High resolution and single cell analyses of PanIN initiation and progression

PanIN 起始和进展的高分辨率单细胞分析

• 批准号: 8095001
• 负责人: Steven D Leach
• 金额: $ 21.4万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8095001
• 关键词: Alleles; Cells; Chemoprevention; Disease; Ductal; Elastases; Epigenetic Process; Event; Experimental Models; Funding; Future; Gene Expression Profile; Genetic; Genetic Variation; Genomic Instability; Goals; Grant; Heterogeneity; Human; Imagery; In Vitro; Individual; Invasive Lesion; Lesion; Malignant neoplasm of pancreas; Maps; Mediating; Modeling; Molecular; Mus; Neoplasms; Oncogenic; Pancreatic Intraepithelial Neoplasia; Pilot Projects; Population; Resolution; Signal Pathway; Staging; Surface; System; Testing; Time; Transgenic Mice; Work; base; cell type; effective therapy; innovation; insight; interest; mouse model; neoplastic cell; novel; response

DESCRIPTION (provided by applicant): Consistent with the multiple objectives of PA-08-208, "Pilot Studies in Pancreatic Cancer", we are proposing to develop a novel experimental model of human pancreatic cancer that will facilitate the identification of novel genetic and epigenetic aberrations underlying PanIN formation. The central hypothesis guiding this work is that by studying molecular and cellular changes at the very earliest time points following oncogenic Kras activation, even prior to the onset of morphologic PanIN formation, we will gain unique insight into the mechanisms underlying the true initiation of pancreatic cancer, in a manner that will allow effective chemoprevention and/or pharmacologic termination of PanIN progression. In pursuit of this goal, we are proposing to study the initiation and progression of pancreatic cancer with unprecedented temporal and spatial resolution. We are currently generating a new mouse model in which a lox-stop-lox-GFP::KrasG12D cassette encoding a GFP-KrasG12D fusion is knocked into the endogenous Kras locus. Unlike prior models, this approach will allow for the direct visualization and FACS-based isolation of specific cell populations in which oncogenic Kras has been activated, even prior to the onset of morphologic change. This will not only allow genetic, epigenetic and functional analyses to be performed with unprecedented temporal resolution, it will also allow for these analyses to be carried out on the level of individual cells, allowing an entirely novel view of cellular heterogeneity within both forming PanINs and invasive later lesions. To test the above hypothesis, we will pursue the following Specific Aims: First, we will visualize the earliest cellular responses to LSL-GFP::KrasG12D activation in the acinar and ductal compartments using a novel in vitro culture system, and will determine the signaling pathways mediating these responses; second, we will generate and compare high resolution temporal mapping of the pre-PanIN and PanIN transcriptomes following cell type-specific activation of LSL-GFP::KrasG12D in acinar and ductal lineages; and third, we will generate and compare high resolution temporal mapping of surface marker expression in individual cells following activation of LSL-GFP::KrasG12D in the acinar and ductal lineages. Together, these Aims will provide highly innovative and important insights into the earliest events in PanIN formation, as well as an initial glimpse at the onset of cellular heterogeneity during Kras-mediated neoplasia. PUBLIC HEALTH RELEVANCE: This application seeks funding to generate a novel murine model of human pancreatic cancer, in a manner that will allow the direct visualization, isolation and characterization of tumor cells with unprecedented spatial and temporal resolution.

描述（由申请人提供）：根据PA-08-208“胰腺癌试点研究”的多重目标，我们建议开发一种新的人类胰腺癌实验模型，以促进鉴定PanIN形成背后的新型遗传和表观遗传畸变。指导这项工作的中心假设是，通过研究致癌Kras激活后最早时间点的分子和细胞变化，甚至在PanIN形成形态开始之前，我们将获得独特的见解，了解胰腺癌真正开始的机制，以一种允许有效的化学预防和/或药物终止PanIN进展的方式。为了实现这一目标，我们提议以前所未有的时间和空间分辨率研究胰腺癌的发生和进展。我们目前正在生成一种新的小鼠模型，其中编码GFP-KrasG12D融合的lox-stop-lox-GFP::KrasG12D盒被敲入内源性Kras位点。与先前的模型不同，这种方法将允许直接可视化和基于facs的特异性细胞群的分离，其中致癌Kras已经被激活，甚至在形态变化开始之前。这不仅将允许以前所未有的时间分辨率进行遗传、表观遗传和功能分析，还将允许在单个细胞水平上进行这些分析，从而在形成PanINs和侵入性后期病变中对细胞异质性进行全新的观察。为了验证上述假设，我们将追求以下具体目标：首先，我们将使用一种新的体外培养系统可视化细胞对腺泡和导管室中LSL-GFP::KrasG12D激活的最早细胞反应，并将确定介导这些反应的信号通路；其次，我们将在细胞类型特异性激活LSL-GFP::KrasG12D后，在腺泡和导管谱系中生成并比较PanIN前和PanIN转录组的高分辨率时间图谱；第三，我们将在腺泡和导管谱系中激活LSL-GFP::KrasG12D后，生成并比较单个细胞中表面标记物表达的高分辨率时间图谱。总之，这些目标将为PanIN形成的早期事件提供高度创新和重要的见解，并初步了解kras介导的瘤变过程中细胞异质性的发生。