Novel High Content Vascular Patterning Assay(RMI)

描述（由申请人提供）：血管生成，即新血管的生长，发生在多种人类疾病中。尽管抗血管生成仍然是药物的一个大市场，但用于血管生成抑制剂发现的基于细胞的高含量高通量测定仍有待开发用于该应用。我们认为，开发新的检测方法作为筛选工具，用于发现靶向内皮细胞分化的抑制剂，可以证明是鉴定新型抗血管生成药物的有效手段。在这里，我们建议开发一种高含量的血管图案化检测，可能被采用的高通量筛选（HTS）。我们计划通过将高通量筛选能力与三维（3-D）基质中内皮细胞图案化的多参数光学成像相结合，建立一个强大的高内容筛选平台的原型。我们将通过以下目标来实现这些目标的创新方法。在具体目标1中，我们将开发和验证3-D内皮细胞图案化测定（3-D ECPA）。我们将通过开发在微载体珠上产生高度稳健的多细胞内皮细胞球状体并将球状体在384孔光学筛选板中的3-D胶原基质中阵列的方法来实现这一点。此外，这些试验将在低氧环境下进行。在具体目标2中，我们计划开发和验证用于3-D ECPA的高内容（HC）图像分析平台。这将通过开发软件来实现，该软件识别3-D矩阵中内皮细胞图案化的新特征，并应用自动化分析来获得形态发生指纹。最后，我们将验证3-DECPA和图像分析软件的高温超导应用筛选天然产物的多样性。我们相信，HC-HTS平台将为筛选大量血管生成的小分子抑制剂和协助药物发现提供独特的机会。

DESCRIPTION (provided by applicant): Angiogenesis, the growth of new blood vessels, occurs in a diverse array of human diseases. Even though anti-angiogenesis remains a large market for Pharmaceuticals, high content high throughput cell based assays for angiogenesis inhibitor discovery remain to be developed for this application. It is our belief that developing new assays as screening tools for discovery of inhibitors that target endothelial cell differentiation can prove to be an efficient means of identifying new classes of anti-angiogenic drugs. Here we propose to develop a high content vascular patterning assay that can likely be adopted for high throughput screening (HTS). We plan to build a prototype of a powerful high content screening platform by interfacing high throughput screening capability with multiparameter optical imaging of endothelial cell patterning in three dimensional (3-D) matrices. Our innovative approach to achieve these goals will be accomplished through the following aims. In Specific aim 1 we will develop and validate a 3-D endothelial cell patterning assay (3-D ECPA). We will accomplish this by developing methods to generate highly robust multicellular endothelial cell spheroids on microcarrier beads and array the spheroids in 3-D collagen matrix in 384 well optical screening plates. In addition, these assays will be performed under hypoxic environment. In Specific Aim 2 we plan to develop and validate a high content (HC) image analysis platform for use with 3-D ECPA. This will be accomplished by developing software that identifies novel features of endothelial cell patterning in 3-D matrix and apply automated analysis to obtain morphogenic fingerprints. Finally, we will validate the 3-DECPA and image analysis software for HTS application by screening a diversity set of natural products. We believe that this HC-HTS platform will offer unique opportunities to screen vast numbers of small molecule inhibitors of angiogenesis and assist in drug discovery.