A Novel Imaging-based High-throughput Screening Approach to Anti-angiogenic Drug Discovery
A Novel Imaging-based High-throughput Screening Approach to Anti-angiogenic Drug Discovery
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DOI：
10.1002/cyto.a.20808
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发表时间：
2010-01-01
期刊：
影响因子：
3.7
通讯作者：
Lorens, James B.
中科院分区：
文献类型：
--
作者：
Evensen, Lasse;Micklem, David R.;Lorens, James B.
用于癌症治疗的血管生成抑制剂的临床成功进展激发了开发新类别的抗血管生成化合物的兴趣。由此产生的可用候选治疗剂的激增突出了对稳健的高通量血管生成筛选系统的需求，该系统充分捕获新血管形成的复杂性，同时提供对这些药剂效力的定量评估。现有的体外血管生成测定要么繁琐，阻碍了对高通量筛选形式的适应，要么不能充分模拟新血管形成的复杂多步骤过程。因此，我们开发了一种器官型内皮细胞-壁细胞共培养测定系统，该系统反映了血管生成的几个方面，同时保持与高通量/高内容图像筛选兼容。原代人内皮细胞（EC）和血管平滑肌细胞（vSMC）的共培养导致被血管基底膜蛋白包封的管状内皮结构的网络的组装，因此，包括血管的三种主要组分。最初，EC依赖于vSMC衍生的VEGF，并且对临床抗血管生成疗法敏感。随后的表型VEGF转换使得EC网络对抗VEGF治疗剂具有抗性，从而划分成熟的血管表型。相反，成熟的EC网络仍然对血管破坏剂敏感。因此，候选的抗血管生成化合物可以针对其对未成熟和成熟网络的相对效力进行询问，并分类为血管正常化剂或血管破坏剂。在这里，我们证明了EC-vSMC共培养试验代表了一个强大的高内容成像高通量筛选系统，用于识别新型抗血管生成剂。一个试点高通量筛选活动被用来定义信息成像参数，并制定了一个后续的密切响应计划击中表征。使用EC-vSMC共培养测定的高通量筛选建立了筛选用于癌症治疗的新型抗血管生成化合物的新平台。(C)2009年国际细胞计数促进学会
The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting Surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network Of tubular endothelial Structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGF-switch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and Mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot high-throughput screening campaign was used to define informative imaging parameters and develop a follow-up close-response scheme for hit characterization. High-throughput screening using the EC-vSMC co-culture assay establishes a new platform to screen for novel anti-angiogenic compounds for cancer therapy. (C) 2009 International Society for Advancement of Cytometry