Rapid Determination of Phenotypic Responses Across Cancer Cell Lines

 描述（由申请人提供）：该资助的目的是通过创新新方法来使用一套全面的条形码细胞系筛选此类表型，从而改变我们研究哺乳动物细胞培养中复杂表型的方法。从正常和转化的人类组织中获得的体外繁殖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理解各种表型的遗传基础的有力方法，但表观遗传水平的变异性和复杂的遗传变异性不能轻易地通过正向遗传学建模。相比之下，细胞系之间的变异性产生模拟生物学相关状态的复杂表型，如化疗耐药性。广泛的项目，如广泛/诺华癌症细胞系百科全书（CCLE），一直致力于提供超过1,000个细胞系的基因组测序，基因表达和拷贝数数据。虽然这些数据已经促进了具有所需遗传改变或基因表达模式的细胞系的鉴定，但是跨大组细胞系的复杂表型的测量仍然是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试验中跟踪它们的丰度。该技术使我们能够准确地同时测量一组28个悬浮细胞系的低葡萄糖敏感性。在这里，我们建议实施基于细胞条形码的测定，以同时确定数百个细胞系如何响应任何所需的刺激。我们将生产一组200个条形码化细胞系，并测试我们在已建立的测定中测量这些细胞系对低葡萄糖的敏感性的能力。然后，我们将扩大可以筛选的经验证的环境和遗传扰动的集合，包括特异性cDNA、shRNA、SgRNA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、基底膜基质或作为肿瘤异种移植物进行这些实验的能力。最后，我们将开发一种基于流式细胞仪的细胞分选（FACS）检测方法，用于荧光标记物和报告基因的高通量分析。这种方法将极大地扩展可以与该技术一起使用的输出的数量，包括天然或修饰的蛋白质和荧光报告物的免疫荧光。为了评估该技术，我们将使用基于FACS的方法来鉴定在氨基酸饥饿和再刺激后对mTOR途径调节有响应或无响应的细胞系，并确定该表型是否可以通过mTOR途径中的突变来预测。如果成功的话，这项资助所支持的技术将允许一个启动的实验室提出类似于主要财团通常提出的问题。

 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.