Microfluidic single-cell analysis of cancer exosomes

大多数真核细胞分泌大量30- 150nm大小的膜源性囊泡，称为外泌体。作为一种新兴的细胞间通讯机制，外泌体最近被发现在广泛的生物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泌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。尽管外泌体具有重要意义，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中释放的外泌体。由于已知肿瘤细胞具有显著的异质性，因此外泌体的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“金标准”方法只能对从大细胞群中释放的外泌体进行整体测量，因为它们的分离率低，分析灵敏度不足，吞吐量低。在本提案中，PI旨在首次开发能够在单细胞水平上探测外泌体分泌和分子组成的高通量单细胞外泌体分析系统（SCEAS）。该目标将通过两个具体目标来实现：1)开发一种微流控外泌体条形码芯片，用于多路、超灵敏的外泌体免疫荧光检测；2)开发并验证单细胞外泌体分析系统（SCEAS），用于单个癌细胞外泌体的定量表型分析。这项工作的成功将为在单细胞水平上研究肿瘤细胞的异质外泌体释放提供一个关键工具，这将有助于更好地理解癌症发展、转移和耐药性背后的细胞间信号通路。

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.