Advanced High-Content-Screening Applications of Clonogenicity in Cancer
Advanced High-Content-Screening Applications of Clonogenicity in Cancer
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DOI：
10.1177/2472555220926921
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发表时间：
2020-06-02
期刊：
影响因子：
3.1
通讯作者：
LaBarbera, Daniel V.
中科院分区：
文献类型：
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作者：
Esquer, Hector;Zhou, Qiong;LaBarbera, Daniel V.
自1956年Puck和Marcus首次报告以来，尽管有许多可用的自动化系统，但克隆生成分析尚未完全适应高含量筛选（HCS）工作流程。最初，克隆测定用于观察辐射对细胞存活的影响，特别是对癌细胞的影响。克隆性测定已经被很好地表征为癌症干细胞（CSC）的干性，表明单个CSC可以产生克隆性菌落。csc具有高度致瘤性，具有无限的增殖潜力和产生恶性肿瘤的能力。此外，CSCs也被认为能够抵抗常规化疗以及更现代的靶向治疗。因此，鉴于CSC的复杂性及其临床相关性，必须采用新的方法来更有效地研究和表征CSC的机制，使其能够增殖和持续存在，并开发能够更有效地针对这些人群的药物和其他疗法。在此，我们提出了一种HCS方法来量化2D和3D培养模型中菌落的数量和大小，并使用Opera phoenix高含量筛选系统基于荧光标记来区分菌落。此外，我们提出了一种在低倍率下扫描和在更高倍率下重新扫描的方法，以捕获更详细的菌落甚至单个感兴趣的细胞。这些方法可以适用于许多应用程序或其他成像系统，以使用高含量分析和高通量药物发现来研究CSC生物学。
Since its first report in 1956 by Puck and Marcus, the clonogenic assay has not been completely adapted into high-content-screening (HCS) workflows despite the numerous automated systems available. Initially, clonogenic assays were used to observe the effects of radiation on cell survival, particularly with cancer cells. The clonogenic assay has since been well characterized as a measure of cancer stem cell (CSC) stemness, demonstrating that a single CSC can generate clonogenic colonies. CSCs are highly tumorigenic with an unlimited proliferation potential and capacity to generate malignant tumors. Furthermore, CSCs are also known to resist conventional chemotherapy as well as more contemporary targeted therapies alike. Therefore, given the complexity of CSCs and their clinical relevance, new methods must follow to more effectively study and characterize CSC mechanisms that allow them to proliferate and persist, and to develop drugs and other therapies that can more effectively target these populations. Herein, we present a HCS method to quantify the number and size of colonies in 2D and 3D culture models and to distinguish colonies based on fluorescent markers using an Opera Phenix high-content-screening system. In addition, we present a method to scan at low magnification and rescan at a higher magnification to capture in greater detail colonies or even single cells of interest. These methods can be adapted to numerous applications or other imaging systems to study CSC biology using high-content analysis and for high-throughput drug discovery.