Developing a Screen for Novel Therapies with Reprogrammed Pancreatic Cancer Cells

描述(由申请人提供)：我们的建议的目标是开发一种创新的分析方法，使用来自原发性胰腺导管腺癌(PDA)的重新编程的干细胞来筛选新的个性化治疗方法。我们的长期目标是发现有效的抗癌药物和治愈PDA。我们的应用与美国国立卫生研究院胰腺癌试点项目计划高度相关，因为我们建议：1)开发一种新的基于细胞的分析，使用直接来自患者肿瘤的重新编程细胞，以及，2)利用重新编程的细胞，通过一个独特的临床准备好的剂库来筛选有效的个性化治疗。我们的创新分析是基于最近的一项发现，即患者活检组织中的肿瘤和非恶性细胞可以在培养中通过在类似于胚胎干细胞的条件下培养来扩增。这一策略在正常上皮细胞或癌细胞中诱导了成体干细胞表型。具体地说，Rho激酶抑制剂(Y-27632)与小鼠饲养层细胞相结合，将上皮细胞重新编程为干细胞 具有以下特性：1)无限自我更新，2)当从饲养层细胞和Y-27632移除时，从起源的组织分化为更成熟的细胞的能力。来自肿瘤的细胞(称为条件性重编程细胞或CRC)在小鼠异种移植中忠实地复制原始的肿瘤组织结构，而来自正常组织的CRC是非致瘤的。在一名患有难治性肺肿瘤(呼吸道乳头状瘤病)的患者中，CRCs被用来确定伏立诺是一种优先杀死肿瘤细胞而不是正常组织的药物。这种疗法随后在几年内导致了戏剧性的临床反应和肿瘤负担的减轻。此外，肿瘤细胞对体外药物(包括敏感和耐药药物)的反应概括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CRCS将成为PDA发现新的有效治疗方法的创新和有用的模式。我们建议通过以下具体目标来检验这一假设：1)从20名患者肿瘤和匹配的对照组织中产生CRC，2)使用一个独特的已被批准用于人类的3,000种药物库来筛选选择性地杀死PDA中的CRC的药物，而不是正常组织。这项建议之所以具有创新性，是因为：1)到目前为止，从PDA样本中还没有培养出CRC，这将为研究PDA提供一个新的工具；2)CRC从未被用于筛选PDA的新治疗药物；3)由于CRC具有干细胞的特性，它们可作为癌症干细胞/肿瘤启动细胞的模型，并有助于确定针对这一独特细胞群的治疗方法；4)与成纤维细胞共培养可重塑肿瘤微环境的间质区块；5)我们的药库种类繁多、规模庞大，且制剂随时可在患者身上使用。在此授权期结束时，我们预计将有一系列对pda细胞具有细胞毒性的新药在临床前研究中进行测试，并转化为由我们的 临床合作者。这一策略可能为PDA的个体化治疗提供新的范式。

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.