Microfluidic single-cell analysis of cancer exosomes

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，外切体最近被发现在广泛的生物学过程中发挥重要作用，包括癌症的发生和转移。例如，越来越多的证据支持癌症来源的外切体可以重新编程受体细胞的行为，以促进肿瘤的生长和转移。尽管外切体具有重要的意义，但我们对其生物发生、分子分类和生物学功能的了解仍然非常有限。其中一个挑战是分析从单个细胞释放的外切体。众所周知，肿瘤中的细胞具有显著的异质性，因此对外切体的单细胞分析对于了解它们在癌症中的病理作用至关重要。然而，目前的“金标准”方法只能对从大细胞群中释放的外切体进行整体测量，因为它们的分离产率较低，分析灵敏度不够，并且吞吐量较低。在这项建议中，PI的目标是首次开发一个高通量的单细胞外切体分析系统(SCEA)，能够在单细胞水平上探测外切体的分泌和分子组成。这一目标将通过两个具体目标来实现：1)开发一种微流控外切体条形码芯片，用于对外切体进行多路、超灵敏的免疫荧光检测；以及2)开发并验证单细胞外切体分析系统(SCEAS)，用于单个癌细胞来源的外切体的定量表型。这项工作的成功将为在单细胞水平上研究肿瘤细胞异质性外切体释放提供关键工具，这将有助于更好地了解癌症发生、转移和耐药的细胞间信号通路。

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 exosome barcode chip for multiplexed, ultrasensitive immunofluorescence detection of exosomes; and 2) develop and validate a Single Cell Exosome Analysis System (SCEAS) for quantitative phenotyping 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.