Adaptation of an Organotypic 3 Dimensional Culture for High-Throughput Screening

器官型 3 维培养的适应高通量筛选

基本信息

批准号：
8182815
负责人：
Ernst Lengyel
金额：
$ 15.6万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8182815
关键词：
3-Dimensional Abdominal Cavity Adhesions Affect Antineoplastic Agents Apoptosis Automation Biological Assay Cancer Biology Cancer cell line Carboplatin Cell Adhesion Cell Count Cell Line Cell Proliferation Cells Chicago Clinical Coculture Techniques Collaborations Collection Complex Core Facility DNA Binding Dimethyl Sulfoxide Dose Epithelial ovarian cancer Extracellular Matrix Fatty acid glycerol esters Fibroblasts Fluorescent Probes Goals Grant Growth Human Human body In Vitro Inhibitory Concentration 50 Intestines Knowledge Label Lead Libraries Malignant - descriptor Malignant Neoplasms Malignant neoplasm of ovary Measures Mesothelial Cell Metastatic to Methods Molecular Bank Neoplasm Metastasis Noise Omentum Organ Paclitaxel Pathology Patients Peritoneum Pharmaceutical Preparations Pharmacologic Substance Physicians Plastics Play Preclinical Drug Evaluation Process Production Reading Reproducibility Role Sampling Screening procedure Signal Transduction Site Specificity Stromal Cells Testing Time Tissues Toxic effect United States National Institutes of Health Universities Variant Work base cancer cell drug discovery drug use screening high throughput screening improved in vitro Assay in vivo matrigel novel programs research clinical testing research study response tumor

项目摘要

DESCRIPTION (provided by applicant): Epithelial ovarian cancer (OvCa) metastasizes early within the abdominal cavity, and rarely spreads hematogenously. The peritoneum and the omentum, which is a large pad of fat tissue covering the bowel, are the most common sites of metastasis. Currently, there are no treatments specific to metastatic OvCa, which affects 80% of all patients, and new drug discovery for this indication has been very slow. Unfortunately, no drug has been approved for the treatment of OvCa since 1999. Current high-throughput drug screening strategies rely on cell lines cultured on plastic, which, since they are separated from the tumor microenvironment, are poorly representative of OvCa pathology. Recently, we developed an organotypic 3D culture assembled from primary human omental fibroblasts, mesothelial cells, and extracellular matrix, which is representative of the tumor microenvironment observed in patients with OvCa. Co-culture of labeled primary human OvCa cells with the 3D culture mimics the early steps of metastasis, adhesion and invasion. We propose to adapt the 3D culture to a robust and reproducible assay in a 384-well format feasible for high-throughput screening (HTS). Our preliminary experiments have shown that using a 3D culture with a 96-well format for HTS is feasible and yields reproducible results. Our first aim focuses on assay optimization, and will be performed using the 450 compound NIH clinical collection in the University of Chicago (U of C) HTS core facility. We will optimize controls, signal-to-background ratio, reproducibility, and work on acquiring a detailed understanding of control parameters (e.g. primary cell purity, incubation times, day-to-day variations) for the adaptation to HTS. Positive hits will be defined as having equal or greater efficiency than carboplatin, currently the most efficient OvCa drug. Plans for the second aim include optimization of the 3D culture for the secondary screening using a ten-point dose response curve. Working with the U of C HTS facility, we will further develop three assays measuring (i) OvCa apoptosis, (ii) proliferation, and (iii) adhesion/invasion through matrigel in order to confirm the efficacy of the compounds identified. Our goal is to establish a robust and reproducible assay that is amenable to automated analysis of the 300,000 compounds available through the MLPCN centers. Compounds which prevail through the primary and secondary screenings and 2 of the 3 confirmatory assays will ultimately be developed further. We believe that HTS using primary cancer cells growing in an organ-specific microenvironment has the potential to identify lead compounds and improve our knowledge of OvCa biology. Furthermore, the successful adaption of a 3D culture assay for HTS will serve as a proof- of-concept for other drug screens using organotypic cultures. PUBLIC HEALTH RELEVANCE: "Adaptation of an Organotypic 3 Dimensional Culture for High-Throughput Screening" There are few drugs that are effective against ovarian cancer, and their action is generally temporary. One reason that progress finding new drugs has been slow is because pharmaceutical programs use cancer cell lines cultured on plastic, often decades old, for screening. We propose to adapt an organotypic 3D culture of the human omentum, with primary ovarian cancer cell lines for screening compounds through the NIH Molecular Libraries Roadmap Initiative in collaboration with the Molecular Libraries Production Centers Network (MLPCN). Once efficacy of a compound is confirmed in vitro and in vivo a lead compound will be further developed in close collaboration between chemists, biologists and physicians.

描述（由申请人提供）：上皮卵巢癌（OVCA）在腹腔内早期转移，很少会血源性传播。腹膜和大脑是最常见的转移部位。当前，尚无针对转移性OVCA的特定治疗方法，它影响了所有患者的80％，而且这种适应症的新药物发现非常慢。不幸的是，自1999年以来，没有批准任何药物来治疗OVCA。当前的高通量药物筛查策略依赖于在塑料上培养的细胞系，因为它们与肿瘤微环境分离，因此代表了OVCA病理学。最近，我们开发了一种从原发性人性成纤维细胞，间皮细胞和细胞外基质组装的器官3D培养物，该基质代表了OVCA患者观察到的肿瘤微环境。与3D培养的标记原代人OVCA细胞的共同培养模仿转移，粘附和侵袭的早期步骤。我们建议以384孔格式适应3D培养物，可用于高通量筛选（HTS）。我们的初步实验表明，使用96孔格式的HTS培养物是可行的，并且可以产生可重现的结果。我们的第一个目标重点是测定优化，并将使用芝加哥大学（U of C）HTS核心设施的450综合NIH临床收集进行。我们将优化控件，信噪比的比率，可重复性，并为获得对控制参数的详细了解（例如，初级细胞纯度，孵化时间，日常变化），以适应HTS。正命中率将被定义为比卡铂相等或更高的效率，这是目前最有效的OVCA药物。第二个目标的计划包括使用十分剂量响应曲线优化3D培养物进行次级筛选。与C HTS设施U的U合作，我们将进一步开发三种测量（I）OVCA凋亡，（ii）增殖和（iii）通过Matrigel的粘附/侵袭，以确认已确定的化合物的功效。我们的目标是建立一个可重复的测定法，该测定法可以自动分析通过MLPCN中心提供的300,000种化合物。通过初级和次级筛选盛行的化合物以及三种验证性测定中的2种最终将进一步开发。我们认为，使用在器官特异性微环境中生长的原代癌细胞的HTS具有鉴定铅化合物并提高我们对OVCA生物学的了解的潜力。此外，使用器官型培养物的3D培养测定成功适应了HTS的3D培养测定法对其他药物筛查的证明。公共卫生相关性：“适应了高通量筛查的器官3维培养物”，几乎没有有效的药物针对卵巢癌，它们的作用通常是暂时的。寻找新药物的进展缓慢的原因之一是因为药物程序使用在塑料上培养的癌细胞系（通常是数十年）进行筛查。我们建议通过与分子图书馆生产中心网络（MLPCN）合作，通过NIH分子文库路线图（MLPCN）合作，以原代卵巢癌细胞系来适应人类膜的器官3D培养。一旦在体外确认了化合物的功效，并且在体内铅化合物将在化学家，生物学家和医生之间的密切合作中进一步发展。