Developing a Screen for Novel Therapies with Reprogrammed Pancreatic Cancer Cells

描述（由申请人提供）：我们提案的目标是开发一种创新的检测方法，使用来自原发性胰腺导管腺癌（PDA）的重编程干细胞样细胞筛选新的个性化治疗。我们的长期目标是发现有效的抗癌药物，治愈动脉导管未闭.我们的应用与NIH胰腺癌试点项目倡议高度相关，因为我们建议：1）开发一种新的基于细胞的检测方法，直接来自患者肿瘤的重编程细胞，以及2）使用独特的临床现成试剂库筛选重编程细胞以筛选有效的个性化治疗。我们的创新试验是基于最近的发现，即来自患者活检的肿瘤和非恶性细胞可以通过在与胚胎干细胞相似的条件下生长细胞来在培养中扩增。该策略在正常上皮细胞或癌细胞中诱导成体干细胞表型。具体而言，Rho激酶抑制剂（Y-27632）与小鼠饲养细胞的组合将上皮细胞重编程为干细胞样细胞 具有以下属性：1）无限期自我更新，2）当从饲养细胞和Y-27632中取出时，从来源组织分化成莫尔成熟细胞的能力。来自肿瘤的细胞（称为条件重编程细胞或CRC）忠实地复制小鼠异种移植物中的原始肿瘤组织学，而来自正常组织的CRC是非致瘤性的。在患有难治性肺肿瘤（呼吸道乳头状瘤病）的患者中，使用CRC来鉴定伏立诺他作为优先杀死肿瘤细胞而不是正常组织的药物。这种疗法随后导致了显着的临床反应和肿瘤负荷的减少了几年。此外，肿瘤CRC对体外药物（包括敏感和耐药药物）的反应重现了患者对体内治疗的反应。基于这些令人兴奋的结果，我们假设CRC将是一个创新的和有用的模型，为PDA发现新的，有效的治疗。我们提出用以下具体目的来检验这一假设：1）从20个患者肿瘤和匹配的对照组织中产生CRC，以及，2）使用已经批准用于人类的> 3，000种药物的独特文库来筛选选择性地杀死来自PDA而不是正常组织的CRC的药剂。这一建议具有创新性，因为：1）到目前为止，CRC还没有从PDA样品中产生，并将提供一种新的工具来研究PDA，2）CRC从未用于筛选PDA的新治疗剂，3）因为CRC具有干细胞的性质，它们可以作为癌症干细胞/肿瘤起始细胞的模型，并有助于鉴定针对这种独特细胞群的治疗，4）与成纤维细胞的共培养概括了肿瘤微环境的基质区室，5）我们的药物库是多样的、大的，并且试剂准备好用于患者。在这个资助期结束时，我们预计将有一个对PDA细胞有细胞毒性的新药清单，用于临床前研究，并转化为由我们领导的临床试验。 临床合作者这一策略可能为PDA的个性化治疗提供一个新的范例。

DESCRIPTION (provided by applicant): The goal of our proposal is to develop an innovative assay using reprogrammed, stem-like cells derived from primary pancreatic ductal adenocarcinomas (PDAs) to screen for new, personalized therapy. Our long- term goal is to discover effective anticancer drugs and cure PDA. Our application is highly relevant to the NIH Pancreatic Cancer Pilot Project initiative because we propose to: 1) Develop a novel cell-based assay with reprogrammed cells derived directly from patient tumors, and, 2) Harness the reprogrammed cells to screen for effective, personalized therapy with a unique library of clinic ready agents. Our innovative assay is based on the recent discovery that tumor and nonmalignant cells from patient biopsies can be expanded in culture by growing the cells under conditions similar to those used for embryonic stem cells. This strategy induces an adult stem cell phenotype in normal epithelial or carcinoma cells. Specifically, the combination of Rho kinase inhibitor (Y-27632) with mouse feeder cells reprograms epithelial cells into stem-like cells with the following properties: 1) indefinite self-renewal, 2) the ability to differentiate into mor mature cells from the tissue of origin when removed from the feeder cells and Y-27632. The cells (called conditionally reprogrammed cells or CRCs) derived from tumors faithfully replicate the original tumor histology in mouse xenografts, while CRCs from normal tissue are nontumorigenic. In a patient with refractory lung tumors (respiratory papillomatosis), CRCs were used to identify vorinostat as a drug that preferentially killed the tumor cells, but not normal tissue. This therapy subsequently led to a dramatic clinical response and reduction in tumor burden for several years. Moreover, the response of the tumor CRCs to drugs in vitro (including both sensitive and resistant agents) recapitulated the patient's responses to therapy in vivo. Based on these exciting results, we hypothesize that CRCs will be an innovative and useful model for PDA to uncover new, effective therapies. We propose to test this hypothesis with the following Specific Aims: 1) To generate CRCs from 20 patient tumors and matched control tissue, and, 2) To use a unique library of >3,000 drugs already approved for use in humans to screen for agents that selectively kill CRCs from PDA, but not normal tissue. This proposal is innovative because: 1) Until now, CRCs have not been generated from PDA samples and will provide a novel tool to study PDA, 2) CRCs have never been used to screen PDA for new therapeutic agents, 3) Because CRCs have properties of stem cells, they may serve as a model for cancer stem cells/tumor-initiator cells and help to identify therapy directed at this unique population of cells, 4) Co-culture with fibroblasts recapitulates the stromal compartment of the tumor microenvironment, 5) Our drug library is diverse, large, and agents are ready for use in patients. At the completion of this grant period, we expect to have a list of new drugs that are cytotoxic to PDA cells to test in preclinical studies and translate into clinical trials led by our clinical collaborators. This strategy could provide a new paradigm for personalized therapy for PDA.