(1) Intestinal Organoid Models for APC LOH

项目总结/摘要 结肠癌发展的一个关键步骤发生在Apc区域内的细胞， 杂合子突变细胞经历Apc杂合性丢失（洛）。理解 Apc洛缺失事件的机制和细胞后果可以提供重要的 关于如何抑制这一事件以降低癌症风险的信息。关键 尚未回答的问题包括：导致APC洛的事件是否包括 胞质分裂失败？是否所有发生洛缺失事件的细胞都具有相同的 进展？哪些遗传和环境因素会影响Apc洛？ 我们建议开发一个肠道类器官系统作为一个可延展的实验 研究Apc洛缺失的平台。我们将利用从ESC产生的肠类器官， 这些“迷你肠”可以通过基因操作， 检测相对罕见的Apc洛缺失事件所必需的。我们建议开发这一 类器官系统，通过将ApcMin等位基因与GFP连接来实时研究Apc洛， RFP报告基因和白喉毒素受体（DTR）的APC-野生型等位基因。我们将 使用这些类器官来计数和分离已经经历Apc洛的细胞。我们 将首先确定p16和p53如何影响Apc洛频率，但设想这些 类器官被用于评估广泛的突变的影响， 多态性我们将研究从Apc洛事件中出现的细胞的性质， 关注其增殖能力及其与正常ISCs和癌症的关系 干细胞最后，我们将评估四倍体细胞作为Apc洛缺失前体的作用 并确定是否有一种新的有丝分裂靶向剂，选择性诱导细胞凋亡， 四倍体细胞可降低Apc洛缺失频率。 这些探索性研究预计将建立一个实验系统， 研究结肠癌发展中的一个关键事件：APC突变细胞的出现， 在癌前组织区域从长远来看，我们设想建立一个综合的 了解环境和遗传因素如何影响Apc的实验管道 洛，经历过Apc洛的细胞如何发展为癌症干细胞，以及 最终，如何降低个体患结肠癌的风险。

Project Summary/Abstract A critical step in colon cancer development occurs when a cell within a field of Apc- heterozygote mutant cells undergoes Apc loss of heterozygosity (LOH). Understanding the mechanism and cellular consequences of an Apc LOH event could provide important information on how this event might be suppressed to reduce cancer risk. Critical questions that remain unanswered include: Do the events leading to Apc LOH include a failure of cytokinesis? Are all cells that undergo an LOH event at equivalent risk of progression? What genetic and environmental factors can impact Apc LOH? We propose to develop an intestinal organoid system as a malleable experimental platform to study Apc LOH. We will utilize intestinal organoids generated from ESCs since these “mini-intestines” can be genetically manipulated and grown in the quantities necessary to detect the relatively rare Apc LOH event. We propose to develop this organoid system to study Apc LOH in real-time by linking the ApcMin allele to GFP and the Apc-wild type allele to an RFP reporter and the diphtheria toxin receptor (DTR). We will use these organoids to enumerate and isolate cells that have undergone Apc LOH. We will initially determine how p16 and p53 affect Apc LOH frequency, but envision these organoids being used to assess the impact of a broad range of mutations and polymorphisms. We will study the nature of cells emerging from an Apc LOH event, focusing on their proliferative capacity and their relationship to normal ISCs and cancer stem cells. Finally, we will assess the role of tetraploid cells as precursors for Apc LOH and determine if a novel mitosis-targeting agent that selectively induces apoptosis of tetraploid cells can reduce the frequency of Apc LOH. These exploratory studies are anticipated to establish an experimental system for studying a critical event in colon cancer development; the emergence of Apc-mutant cells in a pre-malignant tissue field. In the long term we envision establishing an integrated experimental pipeline to understand how environmental and genetic factors impact Apc LOH, how cells that have undergone Apc LOH progress to cancer stem cells, and ultimately, how an individual's risk of colon cancer risk can be reduced.