High Content Multiplexed Cellular Screening

描述(申请人提供)：癌症的顽固性驱使人们寻找更好、更有效的抗肿瘤药物。搜索通常从组合化学库的高通量筛选(HTS)开始。HTS技术的技术进步极大地提高了这些筛查的效率，导致候选药物的数量激增。HTS活动产量的增加揭示了药物发现过程中的一个新瓶颈：Hit化合物的生物学特性。更多的候选化合物和对更多信息分析的渴望需要更高的吞吐量、高含量的基于细胞的二次分析。由于这种疾病固有的多样性，癌症治疗方法的测试设计面临着更大的障碍。在一种特定的癌症类型中，数千种不同的突变和基因表达谱组合可能导致致癌转化，使特定的细胞类型没有代表性。在这个第一阶段的SBIR提案中，我们概述了通过实施一种新的基于细胞的分析来解决这些问题的解决方案，该方法多路传输实验读数和细胞类型的文库。通过在高含量筛选格式中对细胞系进行多路复用，我们能够在减少样本数量的同时获得复合作用的系统级视图，从而提供更高吞吐量和更多信息的二次筛选平台。这项工作的基础是细胞的选择性标记或条形码与基于多参数细胞的分析相结合。在AIM I中，27个乳腺癌细胞株将被标记上独特的荧光条形码签名。细胞系作为一个样本进行处理；然而，数据可以在分析时分离，以确定化合物对每种单独细胞类型的影响。在AIM II中，优化了五种细胞分析，以便与条形码结合使用，并为27种细胞株中的每一种提供磷蛋白、细胞周期和凋亡的同时分析。作为这个系统的力量和验证技术的一个例子，乳腺癌小组将使用AIM III中的五个化验读数与八种常用化疗药物进行对比。这些研究的完成将提供一种跨多个细胞系的高含量二次筛选的独特方法，从根本上减少样本数量，使这些类型的筛查能够更早地进行，从而促进更高质量的先导化合物的选择。与公共健康相关：该提案中描述的平台技术使科学家能够同时评估药物对数十个细胞系的影响，极大地扩大了关于候选药物的相关生物学信息量。这将导致选择更好的药物，更快，极大地减少寻找治疗分子所需的资源，并增加成功的机会。

DESCRIPTION (provided by applicant): Cancer's intractable nature is driving the search for better, more effective anti-neoplastic agents. The search typically begins with high throughput screening (HTS) of combinatorial chemical libraries. Technological advances in HTS technology have greatly increased the efficiency of these screens, leading to an explosion of drug candidates. The increased production of HTS campaigns has uncovered a new bottleneck in the drug discovery process: the biological characterization of hit compounds. The larger volume of candidate compounds and the desire for more informative assays necessitates higher-throughput, high-content cell-based secondary assays. The design of assays for cancer therapeutics faces even larger obstacles due to the inherent diversity of this disease. Within a specific cancer type, thousands of different combinations of mutations and gene expression profiles can lead to oncogenic transformation making no specific cell type representative. In this Phase I SBIR proposal we outline a solution to these problems with the implementation of a novel cell-based assay that multiplexes experimental readouts and libraries of cell types. By multiplexing the cell lines in a high-content screening format we are able to obtain a systems level view of compound action while reducing the number of samples, providing a higher-throughput and more informative secondary screening platform. The foundation of the work is a combination of selective labeling, or barcoding, of cells and multiparameter cell-based assays. In Aim I, 27 breast cancer cell lines will be labeled with unique fluorescent barcode signatures. The cell lines are manipulated as one sample; however the data can be separated upon analysis to determine the effects of compounds on each individual cell type. In Aim II, five cellular assays are optimized to be used in conjunction with the barcoding and provide the simultaneous analysis of phosphoproteins, cell cycle, and apoptosis for each of the 27 cell lines. As an example of the power of this system and to validate the technology, the breast cancer panel will be profiled against eight commonly used chemotherapy drugs using the five assay readouts in Aim III. The completion of these studies will provide a unique method of high-content secondary screening across multiple cell lines that radically reduces the number of samples, enabling these type of screens to be performed earlier thereby facilitating the selection of higher quality lead compounds. PUBLIC HEALTH RELEVANCE: The platform technology described in this proposal gives scientists the power to assess the effects of drugs on dozens of cell lines simultaneously, greatly expanding the amount of pertinent biological information available on drug candidates. This will lead to the selection of better drugs, faster, drastically reducing the resources needed to find therapeutic molecules and increasing the chances of success.