A SYNTHETIC BIOMARKER TO UNIVERSALLY ASSESS THE RELATIVE CONTRIBUTION OF HEATHY AND CANCEROUS TISSUE TO CIRCULATING EV POOL

项目摘要/摘要 细胞外小泡(EVS)是细胞衍生的膜结合结构，释放到细胞外空间， 通过体液导航，似乎支持细胞间的通讯。癌细胞显著释放 与普通电动汽车相比，电动汽车的数量更多。因为EV内容派生自 循环EVS的分子图谱作为早期癌症的一种非侵入性手段正在被仔细研究 诊断、监测疾病进展和评估治疗反应。然而，基于EV的临床 诊断受到样本不够严谨和重复性不足以具体区分的限制， 分离并鉴定正常和疾病相关的EV。 三个分析和概念挑战阻碍了特定和可重复的EV的识别- 与临床相关的癌症生物标志物：1)还没有开发出公正的策略来评估 EVS检测癌症的限度；2)健康组织对循环池的相对贡献 EVS未知；以及3)对循环中的癌症来源EVS的数量和组成进行系统分析 在肿瘤发展过程中还没有进行过手术。 这些复杂因素促使我们设计了一个通用平台，能够评估 与患癌症的动物相比，其他健康动物的循环EV池中的特定组织 发展。该系统是基于从Tetraspanin蛋白CD63开发的工程EV标记 (EnCD63)，这有助于收集、可视化和量化特定细胞和 纸巾。 通过限制enCD63在特定的正常或肿瘤细胞和组织中的表达 通过基因工程的小鼠模型，我们将毫不含糊地检验EVS作为生物标志物的有效性。我们建议 使用我们的创新平台：1)执行将电动汽车数量与其电池相关联的无偏校准 来源(目标1)；2)确定健康组织对循环EV池的相对贡献(目标2)；以及 3)评价EVS在癌症检测中的特异性和敏感性(目标3)。 这项应用主要针对胰腺导管腺癌(PDAC)，这是一种致命的肿瘤性疾病，具有低血压 缺乏特定和敏感的诊断测试的存活率。完成拟议的研究将有助于 制定以电动汽车为基础的准备、选择和分析的标准化程序 生物标志物。

PROJECT SUMMARY / ABSTRACT Extracellular vesicles (EVs) are cell-derived membrane-bound structures released into extracellular spaces that navigate the bodily fluids and appear to support intercellular communication. Cancer cells release significantly higher numbers of EVs then their normal counterparts. Because the EV contents are derived from the cell of origin, molecular profiling of circulating EVs are being scrutinized as a non-invasive means for early cancer diagnosis, monitoring disease progression, and assessing response to treatment. However, EV-based clinical diagnostics have been limited by inadequate rigor and reproducibility of samples to specifically discriminate, isolate, and characterize normal and disease-associated EVs. Three analytical and conceptual challenges have prevented the identification of specific and reproducible EV- associated cancer biomarkers with clinical relevance: 1) no unbiased strategy has been developed to evaluate the limits of cancer detection using EVs; 2) the relative contribution of healthy tissues to the pool of circulating EVs is not known; and 3) a systematic analysis of the number and composition of circulating cancer-derived EVs during tumor development has not been performed. These complications have prompted us to design a general platform capable of evaluating the contribution of specific tissues to the pool of circulating EVs in otherwise health animals compared to animals undergoing cancer development. The system is based on an engineered EV marker developed from the tetraspanin protein CD63 (enCD63), which facilitates collection, visualization, and quantification of EVs released by specific cells and tissues. By restricting the expression of enCD63 to specific normal or neoplastic cells and tissues of genetically engineered mouse models, we will unambiguously examine the efficacy of EVs as biomarkers. We propose to use our innovative platform to: 1) perform an unbiased calibration correlating the number of EVs with their cells of origin (Aim 1); 2) define the relative contribution of healthy tissues to the pool of circulating EVs (Aim 2); and 3) to assess the specificity and sensitivity of EVs in cancer detection (Aim 3). This application focuses on Pancreatic Ductal Adenocarcinoma (PDAC), a deadly neoplastic disease with low survival rate that lacks specific and sensitive diagnostic tests. Completion of the proposed studies will contribute to the development of standardized procedures for the preparation, selection, and analysis of EV-based biomarkers.