Transformative HTS cell migration assay for rapid screening of cancer therapeutic

用于快速筛选癌症治疗药物的转化 HTS 细胞迁移测定

• 批准号: 7855677
• 负责人: Keren Isaac Hulkower
• 金额: $ 14.92万
• 依托单位: PLATYPUS TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-07 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7855677
• 关键词: Adoption; Affect; Antineoplastic Agents; Area; Atherosclerosis; Automation; Benchmarking; Biological Assay; Cell Count; Cell Line; Cell Survival; Cell-Matrix Junction; Cells; Cellular Morphology; Chemicals; Collaborations; Collection; Communities; Data; Data Analyses; Data Storage and Retrieval; Deposition; Detection; Development; Eligibility Determination; Ensure; Excision; Exclusion; Fluorescence; Funding; Generations; Goals; Hand; Housing; Image; Imagery; In Vitro; Institutes; Knowledge; Laboratories; Lead; Legal patent; Letters; Libraries; Life; Liquid substance; Malignant Neoplasms; Marketing; Measurable; Measures; Membrane; Migration Assay; Molecular Bank; Morphology; Neoplasm Metastasis; Optics; Ornithorhynchus anatinus; Pattern; Pharmacologic Substance; Phase; Polymers; Process; Reader; Reporting; Research; Research Infrastructure; Research Personnel; Robotics; Sales; Screening for cancer; Screening procedure; Seeds; Silicones; Staining method; Stains; System; Techniques; Technology; Testing; Therapeutic; Time; Training; United States National Institutes of Health; Universities; Validation; Wound Healing; anticancer research; base; biocompatible polymer; cancer cell; cell motility; combinatorial chemistry; cost; cost effective; culture plates; drug development; drug discovery; experience; follow-up; high throughput screening; human disease; improved; inhibitor/antagonist; innovation; instrument; migration; miniaturize; skills; small molecule; small molecule libraries; success; therapeutic development; tissue culture

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.

描述（由申请人提供）：该项目的目标是开发一种适用于癌症治疗化学库高通量筛选 (HTS) 的 384 孔细胞迁移测定。目前的细胞迁移测定在 HTS 环境中不可行；它们的执行不稳健、可重复或不具有成本效益。如本提案中所述，对 Platypus Technologies 的 OrisTM 96 孔细胞迁移测定的根本性改变将使拟议的 HTS 384 孔测定与自动液体处理系统和高内涵筛选 (HCS) 平台兼容。这些功能将显着提高测试吞吐量（允许增加待测试化合物的数量）和效率（减少实际操作时间），同时显着降低每次测定的成本。 384 孔 HTS 细胞迁移测定的可用性，需要最少数量的细胞、微量的测试化合物并减少操作时间，这将改变癌症治疗的药物开发研究。所提出的检测方法对现有技术进行了革命性的改进，将适用于细胞迁移抑制剂的初级筛选，并且还允许在同一检测孔中进行后续的二级筛选，以使用高内涵筛选多重探测抑制剂对靶分子、活力和形态的影响仪器。该 HTS 测定基于临时细胞禁区的创新使用，该临时细胞禁区由无毒、生物相容性聚合物组成，将沉积在组织培养孔底部的指定中心区域。细胞被接种并附着在孔的周边，聚合物禁区溶解，露出现在允许细胞迁移的区域。第一代 OrisTM 细胞迁移检测目前提供 96 个孔，填充硅胶塞以创建禁区，用于研究细胞运动调节剂的影响。使用 Oris" 测定，我们已经证明了 A-549 细胞的可测量迁移，并报告了 > 0.46 的 z 因子，以支持测定稳健性的主张。我们已经表明，该测定适合于测试细胞运动调节剂。我们还进一步表明从单个测试孔收集多条信息的能力（即高内涵筛选能力）以及与荧光酶标仪和成像平台兼容的数据分析最后，我们提供了最新数据来支持创建。可溶解的聚合物禁区，消除了对硅胶塞的需要，并且使得该测定非常适合与 HTS 实验室使用的自动液体处理机一起使用。对细胞活力或测试化合物没有明显的有害影响。这些数据有力地支持了将当前基于塞子的 Oris 96 孔细胞迁移测定修改为 384 孔高通量细胞迁移测定的可行性。在这个为期 2 年的项目中，我们建议开发一种 384 孔细胞迁移测定，通过使用具有不同荧光团缀合物的多重染色技术，可以从单个测定孔中获得更多的初级和次级数据。该测定将与匹兹堡大学药物发现研究所（HTS 设施）的 Andreas Vogt 博士合作进行验证。该检测与各种 HCS 平台具有广泛的兼容性，可实现快速数据检索。我们的预期产品将极大地简化药物发现过程，以促进分子库的快速筛选，以开发阻止癌细胞转移的疗法。