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

项目总结/摘要 细胞外囊泡（EV）是释放到细胞外空间的细胞来源的膜结合结构， 引导体液并支持细胞间的通讯。癌细胞大量释放 电动汽车的数量比正常的电动汽车要多。因为EV内容来自于 循环EV的分子分析正在被仔细研究，作为早期癌症的非侵入性手段 诊断、监测疾病进展和评估对治疗的反应。然而，基于EV的临床 诊断受到样本的精确性和再现性不足的限制， 分离并表征正常和疾病相关EV。 三个分析和概念上的挑战阻碍了识别特定的和可重复的EV- 具有临床相关性的相关癌症生物标志物：1）尚未开发出无偏倚的策略来评估 使用EV的癌症检测的限制; 2）健康组织对循环肿瘤池的相对贡献; EV未知;以及3）循环癌症衍生EV的数量和组成的系统分析 在肿瘤发展过程中，没有进行过。 这些复杂性促使我们设计一个通用平台，能够评估 与经历癌症的动物相比，在其他健康动物中， 发展该系统基于从四跨膜蛋白CD 63开发的工程EV标记物 （enCD 63），其促进由特定细胞释放的EV的收集、可视化和定量，以及 组织中 通过将enCD 63的表达限制在特定的正常或肿瘤细胞和组织中， 在工程小鼠模型中，我们将明确地检查EV作为生物标志物的功效。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 2）定义健康组织对循环EV池的相对贡献（目标2）;以及 3)评估EV在癌症检测中的特异性和灵敏度（目的3）。 该应用程序的重点是胰腺导管腺癌（PDAC），一种致命的肿瘤性疾病， 缺乏特异性和敏感性诊断测试的存活率。完成拟议的研究将有助于 制定标准化程序，用于制备，选择和分析基于EV的 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.