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

描述（由申请人提供）：上皮性卵巢癌（OvCa）在腹腔内早期转移，很少通过血源性扩散。腹膜和大网膜是最常见的转移部位，大网膜是覆盖肠的一大块脂肪组织。目前，没有针对转移性OvCa的治疗方法，这影响了80%的患者，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，没有药物被批准用于治疗OvCa。 目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，因此不能很好地代表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的器官型3D培养物，其代表了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培养适应于384孔格式的高通量筛选（HTS）中的稳健和可重现的测定。我们的初步实验表明，使用具有96孔格式的3D培养物用于HTS是可行的，并且产生可重复的结果。我们的第一个目标集中在分析优化，并将使用450化合物NIH临床收集在芝加哥大学（U of C）HTS核心设施。我们将优化控制，信号-背景比，再现性，并努力获得控制参数（例如原代细胞纯度，孵育时间，日间变化）的详细了解，以适应HTS。阳性命中将被定义为具有等于或大于卡铂的效率，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用于二次筛选的3D培养。与加州大学HTS设施合作，我们将进一步开发三种测定（i）OvCa细胞凋亡，（ii）增殖和（iii）通过基质胶的粘附/侵袭的试验，以确认所鉴定化合物的疗效。 我们的目标是建立一个强大的和可重复的测定，适合通过MLPCN中心提供的300，000种化合物的自动化分析。最终将进一步开发通过一级和二级筛选以及3项确证性试验中的2项获得优势的化合物。我们认为，HTS使用在器官特异性微环境中生长的原代癌细胞，有可能识别先导化合物并提高我们对OvCa生物学的认识。此外，HTS的3D培养测定的成功适应将用作使用器官型培养物的其他药物筛选的概念验证。 公共卫生相关性：“器官型三维培养用于高通量筛选的适应性”很少有药物对卵巢癌有效，而且它们的作用通常是暂时的。发现新药进展缓慢的一个原因是，制药项目使用在塑料上培养的癌细胞系进行筛选，这些细胞系通常有几十年的历史。我们建议通过NIH分子库路线图计划与分子库生产中心网络（MLPCN）合作，采用原发性卵巢癌细胞系对人类网膜进行器官型3D培养，以筛选化合物。一旦一种化合物的功效在体外和体内得到证实，化学家、生物学家和医生将密切合作，进一步开发一种先导化合物。

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.