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

描述(申请人提供)：上皮性卵巢癌(OvCa)早期在腹腔内转移，很少通过血液扩散。腹膜和大网膜是覆盖在肠道上的一大块脂肪组织，是最常见的转移部位。目前，还没有针对转移性OvCa的治疗方法，转移性OvCa影响了80%的患者，而且针对这一适应症的新药发现非常缓慢。不幸的是，自1999年以来，没有任何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塑料上培养的细胞系，因为它们与肿瘤微环境分离，不能很好地代表卵巢癌的病理。最近，我们开发了一种器官类型的3D培养，它由原代人大网膜成纤维细胞、间皮细胞和细胞外基质组装而成，代表了Ovca患者观察到的肿瘤微环境。标记的原代人OvCa细胞与3D培养的共培养模拟了转移、黏附和侵袭的早期步骤。我们建议将3D培养适应于适用于高通量筛选(HTS)的384孔格式的稳健和可重复性的分析。我们的初步实验表明，对HTS使用96孔格式的3D培养是可行的，并产生可重复性的结果。我们的第一个目标是优化分析，并将使用芝加哥大学(U Of C)HTS核心设施中的450个化合物NIH临床样本进行测试。我们将优化控制、信号与背景比、重复性，并努力详细了解适应HTS的控制参数(例如原代细胞纯度、孵化时间、日变化)。阳性点击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卵巢癌药物。第二个目标的计划包括使用十点剂量反应曲线优化二次筛查的3D培养。我们将与华中高温超导研究所合作，进一步开发三种检测方法，通过Matrigel检测(I)OvCa凋亡、(Ii)增殖和(Iii)黏附/侵袭，以证实所鉴定的化合物的有效性。我们的目标是建立一种稳健和可重复性的分析方法，能够对MLPCN中心提供的300,000种化合物进行自动化分析。通过第一次和第二次筛选的化合物以及3种确证分析中的2种最终将得到进一步开发。我们相信，HTS使用在器官特有的微环境中生长的原代癌细胞，具有识别铅化合物和提高我们对OvCa生物学的知识的潜力。此外，HTS 3D培养试验的成功应用将为其他使用器官型培养的药物筛选提供概念验证。 公共卫生相关性：适应器官类型的三维培养进行高通量筛选“很少有药物能有效对抗卵巢癌，而且它们的作用通常是暂时的。寻找新药进展缓慢的一个原因是，制药项目使用培养在塑料上的癌细胞株进行筛查，塑料通常有几十年的历史。我们建议通过与分子图书馆生产中心网络(MLPCN)的合作，通过NIH分子图书馆路线图倡议，采用原代卵巢癌细胞系对人大网膜进行器官型3D培养，以筛选化合物。一旦一种化合物在体外和体内的有效性得到证实，一种先导化合物将在化学家、生物学家和内科医生的密切合作下进一步开发。

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.