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。我们不知道转移是否存在。 是由随机播散的细胞形成，还是分子定义的具有卓越能力的转移性克隆 在小鼠的转移实验中，（罕见）细胞亚群的数量增加。 转移潜力已被证明存在，这种潜力是有丝分裂遗传的并且与特定的相关。 在人类中，不存在类似的令人信服的数据，并且没有统一的理论。 协调动物和人类观察的转移，并根据临床结果做出可测试的预测 通过对数百个人类结直肠癌样本进行系统发育分析，我们发现相关性仍然缺失。 最近表明，解剖学上不同的远处转移通常仅由一个原发肿瘤形成 在这里，我们提出了一种新颖的实验设计，旨在识别。 在具体目标 1 中，我们将进行严格的搜索。 我们将从新的转移创始者克隆中收集网格活检。 切除原发性结直肠癌和匹配的同步肝转移瘤，重建进化树 从微卫星突变数据中精确注释转移起始区域相对于其余区域 然后，我们将进行 RNA 测序来识别原发肿瘤（“旁观者区域”）。 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 在特定目标 2 中，我们将研究原发性肿瘤内具有优异转移能力的细胞群。 有丝分裂遗传的分子改变可能会产生促进转移的基因 我们优先考虑体细胞拷贝数改变 (SCNA) 和 DNA 甲基化模式。 此类改变的候选者，但为了全面起见，我们还将评估驱动程序突变。 浅层全基因组、外显子组和创始人的简化代表性亚硫酸氢盐测序，选择 旁观者和匹配的转移以确定创始人是否丰富了特定的子集 SCNA、驱动突变和/或甲基化变化在具体目标 3 中，我们将在机制上进行验证。 通过患者来源的异种移植测定探索转移创始人克隆的生物学特性 在目标 3A 中，我们将部署 PDO 技术来检验 PDO 来自转移的假设。 与来自旁观者区域的 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。 异种移植系统探索目标中与转移相关的基因的生物学功能 1 和 2 通过功能丧失和获得的遗传筛选，该体内实验框架将实现深入。 对目标 1 和 2 中获得的线索进行机械跟进。总的来说，该提案概述了实现目标的第一步 临床迫切需要确定结直肠癌转移起始克隆的分子特征。