Isolation of miRNA-rich extracellular vesicles for liquid biopsy

项目摘要/摘要 细胞外小泡(EV)由一组不同种类的分泌膜结构组成，这些膜结构含有 各种生物分子货物，如核酸、蛋白质和脂类。EVS是液体活组织检查的理想选择，因为 癌细胞通常比正常细胞分泌更多的EV，可以在癌症的体液中检测到 病人，并保护他们的货物不会变质。因为电动汽车的货物往往反映了遗传和生物 EV货物来源细胞状态和成分是癌症诊断、预后的有希望的生物标志物 和复发。在这些成分中，miRNAs作为潜在的生物标志物引起了人们的极大关注。 然而，几项研究表明，相当大比例的EV不包含显著的miRNA拷贝 数字。这些研究表明，存在富含miRNA的电动汽车亚群(S)， 但这一亚群尚未确定。这项研究的总体目标是开发方法来 分离富含miRNAs的EV亚群，用于液体活检。我们的初步研究 根据EV的表面蛋白表达确定了几个不同的EV亚群，并涉及 表面蛋白谱系可以识别富含miRNAs的EV。在这项研究中，我们将首先 评估由人类癌细胞分泌并存在于三种主要疾病患者体液中的EVS 肿瘤类型(卵巢癌、结直肠癌、肾癌)的EV亚群的流行情况 蛋白质曲目。其次，我们将确定每个EV亚群中的总miRNA含量，这些亚群是 由人类癌细胞分泌，存在于癌症患者的体液中。第三，我们将对检测进行评估 通过免疫捕获肿瘤体液中不同的EV表面蛋白而分离出的EV中的miRNAs- 孕育老鼠和癌症患者。如果成功，我们的研究将为以下领域的关键知识差距提供答案 EV生物标记领域，重要的是，开发了一种分离富含miRNA的EV的方法，这种方法可以很容易地 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.