Decoding Individual Exosomes in Cancer

项目摘要 越来越多的人认识到，细胞外囊泡（EV）是微米或纳米尺寸的脂质， 含有蛋白质和核酸货物的颗粒（信息） 诊断学/病理学，甚至具有治疗价值。然而，存在对能够 解决生物学和医学中EV信息开发的一个基本问题 是天然高度不均匀的颗粒。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）。具体地说， 我们的新技术平台旨在解决解决大量外泌体亚群的问题， 直接关联单个外来体的表面蛋白和核酸（微小RNA或miRNA）货物， 进行高度多重荧光成像分析。我们的最终目标是开发一种独特的成像技术 用于高通量、高含量分析单个细胞的蛋白质和核酸货物的平台 外泌体（和其他EV）。这个新平台可以使小说 诊断/预后"液体活组织检查"测试，用于管理癌症以及其他病理，例如 神经变性和心血管疾病。 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的实验性多重分析平台 货物。这些目标将验证我们的平台，用于癌细胞单个外泌体的原位miRNA分析 有记录的外泌体miRNA特征。在目标3中，我们将使用经过验证的平台来关联 人乳腺癌细胞外泌体表面蛋白展示和miRNA运载 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 测试我们的平台在原始批量样品中解析外泌体亚群的能力，基于 单个外泌体的表面蛋白和miRNA货物的相关信号。 我们认为，我们的新型成像平台有可能成为一种新的诊断/预后工具 用于乳腺癌、其他肿瘤类型和EV/外泌体感染的其他病理的临床管理 分析可以提供临床有用的信息。最近的研究表明，乳腺癌细胞， 衍生的外来体可以运输促进致癌或恶性表型的货物。因此， 从乳腺肿瘤中释放出来的信息可能具有重要的诊断/预后价值， 从血液或其他患者液体中取样（"液体活检"）。这种能力还可以帮助开发 基于外泌体的治疗人类肿瘤类型。

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.