Somatic Engineering-based Models of Pancreatic Cancer

 描述（由申请方提供）：胰腺导管腺癌（PDAC）是一种几乎一致的致命性恶性肿瘤，临床上通常表现为晚期浸润性和转移性疾病。癌细胞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成为了解胰腺癌发展早期阶段、基础癌症生物学的许多方面以及评估新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，并且缓慢，只有很小的努力才能产生更准确，快速和灵活的替代方案。此外，尽管在已建立的胰腺癌中改变感兴趣的基因的明显重要性，但这仍然是一个主要的未解决的挑战。在这个高风险高回报的建议中，我们记录了初步数据并概述了系统，该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。我们将使用基于逆行胰管注射病毒表达Cre载体的肿瘤诱导方法，生成灵活的基于病毒的胰腺癌本地小鼠模型。这将允许在体内胰腺癌发展期间快速询问基因功能。掺入含有靶向感兴趣基因的cDNA或shRNA的慢病毒载体将使得PDAC的体内遗传操作不会比改变培养物中的细胞系更困难。 为了允许在人胰腺癌的小鼠模型中进行简化的基因组改变，我们还产生了具有Cre可调节的Cas9表达的小鼠。与常规条件KrasG 12 D和肿瘤抑制等位基因组合，这将允许携带Cre和向导RNA的慢病毒载体在胰腺癌中诱导CRISPR介导的基因组工程。最后，我们将结合联合收割机病毒-FLP诱导的胰腺癌的启动与继发性病毒-Cre感染，使时间上分离的遗传和基因组改变。这些方法将大大简化和加速体内基因功能的研究，并提供了一条通往多重组合遗传操作以及合并胰腺癌体内功能获得和丧失筛选的途径。我们拥有广泛的专业知识，使用基因工程方法来生成疾病模型，在PDAC方面的努力越来越多，在使用其他病毒诱导的肿瘤模型方面有相当多的经验，并且很好地融入了斯坦福大学胰腺癌基础科学和临床研究社区。PDAC患者的5年生存率约为5%，强调需要真正的新方法来加速这种疾病的分子表征。随着人们对胰腺癌细胞与免疫系统和其他基质成分在癌变过程中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.