Decoding Individual Exosomes in Cancer

项目概要 人们越来越认识到细胞外囊泡 (EV) 是微米或纳米大小的脂质 含有蛋白质和核酸货物的颗粒（信息）在新产品中非常有前景 诊断/预后，甚至具有治疗价值。然而，对于能够 解决生物学和医学中电动汽车信息开发的基本问题 是天然高度异质的颗粒。在这个试点 (R21) 项目中，我们的实验目标是解决 需要通过关注称为外泌体的颗粒来分析 EV 异质性（亚群）。具体来说， 我们的新技术平台旨在通过以下方式解决大量外泌体亚群的解析问题 通过直接关联单个外泌体的表面蛋白和核酸（microRNA 或 miRNA）货物 执行高度多重荧光成像分析。我们的最终目标是开发独特的成像 用于对单一蛋白质和核酸货物进行高通量、高内涵分析的平台 从任何生物样本中获得的外泌体（和其他 EV）。这个新平台可以实现新颖的 用于管理癌症以及其他病理的诊断/预后“液体活检”测试，例如 神经退行性疾病和心血管疾病。 为了实现我们的实验目标，在目标 1 和 2 的工作中，我们将开发和优化我们的成像 人乳腺癌细胞外泌体及其 miRNA 实验多重分析平台 货物。这些目标将验证我们对癌细胞单个外泌体进行原位 miRNA 分析的平台 具有记录的外泌体 miRNA 特征。在目标 3 期间，我们将使用经过验证的平台来关联 人乳腺癌细胞释放的单个外泌体的表面蛋白展示和 miRNA 货物 代表早期和晚期癌症的细胞系，并将其与正常细胞系对照进行比较。目标3将 测试我们的平台解析原始批量样品中外泌体亚群的能力，基于 单个外泌体表面蛋白和 miRNA 货物的相关信号。 我们认为我们的新型成像平台有潜力成为一种新的诊断/预后工具 用于乳腺癌、其他肿瘤类型以及 EV/外泌体的其他病理的临床管理 分析可以提供临床上有用的信息。最近的研究表明，乳腺癌细胞 衍生的外泌体可以运输促进致癌或恶性表型的货物。因此，外泌体 从乳腺肿瘤释放的信息可能携带具有关键诊断/预后价值的信息，这可能是 从血液或其他患者体液中取样（“液体活检”）。这种能力也可能有助于开发 针对人类肿瘤类型的外泌体疗法。

PROJECT SUMMARY There is increasing recognition that extracellular vesicles (EVs)¾micrometer- or nanometer-sized lipid particles containing protein and nucleic acid cargoes (information)¾are highly promising for new diagnostics/prognostics and even have therapeutic value. There is, however, an unmet need for methods able to solve a fundamental problem confounding the exploitation of EV information in biology and medicine¾EVs are naturally highly heterogeneous particles. In this pilot (R21) project, our experimental goal is to address the need for analyzing EV heterogeneity (subpopulations) by focusing on particles called exosomes. Specifically, our new technology platform is designed to address the problem of resolving bulk exosomal subpopulations by directly correlating surface protein and nucleic acid (microRNA or miRNA) cargoes of single exosomes by performing highly multiplexed fluorescence imaging analysis. Our ultimate goal is to develop a unique imaging platform for the high-throughput, high-content analysis of the protein and nucleic acid cargoes of single exosomes (and other EVs) obtained from any biological sample. This new platform could enable novel diagnostic/prognostic “liquid biopsy” tests for managing cancers as well as other pathologies, such as neurodegenerative and cardiovascular diseases. To achieve our experimental goal, in the work for Aims 1 and 2 we will develop and optimize our imaging platform for experimental multiplexed analysis of human breast cancer cell exosomes and their miRNA cargoes. These Aims will validate our platform for in situ miRNA analysis of single exosomes from cancer cells with documented exosomal miRNA signatures. During Aim 3, we will use the validated platform to correlate the surface protein display and miRNA cargo of individual exosomes released by human breast cancer cell lines representing early- and late-stage cancers and compare these with a normal cell line control. Aim 3 will test the capability of our platform to resolve exosomal subpopulations in an original bulk sample, based on correlated signals for surface proteins and miRNA cargoes of single exosomes. We propose that our novel imaging platform has the potential to become a new diagnostic/prognostic tool for the clinical management of breast cancer, other tumor types, and other pathologies in which EV/exosomal analysis could provide clinically useful information. Recent research demonstrates that breast-cancer-cell- derived exosomes can transport cargoes that promote oncogenic or malignant phenotypes. Thus, exosomes released from breast tumors could carry information with critical diagnostic/prognostic value, which could be sampled from blood or other patient fluids (a “liquid biopsy”). This capability may also assist in developing exosome-based therapies for human tumor types.