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.

人类转移形成的机械基础仍然难以捉摸。我们不知道转移是否 由随机散布细胞形成，或分子定义的转移克隆是否具有较高的能力 存在定居特定的器官。在小鼠的转移实验中，（罕见的）细胞的亚群随着增加 转移潜力已证明存在。这种潜力在有丝分裂上是可遗传的，并且与特定有关 基因表达程序。在人类中，同样令人信服的数据也不存在，并且是一个统一的理论 转移统一动物和人类的观察结果，并通过临床进行可测试的预测 相关性仍然缺失。使用数百种人类结肠癌样本的系统发育分析，我们有 最近表明，解剖上不同的远处转移通常仅由一个原发性肿瘤形成 亚种群（“转移创始人克隆”）。在这里，我们提出了一种新颖的实验设计，旨在确定 人类转移创始人克隆的分子特征。在特定目标1中，我们将进行严格的搜索 用于转移创建者克隆中的复发基因表达模式。我们将从新的 切除的主要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准确注释转移的创建者区域相对于其余的 原发性肿瘤（“旁观者区域”）。然后，我们将执行RNA测序以识别创建者特异性基因 70名患者的表达模式，目的是定义通用的“创始人签名”，以识别 原发性肿瘤内具有较高转移性的细胞群。在特定目标2中，我们将调查 有丝分裂性可遗传的分子改变，可能导致增强基因 表达签名。我们优先考虑体拷贝数变化（SCNA）和DNA甲基化模式为 候选人进行此类改变，但要全面，我们还将评估驾驶员突变。我们将表演 整个基因组，外显子组和降低表示的基硫酸盐测序，选定 旁观者和匹配的转移，以确定创始人是否富含特定子集 SCNA，驱动突变和/或甲基化变化。在特定的目标3中，我们将验证并机械验证 探索具有异种移植试验的转移创始人克隆的生物学特性 器官（PDOS）。在AIM 3A中，我们将部署PDO技术来检验以下假设。 与旁观者区域衍生的PDO相比，创始人区域更有可能转移到小鼠肝脏， 提供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 异种移植系统探索以转移相关为目标的基因的生物学功能 1和2通过损失和功能获得的遗传筛选。这个体内实验框架将深入 在目标1和2中获得的潜在客户的机械随访。总共，该提案概述了迈向的第一步 紧急临床必须识别大肠癌中转移创始人克隆的分子特征。