Rapid Determination of Phenotypic Responses Across Cancer Cell Lines

 描述(申请人提供)：这项资助的目的是改变我们研究哺乳动物细胞培养中复杂表型的方式，通过创新使用一套全面的条形码细胞系来筛选这种表型的新方法。来源于正常和转化的人体组织的体外扩增系为研究细胞生物学和基因功能提供了重要的模型。虽然正向遗传筛选是了解各种表型的遗传基础的一种强有力的方法，但表观遗传水平的变异性和复杂的遗传变异性不容易被正向遗传学建模。相比之下，细胞系之间的差异产生了复杂的表型，模仿了生物相关的状态，如化疗耐药。博德/诺华癌症细胞系百科全书(CCLE)等综合性项目致力于提供1,000多个细胞系的基因组测序、基因表达和拷贝数数据。虽然这些数据有助于识别具有所需基因改变或基因表达模式的细胞系，但测量大量细胞系的复杂表型仍然是一项劳动密集型和昂贵的任务。为了解决这个问题，我们设计了一种基于慢病毒的策略，将DNA条形码传递到单个细胞系，使我们能够在细胞竞争分析中跟踪它们的丰度。这项技术使我们能够同时准确地测量28个悬浮细胞系的低糖敏感度。在这里，我们建议实施基于细胞条形码的分析，以同时确定数百个细胞系对任何所需刺激的反应。我们将生产一组200个条形码的细胞系，并在已建立的检测中测试我们测量这些细胞系对低糖的敏感性的能力。然后，我们将扩大可以筛选的有效环境和遗传扰动的集合，包括特定cDNA、shRNAs、sgRNAs的表达、药物治疗和环境扰动。此外，我们将评估我们在软琼脂、基底膜基质或肿瘤异种移植中进行这些实验的能力。最后，我们将开发一种基于荧光激活细胞分类(FACS)的分析方法，用于混合高通量分析荧光标记物和记者。这一方法将极大地扩大可用于这项技术的产量的数量，包括天然或修饰蛋白质和荧光报告的免疫荧光。为了评估这项技术，我们将使用基于FACS的方法来识别在氨基酸饥饿和重新刺激后对mTOR途径调制有反应或无反应的细胞系，并确定是否可以通过mTOR途径的突变来预测这种表型。如果成功，这项资助带来的技术将允许启动实验室提出与主要财团工作通常提出的问题类似的问题。

 DESCRIPTION (provided by applicant): The purpose of this grant is to transform the way we approach studying complex phenotypes in mammalian cell culture by innovating new ways to screen for such phenotypes using a comprehensive set of barcoded cell lines. In vitro propagated lines derived from normal and transformed human tissues have provided an important model for studying cell biology and gene function. While forward genetic screening has been a powerful approach to understand the genetic underpinnings of various phenotypes, variability at the epigenetic level, and complex genetic variability cannot easily be modeled by forward genetics. In contrast, the variability between cell lines produces complex phenotypes that mimic biologically relevant states, such as chemotherapeutic resistance. Comprehensive projects, such as the Broad/Novartis Cancer Cell Line Encyclopedia (CCLE), have been devoted to providing genome sequencing, gene expression, and copy number data for over 1,000 cell lines. While these data have facilitated the identification of cell lines with desired genetic alterations or gene expression patterns, the measurement of complex phenotypes across large panels of cell lines remains a labor intensive and expensive task. To address this problem, we devised a lentiviral-based strategy to deliver DNA barcodes to individual cell lines, allowing us to follow their abundance in a cell competition assay. This technique allowed us to accurately and simultaneously measure the low glucose sensitivity of a panel of 28 suspension cell lines. Here, we propose implementing cell barcoding based assays to simultaneously determine how hundreds of cell lines respond to any desired stimulus. We will produce a set of 200 barcoded cell lines and test our ability to measure the sensitivity of these cell lines to low glucose in established assays. We will then expand the set of validated environmental and genetic perturbations that can be screened, including expression of specific cDNAs, shRNAs, sgRNAs, drug treatment, and environmental perturbations. Moreover, we will evaluate our ability to perform these experiments in soft agar, basement membrane matrix, or as tumor xenografts. Finally, we will develop a Fluoresce-Activated Cell Sorting (FACS) based assay for pooled high-throughput analysis of fluorescent markers and reporters. This approach will greatly expand the number of outputs which can be used with this technology, including immunofluorescence of native or modified proteins and fluorescent reporters. To evaluate this technology, we will use FACS based approaches to identify cell lines that are responsive or non- responsive to mTOR pathway modulation following amino acid starvation and re-stimulation and determine whether this phenotype can be predicted by mutations in the mTOR pathway. If successful, the technology enabled by this grant will allow a starting lab to ask questions similar to those typically posed by major consortium efforts.