Integration of multiple lineage measurements from the same single cell reconstructs parallel tumor evolution

The dynamics of tumorigenesis shares many similarities with Darwinian evolution. Elevated mutation rates result in a genetically diverse population of cells competing for limited resources in the tumor. Natural selection ultimately leads to clonal expansion of the fittest genotypes resulting in aggressive tumor outgrowth and dissemination. Here we mimic clonal expansion by evolving a colon cancer organoid model for over 100 cellular generations while simultaneously monitoring clone size, copy number variants (CNVs), and single nucleotide variants (SNVs) in thousands of individual cells. This offers the unique opportunity to combine multiple lines of evidence to establish reliable clonal evolution trees that reveal the order of events in which chromosomal deletions and amplifications occur and allow the identification of chromosomal aberrations that recur multiple times within the same population. For example, we observe recurrent sequential loss of chromosome 4 after loss of chromosome 18 in multiple unique tumor clones. Reanalysis of clinical data indeed suggests that this sequential loss is often observed in colon tumors and is associated with poor survival. Our results suggest that organoid evolution models complemented with integrated single-cell sequencing technology provide a powerful platform to study and ultimately control tumor evolution in detail.

肿瘤发生的动力学过程与达尔文进化存在诸多相似之处。突变率升高会促使肿瘤内形成遗传多样性丰富的细胞群体，这些细胞在肿瘤微环境中争夺有限资源。自然选择最终将使最适配的基因型发生克隆扩增，进而引发侵袭性肿瘤生长与扩散。本研究通过对结肠癌类器官模型进行超过100个细胞世代的传代培养以模拟克隆扩增过程，同时对数千个单个细胞的克隆大小、拷贝数变异（copy number variants, CNVs）以及单核苷酸变异（single nucleotide variants, SNVs）进行同步监测。该实验体系为我们提供了独特的研究契机，可整合多维度证据以构建可靠的克隆进化树，进而揭示染色体缺失与扩增事件的发生顺序，并能识别在同一细胞群体中反复出现的染色体畸变。例如，我们在多个独特的肿瘤克隆中观察到，在18号染色体缺失后，会相继出现4号染色体的重复性缺失。对临床数据的重新分析同样表明，这类序贯染色体缺失在结肠癌患者中较为常见，且与不良预后密切相关。本研究结果表明，结合整合式单细胞测序技术的类器官进化模型，可为深入研究并最终实现肿瘤进化的精准调控提供强有力的研究平台。