Panagopoulos Andreas, Stout Merula, Kilic Sinan, Leary Peter, Vornberger Julia, Pasti Virginia, Galarreta Antonio, Lezaja Aleksandra, Kirschenbühler Kyra, Imhof Ralph, Rehrauer Hubert, Ziegler Urs, Altmeyer Matthias
Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland.
The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.
Nature. 2025 May 21. doi: 10.1038/s41586-025-08986-0.
Cell heterogeneity is a universal feature of life. Although biological processes affected by cell-to-cell variation are manifold, from developmental plasticity to tumour heterogeneity and differential drug responses, the sources of cell heterogeneity remain largely unclear. Mutational and epigenetic signatures from cancer (epi)genomics are powerful for deducing processes that shaped cancer genome evolution. However, retrospective analyses face difficulties in resolving how cellular heterogeneity emerges and is propagated to subsequent cell generations. Here, we used multigenerational single-cell tracking based on endogenously labelled proteins and custom-designed computational tools to elucidate how oncogenic perturbations induce sister cell asymmetry and phenotypic heterogeneity. Dual CRISPR-based genome editing enabled simultaneous tracking of DNA replication patterns and heritable endogenous DNA lesions. Cell lineage trees of up to four generations were tracked in asynchronously growing cells, and time-resolved lineage analyses were combined with end-point measurements of cell cycle and DNA damage markers through iterative staining. Besides revealing replication and repair dynamics, damage inheritance and emergence of sister cell heterogeneity across multiple cell generations, through combination with single-cell transcriptomics, we delineate how common oncogenic events trigger multiple routes towards polyploidization with distinct outcomes for genome integrity. Our study provides a framework to dissect phenotypic plasticity at the single-cell level and sheds light onto cellular processes that may resemble early events during cancer development.
细胞异质性是生命的一个普遍特征。尽管受细胞间差异影响的生物学过程多种多样，从发育可塑性到肿瘤异质性以及不同的药物反应，但细胞异质性的来源在很大程度上仍不清楚。癌症（表观）基因组学中的突变和表观遗传特征对于推断塑造癌症基因组进化的过程很有帮助。然而，回顾性分析在解决细胞异质性如何出现并传播到后续细胞世代方面面临困难。在这里，我们使用基于内源性标记蛋白的多代单细胞追踪和定制设计的计算工具，来阐明致癌扰动如何诱导姐妹细胞不对称性和表型异质性。基于双CRISPR的基因组编辑能够同时追踪DNA复制模式和可遗传的内源性DNA损伤。在异步生长的细胞中追踪了多达四代的细胞谱系树，并通过迭代染色将时间分辨的谱系分析与细胞周期和DNA损伤标记物的终点测量相结合。除了揭示复制和修复动态、损伤遗传以及多代姐妹细胞异质性的出现外，通过与单细胞转录组学相结合，我们还描绘了常见的致癌事件如何触发多条通向多倍体化的途径，这些途径对基因组完整性具有不同的结果。我们的研究提供了一个在单细胞水平剖析表型可塑性的框架，并揭示了可能类似于癌症发展早期事件的细胞过程。
