Uncovering exRNA and protein determinants of secreted vesicle heterogeneity by flow cytometric purification of vesicle subsets from cells and plasma

通过流式细胞仪纯化细胞和血浆中的囊泡子集，揭示分泌囊泡异质性的 exRNA 和蛋白质决定因素

• 批准号: 10470428
• 负责人: Alain Charest
• 金额: $ 104.97万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470428
• 关键词: Uncovering; exRNA; protein; determinants; secreted

PROJECT SUMMARY All cells secrete small portions of their protein and RNA contents as lipid vesicles called extracellular vesicles (EVs). In various diseases, normal EV cargos change as disease initiate and progress, altering what proteins and RNAs are packaged into them. Small EVs, called exosomes, and larger EVs called microvesicles carry many of these disease-associated cargos and we have shown that both protein and RNA exosomal and microvesicle constituents change with cancer progression; such EVs can end up in the biofluids of the body including blood, cerebral spinal fluid, urine and saliva providing a non invasive and readily available source of biomarkers. Recently it has been shown that EVs released from cells are highly heterogeneous in nature and that only small fractions are disease associated. Furthermore many extracellular constituents that were thought to be associated with vesicles are not, either arising from non-vesicular or other lipoprotein complexes of exRNAs and proteins. To advance the field beyond incremental science, we require a superior understanding of the relationship between molecular heterogeneity (cargo composition) and physical heterogeneity for the various types of vesicles secreted by cells and tissues. To this we developed, Fluorescence-Activated Vesicle Sorting (FAVS), as a means to analyze and purify small and large EVs, on a per vesicle basis, from various biofluids. FAVS is generally accessible since it uses a flow sorter available at many research institutions, so it is an ideal method to be applied by this consortium. In this proposal we will demonstrate the capability of FAVS to purify small EVs derived from colorectal cancer (CRC) and Glioblastoma Multiforme (GBM) models, including cell line, PDX, mouse plasma and patient plasma sources of EVs. Both cancers are significant health risks. GBMs are a common, yet incurable, malignant brain tumor (over 12,000 new cases predicted in 2018) and CRC is the third leading cause of cancer deaths in the US. In the first Aim of this proposal we will optimize the FAVS pipeline by: validating preprocessing steps that separate EVs based on their physical heterogeneity (size and density), before performing FAVS; testing new candidate reagents for use with FAVS that more clearly delineates EV subgroups; and uncovering new RNA and protein markers of EV heterogeneity. Because such cancers are often associated with increased expression and activation of Epidermal Growth Factor Receptor (EGFR) we will use EGFR-targeted antibodies, along with other EV cargo binding antibodies, to purify EV subsets from these cancers. We will use EGFR antibodies to analyze CRC and GBM associated EVs as we have done previously, using antibodies that bind total and active EGFR. The second Aim of the grant is to uncover tissue specific markers of EV production by using a cell specific EV-tagging methodology in mouse genetic models. We will also use orthotopically implanted GBM and CRC PDX xenografts to purify circulating EV subsets to compare to EVs purified from patient plasmas. In the third Aim we will use our FAVS pipeline to purify patient derived EVs from plasma to credentialize EV RNA/protein constituents discovered by this work.

项目摘要 所有的细胞都分泌一小部分蛋白质和RNA作为脂囊泡，称为细胞外囊泡 （电动汽车）。在各种疾病中，正常的EV货物随着疾病的开始和进展而改变， RNA被包装在其中。称为外泌体的小EV和称为微泡的较大EV携带 许多这些疾病相关的货物，我们已经表明，蛋白质和RNA外泌体， 微囊泡成分随着癌症进展而变化;这种EV可以最终进入身体的生物流体中 包括血液、脑脊髓液、尿液和唾液，提供了非侵入性和容易获得的 生物标志物。最近已经表明，从细胞释放的EV在性质上是高度异质的， 只有一小部分与疾病有关。此外，许多细胞外成分被认为 与囊泡相关的脂蛋白不是由非囊泡或其他脂蛋白复合物引起的， exRNA和蛋白质。为了超越增量科学来推进该领域，我们需要上级的理解 分子异质性（货物组成）和物理异质性之间的关系， 由细胞和组织分泌的各种类型的囊泡。为此，我们开发了荧光激活囊泡 分选（FAVS），作为一种在每个囊泡的基础上分析和纯化来自各种囊泡的小和大EV的手段， 生物流体FAVS通常是可访问的，因为它使用许多研究机构提供的流分类器，所以它 是该联合体应用的理想方法。在本提案中，我们将展示FAVS的能力 为了纯化源自结肠直肠癌（CRC）和多形性胶质母细胞瘤（GBM）模型的小EV， 包括EV的细胞系、PDX、小鼠血浆和患者血浆来源。这两种癌症都是重要的健康 风险GBM是一种常见但无法治愈的恶性脑肿瘤（预计2018年将有超过12，000例新发病例） CRC是美国癌症死亡的第三大原因。在本提案的第一个目标中，我们将优化 FAVS流水线通过：验证预处理步骤，该预处理步骤基于EV的物理异质性来分离EV (size和密度）;测试与FAVS一起使用的新候选试剂， 明确划定EV亚群;并发现EV异质性的新RNA和蛋白质标记物。因为 这些癌症通常与表皮生长因子的表达和活化增加有关 我们将使用EGFR靶向抗体，连同其他EV货物结合抗体，沿着， 从这些癌症中纯化EV亚群。我们将使用EGFR抗体分析CRC和GBM相关EV 正如我们以前所做的那样，使用结合总EGFR和活性EGFR的抗体。拨款的第二个目的是 通过在小鼠中使用细胞特异性EV标记方法来揭示EV产生的组织特异性标志物 基因模型我们还将使用原位植入的GBM和CRC PDX异种移植物来纯化循环中的 EV亚组与从患者血浆中纯化的EV进行比较。在第三个目标中，我们将使用我们的FAVS管道， 从血浆中纯化患者来源的EV，以验证通过这项工作发现的EV RNA/蛋白质成分。