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: 9811990
• 负责人: Alain Charest
• 金额: $ 53.37万
• 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 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9811990
• 关键词: Animal Model; Antibodies; Benchmarking; Binding; Biological Markers; Biology; Blood; Brain; CD81 gene; Cancer Etiology; Cancer Model; Catalogs; Cell Line; Cell membrane; Cells; Cerebrospinal Fluid; Cessation of life; Classification; Clinical; Colon; Colorectal Cancer; Complex; Complex Mixtures; Coupled; Data; Disease; Enterobacteria phage P1 Cre recombinase; Epidermal Growth Factor Receptor; Fc Receptor; Filtration; Flow Cytometry; Fluorescence; Genetic Models; Glioblastoma; Goals; Grant; Health; Hematopoietic Neoplasms; Heterogeneity; Human; Implant; Individual; Institution; Knowledge; Label; Lipids; Lipoproteins; Malignant Neoplasms; Malignant neoplasm of brain; Mass Spectrum Analysis; Mediating; Membrane Proteins; Methodology; Methods; Modeling; Molecular; Monitor; Mus; Nature; Normalcy; Nucleic Acids; Organoids; Patient-Focused Outcomes; Patients; Plasma; Production; Proteins; RNA; Reagent; Receptor Activation; Receptor Signaling; Reporter; Reproducibility; Research; Risk; Saliva; Sampling; Science; Sorting - Cell Movement; Source; Standardization; Subgroup; Techniques; Testing; Therapeutic; Tissues; Urine; Vesicle; Work; Xenograft procedure; base; biomarker development; cancer biomarkers; cancer cell; cell type; colon cancer patients; density; exosome; extracellular; extracellular vesicles; glycosyltransferase; improved outcome; microvesicles; mortality; neoplastic cell; protein biomarkers; tool; transcriptome sequencing; transmission process; tumor; tumor progression; ubiquitin-protein ligase; vesicular release

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.

项目概要 所有细胞都会分泌一小部分蛋白质和 RNA 成分，作为脂质囊泡，称为细胞外囊泡 （电动汽车）。在各种疾病中，正常的 EV 货物会随着疾病的发生和进展而发生变化，从而改变哪些蛋白质 RNA 被包装到其中。小型 EV（称为外泌体）和较大的 EV（称为微泡）携带 许多这些与疾病相关的货物，我们已经证明蛋白质和 RNA 外泌体和 微泡成分随癌症进展而变化；这种电动汽车最终可能会进入人体的生物体液中 包括血液、脑脊液、尿液和唾液，提供非侵入性且易于获得的来源 生物标志物。最近的研究表明，从细胞中释放的电动汽车本质上具有高度异质性，并且 只有一小部分与疾病相关。此外，许多细胞外成分被认为 与囊泡无关，无论是由非囊泡或其他脂蛋白复合物产生 exRNA 和蛋白质。为了推动该领域超越增量科学，我们需要更好的理解 分子异质性（货物成分）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囊泡 分选 (FAVS)，作为一种分析和纯化小型和大型 EV 的方法，以每个囊泡为基础，从各种 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式分类器，因此它 是该联盟应用的理想方法。在本提案中，我们将展示 FAVS 的功能 纯化源自结直肠癌 (CRC) 和多形性胶质母细胞瘤 (GBM) 模型的小型 EV， 包括 EV 的细胞系、PDX、小鼠血浆和患者血浆来源。这两种癌症都对健康至关重要 风险。 GBM 是一种常见但无法治愈的恶性脑肿瘤（2018 年预计有超过 12,000 个新病例） 结直肠癌是美国癌症死亡的第三大原因。在该提案的第一个目标中，我们将优化 FAVS 流程：验证根据物理异质性分离 EV 的预处理步骤 （尺寸和密度），在执行 FAVS 之前；测试与 FAVS 一起使用的新候选试剂 明确划分 EV 亚组；并发现 EV 异质性的新 RNA 和蛋白质标记。因为 此类癌症通常与表皮生长因子的表达和激活增加有关 受体 (EGFR) 我们将使用 EGFR 靶向抗体以及其他 EV 货物结合抗体来 从这些癌症中纯化 EV 子集。我们将使用 EGFR 抗体来分析 CRC 和 GBM 相关的 EV 正如我们之前所做的，使用结合总 EGFR 和活性 EGFR 的抗体。补助金的第二个目标是 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记 遗传模型。我们还将使用原位植入的 GBM 和 CRC PDX 异种移植物来净化循环 EV 子集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 FAVS 管道来 从血浆中纯化患者来源的 EV，以验证这项工作发现的 EV RNA/蛋白质成分。