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

基本信息

  • 批准号:
    7855677
  • 负责人:
  • 金额:
    $ 14.92万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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高温高温环境下是不可行的;它们不是健壮的、可复制的或具有成本效益的。如本提案所述,对Platypus Technologies的OrisTM 96孔细胞迁移测定法进行了根本性的改变,将使拟议的HTS 384孔测定法与自动化液体处理系统和高含量筛选(HCS)平台兼容。这些功能将显著提高测试吞吐量(允许更多的化合物被测试)和效率(减少手工时间),同时显著降低每次分析的成本。384孔HTS细胞迁移试验的可用性,需要最少的细胞数量、微量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。拟议的分析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,并且还允许在相同的分析井中进行后续的二次筛选,以便使用高含量筛选仪器对抑制剂对目标分子的影响、活力和形态进行多重探测。这个HTS试验是基于一个临时细胞隔离区的创新使用,该隔离区由一种无毒的、生物相容性的聚合物组成,将沉积在组织培养孔底部的一个确定的中心区域。细胞被播种并附着在井周,聚合物隔离区溶解,露出一个允许细胞迁移的区域。第一代OrisTM细胞迁移试验目前提供96个孔,填充硅胶塞以创建隔离区,用于研究细胞运动调节剂的影响。使用Oris"实验,我们已经证明了A-549细胞的可测量迁移,并报告了>.46的z因子,以支持该实验的稳健性。我们已经证明,该试验适用于测试细胞运动的调节剂。我们进一步展示了从单个测试孔中收集多个信息的能力(即高含量筛选能力)以及与荧光微孔板读取器和成像平台兼容的数据分析能力。最后,我们提供了最近的数据来支持可溶解聚合物隔离区的创建,该隔离区消除了对硅胶塞的需要,并使该分析非常适合与HTS实验室采用的自动化液体处理器一起使用。从细胞完全迁移到先前限制的区域来看,聚合物似乎完全溶解,并且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目前基于塞器的Oris“96孔细胞迁移试验改为384孔高通量细胞迁移试验的可行性。在这个为期2年的项目中,我们建议开发384孔细胞迁移测定,通过使用不同荧光偶联物的多重染色技术,从单个测定孔中获得更多的初级和次级数据。将与匹兹堡大学药物发现研究所(HTS的一个机构)的Andreas Vogt博士合作对该检测方法进行验证。该分析将与各种HCS平台广泛兼容,用于快速数据检索。我们的预期产品将极大地简化药物发现过程,以促进分子文库的快速筛选,以开发阻止癌细胞转移的治疗方法。

项目成果

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Keren Isaac Hulkower其他文献

Keren Isaac Hulkower的其他文献

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{{ truncateString('Keren Isaac Hulkower', 18)}}的其他基金

High throughput cell migration assay amenable to high content imaging
高通量细胞迁移测定适合高内涵成像
  • 批准号:
    7800228
  • 财政年份:
    2009
  • 资助金额:
    $ 14.92万
  • 项目类别:

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