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

项目总结 所有细胞都以脂泡的形式分泌一小部分蛋白质和RNA内容物，称为细胞外小泡 (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发展而改变，改变什么蛋白质 RNA被打包到它们里面。被称为外体的小型电动汽车和被称为微囊的较大电动汽车携带 许多这些与疾病相关的货物，我们已经证明了蛋白质和RNA外体和 随着癌症的进展，微泡的成分会发生变化；这样的EV最终会进入人体的生物液中。 包括血液、脑脊液、尿液和唾液，提供了一种非侵入性和现成的来源 生物标志物。最近的研究表明，从细胞中释放的电动汽车在性质上是高度异质性的，并且 只有一小部分人与疾病有关。此外，许多被认为是胞外成分的物质 与囊泡相关的则不是由非囊泡或其他脂蛋白复合体引起的 外源RNA和蛋白质。为了使这一领域超越增量科学，我们需要更好的理解 分子异质性(货物成分)和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(BESS)，作为一种分析和纯化小型和大型电动汽车的手段，以每个水泡为基础，从各种 生物流体。Bavs通常是可以访问的，因为它使用了许多研究机构提供的流分类器，所以它 是这个财团采用的理想方法。在这份提案中，我们将展示BELS的能力 为了纯化来自结直肠癌(CRC)和多形性胶质母细胞瘤(GBM)模型的小EV， 包括EVS的细胞系、PDX、小鼠血浆和患者血浆来源。这两种癌症都很健康 风险。基底节瘤是一种常见但无法治愈的恶性脑瘤(2018年预计将有超过1.2万例新病例) 在美国，CRC是导致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 Faves流水线通过：验证根据电动汽车的物理异构性来分离电动汽车的预处理步骤 (大小和密度)，在执行优势之前；测试用于优势的新候选试剂 清楚地描绘了EV亚群；并发现了EV异质性的新的RNA和蛋白质标记。因为 这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，以 从这些癌症中提纯EV亚群。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那样，使用结合总EGFR和活性EGFR的抗体。这笔赠款的第二个目的是 用细胞特异性EV标记方法在小鼠体内发现EV产生的组织特异性标志物 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 将EV亚群与从患者血浆中纯化的EV进行比较。在第三个目标中，我们将使用我们的优势渠道来 从血浆中提纯患者来源的EV，以证明本工作发现的EV RNA/蛋白质成分。

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.