Somatic Engineering-based Models of Pancreatic Cancer

 描述(申请人提供)：胰腺导管腺癌(PDAC)是一种几乎一致致命的恶性肿瘤，临床上通常表现为晚期侵袭性和转移性疾病。癌细胞的内在和外在因素都有助于胰腺癌的发生和发展，但大多数步骤在分子水平上仍然知之甚少。人类PDAC的基因工程小鼠模型已经成为了解胰腺癌早期发展阶段、基础癌症生物学的许多方面以及评估新的治疗策略的重要工具。使用传统的胰腺癌小鼠模型评估基因功能是昂贵的、劳动密集型的，而且速度慢，只需很少的努力就可以产生更准确、快速和灵活的替代方案。此外，尽管改变已确定的胰腺癌中的相关基因具有明显的重要性，但这仍然是一个主要的、基本上没有得到回答的挑战。在这项高风险、高回报的提案中，我们记录了初步数据并概述了系统，这些系统将允许在胰腺癌进展的任何阶段对体内候选基因进行快速功能询问。我们将使用一种基于逆行胰管注射病毒Cre表达载体的肿瘤诱导方法来建立灵活的基于病毒的自体小鼠胰腺癌模型。这将允许快速询问胰腺癌在体内发展过程中的基因功能。在慢病毒载体中加入针对目的基因的cDNA或shRNA，将使在体内对PDAC进行基因操作不会比改变培养中的细胞系更困难。 为了在人类胰腺癌的小鼠模型中进行简化的基因组改变，我们还培育了一只Cre可调节Cas9表达的小鼠。结合传统的条件性KrasG12D和肿瘤抑制等位基因，这将允许携带Cre和GuideRNA的慢病毒载体在胰腺癌中诱导CRISPR介导的基因组工程。最后，我们将结合病毒-FLP诱导的胰腺癌启动和继发病毒-Cre感染，以实现暂时分离的基因和基因组改变。这些方法将极大地简化和加速体内基因功能的研究，并提供一条通往多路组合遗传操作以及体内胰腺癌功能得失筛查的途径。我们在使用基因工程方法建立疾病模型方面拥有广泛的专业知识，在PDAC方面的努力不断增加，在使用其他病毒诱导的肿瘤模型方面拥有丰富的经验，并与斯坦福大学胰腺癌基础科学和临床研究社区进行了良好的整合。PDAC患者的5年存活率约为5%，这表明需要真正新颖的方法来加速这种疾病的分子特征。随着人们对胰腺癌细胞在癌变的每一步中与免疫系统和其他间质成分的相互作用越来越感兴趣，以及与转移进展相关的机制知识的相对缺乏，我们的高风险项目有可能使许多胰腺癌研究人员受益。

 DESCRIPTION (provided by applicant): Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly fatal malignancy that often presents clinically as late stage invasive and metastatic disease. Both cancer cell intrinsic and extrinsic factors contribute to pancreatic cancer initiation and progression but most steps remain poorly understood at the molecular level. Genetically engineered mouse models of human PDAC have been important tools to understand the early stages of pancreatic cancer development, many aspects of basic cancer biology, and evaluate new treatment strategies. Assessing gene function using conventional mouse models of pancreatic cancer is expensive, labor intensive, and slow with only minor efforts towards generating more accurate, rapid, and flexible alternatives. Furthermore, despite the clear importance of altering genes of interest within established pancreatic cancer this remains a major largely unanswered challenge. In this high-risk high-reward proposal, we document preliminary data and outline systems that will allow rapid functional interrogation of candidate genes in vivo at any stage of pancreatic cancer progression. We will generate flexible virus-based autochthonous mouse models of pancreatic cancer using a method of tumor induction based on retrograde pancreatic duct injection of viral Cre-expressing vectors. This will allow the rapid interrogation of gene function during pancreatic cancer development in vivo. Incorporation of lentiviral vectors containing a cDNA or shRNA targeting a gene of interest will enable genetic manipulation of PDAC in vivo no more difficult than altering a cell line in culture. To allow streamlined genomic alterations in mouse models of human pancreatic cancer we have also generated a mouse with Cre-regulatable Cas9 expression. In combination with the conventional conditional KrasG12D and tumor suppressor alleles this will allow lentiviral vectors carrying Cre and a guideRNA to induce CRISPR- mediated genome engineering in pancreatic cancer. Finally, we will combine viral-FLP induced pancreatic cancer initiation with secondary viral-Cre infection to enable temporally separated genetic and genomic alterations. These methods will greatly simplify and accelerate investigation of gene function in vivo and provide a path towards multiplexed combinatory genetic manipulations as well as pooled gain- and loss-of- function screens in pancreatic cancer in vivo. We have extensive expertise using genetic-engineering approaches to generate disease models, a growing effort in PDAC, considerable experience using other viral induced tumors models, and are well integrated within the Stanford pancreatic cancer basic science and clinical research community. PDAC patients have a 5 year survival rate of ~5% underscoring the need for truly novel approaches to accelerate the molecular characterization of this disease. With growing interest in the interaction of pancreatic cancer cells with the immune system and other stromal components during each step of carcinogenesis as well as the relative void of mechanistic knowledge related to metastatic progression, our high-risk project has the potential to benefit many pancreatic cancer investigators.