腺癌临床前模型中，探究细胞在响应递增浓度阿法替尼时的适应性亚克隆进化过程。阿法替尼是一种可不可逆同时抑制EGFR与人表皮生长因子受体2（HER2）的第二代EGFR酪氨酸激酶抑制剂（EGFR-TKI）。回顾性谱系追踪数据分析发现，预先存在一类罕见的阿法替尼耐受细胞亚群，其具备独特的生物学特征，例如IGFBP2基因的mRNA水平显著升高。此外，我们通过将细胞沿拟时间轨迹（pseudotime trajectory）重排序，探究了具有高复制适应性的耐药克隆中转录程序的时间调控模式。值得注意的是，该分析揭示了脂肪酸代谢等生物学过程的激活，此类过程此前已被证实与EGFR-TKI耐药机制密切相关。本研究整合了商业化的细胞条形码与单细胞转录组学平台，以实现细胞谱系追踪。