Transformative HTS cell migration assay for rapid screening of cancer therapeutic

描述（由申请人提供）：本项目的目标是开发一种384孔细胞迁移试验，适用于癌症治疗化学文库的高通量筛选（HTS）。目前的细胞迁移测定在HTS环境中是不可行的;它们不稳健、可重复或不具有成本效益。如本提案所述，对Platypus Technologies的OrisTM 96孔细胞迁移测定的根本性变更将使拟议的HTS 384孔测定与自动液体处理系统和高含量筛选（HCS）平台兼容。这些实现将显著提高测试通量（允许增加待测试化合物的数量）和效率（减少动手时间），同时显著降低每次测定的成本。384孔HTS细胞迁移试验的可用性，需要最少数量的细胞，微小体积的测试化合物和减少的操作时间，将改变癌症治疗的药物开发研究。所提出的测定法提供了对现有技术的变革性改进，将适用于细胞迁移抑制剂的初步筛选，并且还将允许在相同测定威尔斯孔中进行后续的二次筛选，以使用高含量筛选仪器多重探测抑制剂对靶分子、活力和形态的影响。该HTS测定基于临时细胞排除区的创新使用，该临时细胞排除区由无毒的生物相容性聚合物组成，该聚合物将沉积在组织培养孔底部的限定中心区域中。细胞接种并附着在孔的周边，聚合物排除区溶解以显示现在允许细胞迁移的区域。第一代OrisTM细胞迁移检测试剂盒目前提供96个威尔斯孔，填充硅胶塞以创建排除区，用于研究细胞运动调节剂的作用。使用Oris检测，我们已经证明了A-549细胞的可测量迁移，并报告了z因子> 0.46，以支持检测稳健性的声明。我们已经表明，该测定法适用于测试细胞运动性的调节剂。我们还显示了从单个测试井收集多条信息的能力（即，能够进行高含量筛选）和与荧光酶标仪和成像平台兼容的数据分析。最后，我们提供了最近的数据，以支持创建一个可溶解的聚合物排除区，消除了对硅胶塞的需要，并使测定非常适合与HTS实验室采用的自动液体处理器一起使用。通过细胞完全迁移到先前限制的区域中证明，聚合物似乎完全溶解，并且对细胞活力或供试化合物没有明显的有害影响。这些数据有力地支持了将现有的基于塞子的Oris 96孔细胞迁移分析仪改进为384孔高通量细胞迁移分析仪的可行性。在这个为期2年的项目中，我们建议开发一种384孔细胞迁移试验，通过使用具有不同荧光体缀合物的多重染色技术，可以从单个试验孔获得更多的主要和次要数据。该检测方法将与匹兹堡大学药物发现研究所的Andreas沃格特博士合作进行验证。该检测试剂盒与各种HCS平台具有广泛的兼容性，可快速检索数据。我们的预期产品将极大地简化药物发现过程，以促进快速筛选分子库，用于开发阻断癌细胞转移的疗法。

DESCRIPTION (provided by applicant): The goal of this project is to develop a 384-well cell migration assay suitable for high throughput screening (HTS) of chemical libraries for cancer therapeutics. Current cell migration assays are not feasible in HTS settings; they are not robust, reproducible or cost-effective to perform. Fundamental changes to Platypus Technologies' OrisTM 96-wellcell migration assays, as described in this proposal, will make the proposed HTS 384-well assay compatible with automated liquid handling systems and high content screening (HCS) platforms. These enablements will dramatically improve testing throughput (allowing increased numbers of compounds to be tested) and efficiency (reducing hands- on time) while markedly reducing the cost per assay. The availability of a 384-well HTS cell migration assay that requires minimal numbers of cells, minute volumes of test compounds and reduced operator time will transform drug development research for cancer therapeutics. The proposed assay, offering transformative improvements over existing technologies, will be amenable to primary screens for cell migration inhibitors and will also permit subsequent secondary screens in the same assay wells for multiplexed probing of inhibitor effects on target molecules, viability and morphology using high content screening instruments. This HTS assay is based on the innovative use of a temporary cell exclusion zone comprised of a non-toxic, biocompatible polymer that will be deposited in a defined central area at the bottom of a tissue culture well. Cells are seeded and attach at the perimeter of the well, and the polymeric exclusion zone dissolves to reveal a zone that is now permissible for cell migration. The first generation OrisTM Cell Migration Assay currently provides 96 wells, populated with silicone stoppers to create exclusion zones, for investigating the effects of cell movement modulators. Using the Oris" assay, we have demonstrated measurable migration of A-549 cells and reported z-factors of > 0.46 to support claims of assay robustness. We have shown that the assay is suitable for testing modulators of cell motility. We have further shown the ability to collect multiple pieces of information from a single test well (i.e., high content screening capable) and data analysis compatible with fluorescence microplate readers and imaging platforms. Finally, we provided recent data to support the creation of a dissolvable polymeric exclusion zone that eliminates the need for a silicone stopper and makes the assay highly amenable for use with automated liquid handlers employed by HTS laboratories. It appears that the polymer completely dissolves as evidenced by the full migration of cells into the previously restricted area and has no obvious deleterious effects on cell viability or test compounds. These data strongly support the feasibility of modifying the current, stopper-based Oris" 96-well cell migration assay into a 384-well, high throughput cell migration assay. In this 2-year project, we propose to develop a 384-well cell migration assay that allows for greater amounts of both primary and secondary data to be obtained from a single assay well by using multiplexed staining techniques with different fluorophor conjugates. The assay will be validated in collaboration with Dr Andreas Vogt at the University of Pittsburgh's Drug Discovery Institute, a HTS facility. The assay will enjoy a wide range of compatibility with a variety of HCS platforms for quick data retrieval. Our intended product will dramatically streamline the drug discovery process to facilitate rapid screening of molecular libraries for development of therapeutics that block cancer cell metastasis.