Isolation of miRNA-rich extracellular vesicles for liquid biopsy

项目总结/摘要 细胞外囊泡（EV）包含一组异质的分泌膜结构，其含有一种或多种细胞因子。 各种生物分子货物，如核酸、蛋白质和脂质。EV是液体活检的理想选择， EV通常由癌细胞比正常细胞分泌得更高，可以在癌症的体液中检测到 病人，并保护他们的货物免受降解。因为EV货物往往反映了遗传和生物 起源细胞的状态、EV货物的成分是癌症诊断、预后 和复发。在这些成分中，miRNA作为潜在的生物标志物引起了人们的广泛关注。 然而，一些研究表明，相当大比例的EV不包含显著的miRNA拷贝， 号码这些研究表明存在富含miRNA的EV亚群， 但是这个亚群还没有被确定。本研究的总体目标是制定方法， 分离的EV亚群富含用于液体活检目的的miRNA。我们的初步研究 基于它们的表面蛋白表达，已经鉴定了几种不同的EV亚群，并暗示 表面蛋白库可以识别富含miRNA的EV。在本研究中，我们首先 评估由人类癌细胞分泌并存在于三种主要癌症患者体液中的EV 癌症类型（卵巢癌、结直肠癌、肾癌）的EV亚群患病率， 蛋白库其次，我们将确定每个EV亚群中的总miRNA含量， 由人类癌细胞分泌并存在于癌症患者体液中。第三，我们将评估检测 通过免疫捕获从肿瘤体液中分离的不同EV表面蛋白， 荷瘤小鼠和癌症患者。如果成功，我们的研究将为以下方面的关键知识空白提供答案： EV生物标志物领域，重要的是，开发了一种用于分离富含miRNA的EV的方法， 在临床实验室环境中用于液体活检目的。

PROJECT SUMMARY/ABSTRACT Extracellular vesicles (EVs) comprise a heterogeneous group of secreted membranous structures that contain a variety of biomolecular cargo such as nucleic acids, proteins and lipids. EVs are ideal for liquid biopsy because EVs are often more highly secreted by cancer cells than by normal cells, can be detected in body fluids of cancer patients, and protect their cargo from degradation. Because EV cargo often reflects the genetic and biological status of the cell of origin, constituents of EV cargo are promising biomarkers for cancer diagnosis, prognosis and recurrence. Of these constituents, miRNAs have attracted substantial attention as potential biomarkers. However, several studies indicate that a substantial proportion of EVs do not contain significant miRNA copy numbers. These studies have implicated the existence of a subpopulation(s) of EVs that is enriched in miRNAs, but this subpopulation has not been identified. The overarching goal of this study is to develop approaches to isolate subpopulations of EVs that are enriched in miRNAs for liquid biopsy purposes. Our preliminary studies have identified several distinct subpopulations of EVs based on their surface protein expression, and implicate that the surface protein repertoire could identify EVs that are enriched in miRNAs. In this study, we will firstly evaluate EVs that are secreted by human cancer cells and present in body fluids of patients with three major types of cancers (ovarian, colorectal, renal) for the prevalence of EV subpopulations defined by their surface protein repertoire. Secondly, we will determine the total miRNA content in each subpopulation of EVs that are secreted by human cancer cells and present in cancer patient body fluids. Thirdly, we will evaluate the detection of miRNAs in EVs that are isolated by immunocapture of different EV surface proteins from body fluids of tumor- bearing mice and cancer patients. If successful, our study will provide answers to critical gaps-in-knowledge in the EV biomarker field and, importantly, develop an approach for isolating miRNA-rich EVs that can be readily used in a clinical laboratory setting for liquid biopsy purposes.