High Throughput Single-Cell Phenotype Isolation by Protrusion Analysis Chip (PAC)

 描述(申请人提供)：用突起分析芯片(PAC)分离高通量单细胞表型越来越多的证据表明，实体肿瘤可能由许多具有不同基因类型和表型的细胞亚群组成，这是一种被称为肿瘤内异质性的现象。这种异质性成为有效的癌症治疗和个性化药物的主要障碍。由于这种固有的异质性，从癌细胞群体平均分析中收集的数据可能隐藏了有价值但罕见的事件，例如在单细胞水平上基因表达的巨大变化。因此，从肿瘤生物样本中了解细胞异质性，特别是在表型-基因相关性的研究中，将有助于识别新的细胞亚群，并有助于癌症的预防、诊断和治疗。在这个方案中，我们专注于开发一种基于细胞产生突起的能力的高通量的单细胞分离方法。为了能够回收所需的单个贴壁细胞，提出了一种突起分析芯片(PAC)，其中单个细胞被单个钩子捕获并被屏障物理隔离。PAC具有快速、操作简单、效率高、进样量小等特点。在黏附和铺展后，在显微镜下鉴定细胞的表型，然后取出所需的细胞进行基因分析。拟议的技术发展和研究计划可能会在单细胞水平上加强对表型和基因型之间关系的理解。

 DESCRIPTION (provided by applicant): High Throughput Single-Cell Phenotype Isolation by Protrusion Analysis Chip (PAC) There is increasing evidence that solid tumors are likely comprised of many subpopulations of cells with distinct genotypes and phenotypes, which is a phenomenon termed intratumor heterogeneity. Such heterogeneity becomes a major obstacle to effective cancer treatment and personalized medicine. Because of this inherent heterogeneity, data collected from cancer cell population-averaged assays likely hides valuable but rare events such as dramatic variations in gene expression at the single cell level. Therefore, understanding cellular heterogeneity from cancer biospecimens, especially in the study of phenotype-genotype correlation, will facilitate identification of new cell subsets, and assist in cancer prevention, diagnosis, and therapy. In this proposal, we focus on developing a high throughput approach for single-cell isolation based on cells' capability in generating protrusions. To be able to retrieve the desired single adherent cell, a Protrusion Analysis Chip (PAC) is proposed, in which the single cell is captured by a single hook and physically isolated by a barrier. The PAC is rapid, operationally simple, highly efficient, and requires low-volume sample introduction. After adhesion and spreading, cell phenotype is identified microscopically and then the desired cell is retrieved for genotype analysis. The proposed technology developments and study plans may potentially strengthen the understanding of the relationship between phenotype and genotype at the single cell level.