Multiplexed Profiling of Single-Cell Secreted Exosomes

摘要 大多数真核细胞分泌大量30-150 nm大小的膜来源的小泡，称为外小体。 作为一种新兴的细胞间通讯机制，外切体最近被发现发挥了作用 在广泛的生物过程中发挥重要作用，包括癌症的发展和转移。为 例如，越来越多的证据支持癌症衍生的外切体可以重新编程接受者的行为 细胞促进肿瘤生长和转移。尽管外显体很重要，但我们对 它们的生物发生、分子分类和生物学功能仍然非常有限。其中一个挑战是 分析从单个细胞释放的外切体。因为已知肿瘤中的细胞显著地 外切体的异质单细胞分析对于理解它们在癌症中的病理作用至关重要。 然而，目前的“黄金标准”方法只能对释放的外切体进行整体测量。 从大的细胞群体中分离，因为它们的分离产率低，分析灵敏度不够，并且低 吞吐量。在这项提议中，PI的目标是首次开发高通量的单细胞外切体 能够探测外切体的分泌和分子组成的分析系统(SCEA) 单细胞水平。该目标将通过两个具体目标来实现：1)开发一种微流控数字条形码 用于多重、超灵敏外切体分析的系统；以及2)建立单细胞外切体分析 单个癌细胞外切体定量分析系统(SCEAS)。工作的成功 将产生一个关键的工具，使研究在单个细胞的肿瘤细胞的异质性外切体释放 水平，这将有助于更好地了解癌症潜在的细胞间信号通路 发展、转移和耐药。

SUMMARY Most eukaryotic cells secrete numerous membrane-derived vesicles of 30-150 nm in size termed exosomes. As an emerging mechanism for cell-to-cell communication, exosomes have been recently found to play important roles in a wide range of biological processes, including cancer development and metastasis. For instance, increasing evidences support the cancer-derived exosomes can reprogram the behavior of recipient cells to promote tumor growth and metastasis. Despite the significance of exosomes, our understanding of their biogenesis, molecular classification, and biological functions remain very limited. One of the challenges is to analyze exosomes released from single cells. Because cells in a tumor are known to be remarkably heterogeneous, single-cell analysis of exosomes is crucial to understanding their pathological roles in cancer. However, current “gold standard” methods can only perform ensemble measurements of exosomes released from a large cell population because of their poor isolation yield, insufficient analysis sensitivity and low throughput. In this proposal, the PI aims to develop for the first time a high-throughput single cell exosome analysis system (SCEAS) capable of probing the secretion and molecular composition of exosomes at the single cell level. The goal will be achieved via two specific aims: 1) Develop a microfluidic digital barcode system for multiplexed, ultrasensitive exosome profiling; and 2) Establish a Single Cell Exosome Analysis System (SCEAS) for quantitative profiling of exosomes derived from single cancer cells. Success of the work will yield a key tool to enable the studies of heterogeneous exosome release by tumor cells at the single cell level, which would facilitate better understanding of intercellular signaling pathways underlying cancer development, metastasis, and drug resistance.