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

全面表征结直肠癌转移起始克隆

• 批准号: 10973772
• 负责人: Kamila Naxerova
• 金额: $ 55.68万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2028-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10973772
• 关键词: Towards; complete; characterization; metastasis; founder

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.

人类转移形成的机制基础仍然难以捉摸。我们不知道是否转移 是由随机播散的细胞形成的，还是具有上级能力的分子定义的转移性克隆 在特定的器官中定居在小鼠的转移实验中，（罕见的）细胞亚群增加， 已经显示存在转移潜力。这种潜力是有丝分裂遗传的，并与特定的 基因表达程序。在人类中，类似的令人信服的数据并不存在， 协调动物和人类的观察，并与临床进行可测试的预测， 相关性仍然缺失。通过对数百例人类结直肠癌样本的系统发育分析，我们 最近显示解剖学上不同的远处转移通常仅由一个原发肿瘤形成 亚群（“转移建立者克隆”）。在这里，我们提出了一个新的实验设计，旨在确定 人类转移癌基因克隆的分子特征在具体目标1中，我们将进行严格的搜索 在转移建立者克隆中的复发基因表达模式。我们将收集新的网格活检， 切除原发性结直肠癌和匹配的同步肝转移，重建进化树 从微卫星突变数据和精确注释转移创始人区维斯维斯其余的 原发性肿瘤（“旁观者区域”）。然后，我们将进行RNA测序，以确定特定的基因， 70名患者的表达模式，目的是定义一个通用的“创始人签名”， 原发性肿瘤内具有上级转移能力的细胞群。在具体目标2中，我们将研究 有丝分裂可遗传的分子改变，可能会产生转移使能基因 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， 候选人，但为了全面，我们也将评估驱动突变。我们将执行 浅全基因组，外显子组和简化代表亚硫酸氢盐测序的创始人，选择 旁观者和匹配的转移，以确定创始人是否富集了特定的亚组， SCNA、驱动突变和/或甲基化变化。在具体目标3中，我们将验证和机械地 用患者来源的异种移植试验探索转移建立者克隆的生物学特性， 类器官（PDO）。在Aim 3A中，我们将部署PDO技术来验证转移性PDO的假设， 创始区比来自旁观区的PDO更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。在目标3B中，我们将使用PDO 探索目标中强调与转移相关的基因的生物学功能的异种移植系统 1和2通过功能丧失和获得遗传筛选。这种体内实验框架将使深入 对目标1和2中获得的线索进行机械跟踪。总的来说，该提案概述了实现 临床上迫切需要鉴定结直肠癌转移创始克隆的分子特征。