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 所有细胞都将其蛋白质的一小部分和RNA含量分泌为称为细胞外囊泡的脂质囊泡 （EV）。在各种疾病中，随着疾病的启动和进展，正常的EV货物变化，改变了蛋白质 RNA包装在其中。小型电动汽车，称为外泌体，较大的电动汽车称为微囊泡 这些与疾病相关的尸体中有许多，我们已经表明蛋白质和RNA外泌体以及 微泡成分随癌症进展而变化。这样的电动汽车最终可以进入身体的生物流体 包括血液，脑脊髓液，尿液和唾液，可提供无侵入性且随时可用的来源 生物标志物。最近已经显示，从细胞释放的电动汽车本质上是高度异质的， 只有小部分与疾病有关。此外，许多被认为的细胞外成分 与囊泡有关 exrnas和蛋白质。为了使领域超越渐进科学，我们需要高出的理解 分子异质性（货物组成）与物理异质性之间的关系 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的囊泡 分类（最喜欢），作为通过各种囊泡分析和净化大型和大型EV的一种手段 生物流体。最喜欢的最爱是可以访问的 是该财团应用的理想方法。在此提案中，我们将展示最爱的能力 为了纯化源自结直肠癌（CRC）和胶质母细胞瘤（GBM）模型的小型电动汽车， 包括细胞系，PDX，小鼠血浆和EV的患者血浆来源。两种癌症都很健康 风险。 GBM是一种常见但无法治愈的恶性脑肿瘤（2018年预测的12,000例新病例） CRC是美国癌症死亡的第三主要原因。在本提案的第一个目的中，我们将优化 最喜欢的管道通过：验证基于其物理异质性分开的预处理步骤 （大小和密度），在执行最爱之前；测试与最喜欢的新候选试剂一起使用 显然描述了电动汽车子组；并发现EV异质性的新RNA和蛋白质标记。因为 这样的癌症通常与表达增长和激活相关 受体（EGFR）我们将使用EGFR靶向的抗体以及其他EV货物结合抗体 从这些癌症中净化EV子集。我们将使用EGFR抗体分析CRC和GBM相关的EV 正如我们之前所做的那样，使用结合总EGFR的抗体。赠款的第二个目标是 通过使用小鼠中的细胞特异性EV方法来发现EV产生的组织特异性标记 遗传模型。我们还将使用原始植入的GBM和CRC PDX异种移植物来纯化循环 EV子集比较从患者等离子体中纯化的电动汽车。在第三个目标中，我们将使用最喜欢的管道 纯化的患者从血浆中衍生的EV衍生为这项工作发现的EV RNA/蛋白质成分。