(1) Intestinal Organoid Models for APC LOH

项目摘要/摘要 结肠癌发展的一个关键步骤是当APC视野内的细胞- 杂合子突变细胞经历APC杂合性丢失(LOH)。理解 APC杂合性缺失事件的机制和细胞后果可能提供重要的 关于如何抑制这一事件以降低癌症风险的信息。批判性 仍未回答的问题包括：导致APC Loh的事件是否包括 胞质分裂失败？是否所有经历LOH事件的细胞都具有相同的风险 进展？哪些遗传和环境因素会影响APC LOH？ 我们建议开发一种肠道器官系统作为延展性实验。 研究APC Loh的平台。我们将利用从ESCs产生的肠道器官类物质，因为 这些“迷你肠道”可以通过基因操作并大量生长。 对于检测相对罕见的APC杂合性缺失事件是必要的。我们建议发展这一点 通过将ApcMin等位基因连接到GFP和GFP来实时研究APC杂合性丢失的有机系统 APC-RFP报告和白喉毒素受体(DTR)的野生型等位基因。我们会 使用这些有机化合物来列举和分离经历过APC LOH的细胞。我们 将初步确定p16和p53如何影响APC杂合性缺失频率，但可以设想这些 有机化合物被用来评估广泛的突变和 多态现象。我们将研究从APC杂合性缺失事件中出现的细胞的性质， 关注它们的增殖能力及其与正常胰岛素样干细胞和癌症的关系 干细胞。最后，我们将评估四倍体细胞作为APC杂合性丢失的先兆的作用。 并确定一种新的有丝分裂靶向剂是否选择性地诱导细胞凋亡 四倍体细胞可降低APC杂合性丢失的频率。 这些探索性研究有望建立一个实验系统 研究结肠癌发展中的一个关键事件；APC突变细胞的出现 在癌前组织的视野里。从长远来看，我们设想建立一个完整的 了解环境和遗传因素如何影响APC的实验渠道 LOH，经历了APC LOH的细胞是如何发展成癌症干细胞的，以及 最终，如何降低个人患结肠癌的风险。

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.