Towards a complete characterization of the metastasis founder clones in colorectal cancer

人类肿瘤转移形成的机制基础仍不清楚。我们不知道转移瘤是否 是由随机扩散的细胞形成的，还是具有优越能力的分子定义的转移性克隆 来殖民特定的器官存在。在小鼠的转移实验中，(罕见的)细胞亚群增加 转移潜能已被证明是存在的。这种潜力是有丝分裂可遗传的，并与特定的 基因表达程序。在人类身上，同样令人信服的数据并不存在，而一个统一的理论 转移，使动物和人类的观察结果相一致，并使可检验的预测与临床相符 相关性仍然缺失。利用对数百个人类结直肠癌样本的系统发育分析，我们有 最近的研究表明，在解剖学上不同的远处转移通常只由一个原发肿瘤形成。 亚群(“转移创建者克隆”)。在这里，我们提出了一种新的实验设计，旨在识别 人类肿瘤转移创建者克隆的分子特征。在具体目标1中，我们将进行严格的搜索 了解转移创建者克隆中的重复基因表达模式。我们将从新近收集网格活检 切除原发结直肠癌和匹配的同步肝转移，重建进化树 根据微卫星突变数据，精确地注释转移创建区与其余部分之间的关系 原发肿瘤(“旁观区”)。然后，我们将进行RNA测序，以确定创始人特有的基因 70名患者的表达模式，目标是定义一个通用的“创始人签名”，以识别 原发瘤内具有优越转移能力的细胞群。在具体目标2中，我们将调查 有丝分裂可遗传的分子改变，可能导致肿瘤转移的基因 表达式签名。我们优先考虑体细胞拷贝数改变(SCNAs)和DNA甲基化模式 这些改变的候选者，但为了全面，我们还将评估驱动程序突变。我们将表演 创始人的浅全基因组、外显子组和简化代表性亚硫酸氢盐测序 旁观者，并匹配转移以确定创始人是否为特定子集的富集者 SCNAs、驱动突变和/或甲基化变化。在具体目标3中，我们将机械地验证和 用病人来源的异种移植试验探索转移创建者克隆的生物学特性 有机化合物(PDO)。在Aim 3A中，我们将部署PDO技术来测试来自转移的PDO的假设 创建区比来自旁观区的PDO更有可能转移到小鼠肝脏， 提供了一种独立的方法来证实我们的人类结果。在AIM 3B中，我们将使用PDO 异种移植系统探索AIMS中突出显示的转移相关基因的生物学功能 1和2通过功能丧失和功能获得的基因筛查。这一活体实验框架将使深入 对目标1和目标2中获得的线索采取机械性后续行动。总而言之，本提案概述了实现 确定结直肠癌转移创建者克隆的分子特征是临床的当务之急。

The mechanistic basis of metastasis formation in humans remains elusive. We do not know whether metastases are formed by random disseminated cells, or whether molecularly defined metastatic clones with superior ability to colonize specific organs exist. In metastasis experiments in mice, (rare) subpopulations of cells with increased metastatic potential have been shown to exist. This potential is mitotically heritable and associated with specific gene expression programs. In humans, similarly compelling data does not exist, and a unified theory of metastasis that harmonizes observations in animals and humans and makes testable predictions with clinical relevance is still missing. Using phylogenetic analysis of hundreds of human colorectal cancer samples, we have recently shown that anatomically distinct distant metastases are typically formed by only one primary tumor subpopulation (“metastasis founder clone”). Here, we propose a novel experimental design that aims to identify the molecular traits of metastasis founder clones in humans. In specific aim 1, we will conduct a rigorous search for recurrent gene expression patterns in metastasis founder clones. We will collect grid biopsies from newly resected primary colorectal cancers and matched synchronous liver metastases, reconstruct evolutionary trees from microsatellite mutation data and precisely annotate metastasis founder areas vis-à-vis the remainder of the primary tumor (“bystander areas”). Then, we will perform RNA sequencing to identify founder-specific gene expression patterns across 70 patients, with the goal of defining a universal “founder signature” that identifies cell populations with superior metastatic ability within a primary tumor. In specific aim 2, we will investigate mitotically heritable molecular alterations that could potentially give rise to a metastasis-enabling gene expression signature. We prioritize somatic copy number alterations (SCNAs) and DNA methylation patterns as candidates for such alterations, but to be comprehensive, we will also evaluate driver mutations. We will perform shallow whole genome, exome and reduced representation bisulfite sequencing in founders, selected bystanders, and matched metastases to determine whether founders are enriched for a specific subset of SCNAs, driver mutations and/or methylation changes. In specific aim 3, we will validate and mechanistically explore the biological properties of metastasis founder clones with xenotransplanation assays of patient-derived organoids (PDOs). In Aim 3A, we will deploy PDO technology to test the hypothesis that PDOs from metastasis founder areas are more likely to metastasize to the mouse liver than PDOs derived from bystander areas, providing an independent method for corroborating our human results. In Aim 3B, we will use the PDO xenotransplantation system to explore the biological function of genes highlighted as metastasis-relevant in aims 1 and 2 via loss- and gain-of-function genetic screens. This in vivo experimental framework will enable in depth mechanistic follow-up on leads gained in Aims 1 and 2. In total, this proposal outlines the first steps toward the urgent clinical imperative of identifying the molecular features of metastasis founder clones in colorectal cancer.