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

• 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
• 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

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）：上皮性卵巢癌（OvCa）早期在腹腔内转移，很少发生血行扩散。腹膜和大网膜是覆盖肠道的大片脂肪组织，是最常见的转移部位。目前，尚无针对影响 80% 患者的转移性 OvCa 的特异性治疗方法，并且针对该适应症的新药发现非常缓慢。不幸的是，自 1999 年以来，还没有药物被批准用于治疗 OvCa。当前的高通量药物筛选策略依赖于在塑料上培养的细胞系，由于它们与肿瘤微环境分离，因此很难代表 OvCa 病理学。最近，我们开发了一种由原代人网膜成纤维细胞、间皮细胞和细胞外基质组装而成的器官型3D培养物，它代表了在OvCa患者中观察到的肿瘤微环境。标记的原代人 OvCa 细胞与 3D 培养物的共培养模拟了转移、粘附和侵袭的早期步骤。我们建议将 3D 培养物调整为 384 孔格式的稳健且可重复的测定，适用于高通量筛选 (HTS)。我们的初步实验表明，使用 96 孔格式的 3D 培养物进行 HTS 是可行的，并且可以产生可重复的结果。我们的第一个目标侧重于测定优化，并将使用芝加哥大学 (U of C) HTS 核心设施的 450 种化合物 NIH 临床收藏来进行。我们将优化对照、信号背景比、重现性，并努力详细了解对照参数（例如原代细胞纯度、孵育时间、日常变化）以适应 HTS。阳性命中将被定义为具有与卡铂相同或更高的效率，卡铂是目前最有效的 OvCa 药物。第二个目标的计划包括使用十点剂量反应曲线优化 3D 培养以进行二次筛选。我们将与 C 大学 HTS 机构合作，进一步开发三种检测方法，测量 (i) OvCa 细胞凋亡、(ii) 增殖和 (iii) 通过基质胶的粘附/侵袭，以确认所鉴定化合物的功效。 我们的目标是建立一种稳健且可重复的检测方法，能够对 MLPCN 中心提供的 300,000 种化合物进行自动分析。通过初步和二次筛选以及 3 项验证分析中的 2 项的化合物最终将得到进一步开发。我们相信，使用在器官特异性微环境中生长的原代癌细胞进行 HTS 具有识别先导化合物并提高我们对 OvCa 生物学知识的潜力。此外，HTS 3D 培养测定的成功应用将作为使用器官型培养的其他药物筛选的概念验证。 公共健康相关性：“器官型三维培养的高通量筛选的适应” 很少有药物能够有效对抗卵巢癌，而且它们的作用通常是暂时的。新药研发进展缓慢的原因之一是制药项目使用在塑料上培养的癌细胞系（通常有数十年历史）进行筛选。我们建议采用人类网膜的器官型 3D 培养物，使用原代卵巢癌细胞系，通过 NIH 分子库路线图计划与分子库生产中心网络 (MLPCN) 合作筛选化合物。一旦化合物的功效在体外和体内得到证实，化学家、生物学家和医生之间的密切合作将进一步开发先导化合物。