Cycling cancer persisters arise from lineages with distinct transcriptional and metabolic programs. Cycling cancer persisters arise from lineages with distinct transcriptional and metabolic programs

Non-genetic mechanisms have recently emerged as important drivers of therapy failure in cancer, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment. While most cancer persisters, like their bacterial counterparts, remain arrested in the presence of drug, a rare subset of cancer persisters can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to simultaneously resist therapy and maintain proliferative capacity in the presence of drug. To address this, we developed Watermelon, a new high-complexity expressed barcode lentiviral library for simultaneous tracing each cell's clonal origin, proliferative state, and transcriptional state, and used it to study this rare, transiently-resistant, proliferative persister population and identify what distinguishes it from non-cycling persisters. Analysis of Watermelon-transduced cancer cell lines demonstrated that cycling and non-cycling persisters arise from different pre-existing cell lineages with distinct transcriptional and metabolic programs. The proliferative capacity of persisters is associated with an upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation in specific subpopulations of tumor cells. Overall design: 10X Genomics single cell RNAseq of PC9 cells treated with Osimertinib Lung cancer PC9 cells were treated with osimertinib (300nM) and harvested before treatment, after 3,7, and 14 days of continuous drug treatment. At day 14 prior to sequencing, cells were sorted to three groups based on mCherry expression:cycling (low), moderate cyclers (med) and non-cycling (high) In addition, seven Watermelon lines models from EGFR-driven lung cancer (PC9, HCC827), HER2-driven breast cancer (BT474, EFM192A) and BRAF-driven melanoma (A375, COLO858) and colorectal (HT29) cell lines, treated them with clinically relevant kinase inhibitors for 10 days (300nM osimertinib, 1uM lapatinib or 1uM dabrafenib). At day 10 prior to sequencing, cells were sorted to three groups based om mCherry expression:cycling (low) and non-cycling (high)

近年来，非遗传机制被证实是癌症治疗失败的重要驱动因素：部分癌细胞可在治疗诱导下进入可逆的药物耐受持久态（drug-tolerant persister state）。与细菌中的同类持久细胞类似，多数癌症持久细胞在药物存在下会处于增殖停滞状态，但极少数癌症持久细胞可在持续药物处理下重新进入细胞周期（cell cycle）。目前学界对可使癌症持久细胞在药物存在下同时维持治疗抗性与增殖能力的非遗传机制尚所知甚少。

为解决这一问题，我们开发了Watermelon——一种新型高复杂度表达条形码慢病毒文库（expressed barcode lentiviral library），可同时追踪每个细胞的克隆起源、增殖状态与转录状态，并利用该工具对这种罕见的、短暂耐药的增殖型持久细胞群体展开研究，以明确其与非增殖型持久细胞的差异所在。对转导了Watermelon的癌细胞系的分析显示，增殖型与非增殖型持久细胞源自不同的预先存在的细胞谱系（cell lineage），且各自具有独特的转录与代谢程序。在特定肿瘤细胞亚群中，持久细胞的增殖能力与抗氧化基因程序的上调以及向脂肪酸氧化（fatty acid oxidation）的代谢重编程密切相关。

实验整体设计：
1. 奥希替尼（Osimertinib）处理的PC9细胞的10X Genomics单细胞RNA测序（single cell RNAseq）：对肺癌PC9细胞施加300nM奥希替尼处理，分别在给药前、持续给药3天、7天及14天收集细胞。测序前于第14天将细胞按mCherry表达水平分为三组：增殖型（低表达）、中等增殖型（中等表达）与非增殖型（高表达）。
2. 多癌种细胞系的Watermelon文库分析：选取7株Watermelon模型细胞系，涵盖表皮生长因子受体（EGFR）驱动型肺癌（PC9、HCC827）、人表皮生长因子受体2（HER2）驱动型乳腺癌（BT474、EFM192A）、BRAF驱动型黑色素瘤（A375、COLO858）以及结直肠癌（HT29）细胞系，分别施加临床相关的激酶抑制剂处理（300nM奥希替尼、1μM拉帕替尼或1μM达拉非尼），持续给药10天。测序前于第10天将细胞按mCherry表达水平分为两组：增殖型（低表达）与非增殖型（高表达）。