Effect of methodological and biological variability on molecular profiling of extracellular vesicles in cancer detection

项目摘要 基于识别细胞外信号的发现，最近出现了一种新的旁分泌信号范例 囊泡(EV)在正常和病理条件下都是生物信息的细胞间传输者 比如癌症。我们和其他人已经展示了电动汽车及其货物可以调节基因表达和改变 细胞在各种类型的细胞中发挥功能。此外，在癌症等病理情况下，数量和 EVS的组成改变了宿主的免疫反应，并使继发性肿瘤的行为同步。 肠道病毒的分离和分子图谱(即RNA、蛋白质、翻译后修饰、脂类、 健康和疾病中的代谢产物)对于理解EV的生物发生和效应器功能都是至关重要的。 目前，EV作为与细胞内信号和疾病诊断相关的生物实体的研究是 基于EVS的生物生成和去除以稳定的速率发生的假设，即 主要由宿主的健康/患病状态修改。我们公布的结果表明，这并不是 凯斯。我们的数据表明，组织来源、数量、大小分布以及蛋白质、脂肪、代谢物和 用标准技术分离的EVS的RNA组成不仅取决于采血方法，而且 也是在一天中采集血样的时间。因此，对这些问题的系统评估 EV图谱中的因素和其他变异性来源对于实现基础生物学、临床和 个性化医疗应用。 虽然这超出了这笔赠款的目的，但我们团队的长期目标是建立可再生的 血液采集和样本处理的方法，使我们能够识别特定的EV分子 昼夜循环中的签名，目的是确定理想的、特定于器官的血液时间 这将增加癌症早期检测的可靠性和特异性。我们相信 建立生物学和方法学基线对于正确比较蛋白质组学是绝对至关重要的 和在不同研究中获得的血糖数据，以及解释从患者的 样本。

Project Summary A novel paradigm in paracrine signaling has recently emerged based on the findings identifying extracellular vesicles (EVs) as intercellular conveyors of biological information both in normal and pathological conditions such as cancer. EVs and their cargo have been shown by us and others to regulate gene expression and alter cell function in various cell types. Moreover, during pathological conditions such as cancer, the number and compositions of EVs alter the host immune response as well as synchronize the behavior of secondary tumors. Isolation and molecular profiling of EVs (i.e. RNAs, proteins, post-translational modifications, lipids, metabolites) both in health and disease are critical for understanding EVs' biogenesis and effector functions. Currently, the study of EVs as biological entities relevant for intracellular signaling and disease diagnosis is based on the assumption that the biogenesis and removal of EVs happen at a steady state rate, being modified mostly by the healthy/diseased status of the host. Our published results show that that is not the case. Our data indicate that the tissue-origin, number, size distribution, as well as protein, lipid, metabolite and RNA composition of EVs isolated by standard techniques depend not just on the blood collection methods, but also on the time of the day the blood samples were collected. Therefore, systematic assessment of these factors and other sources of variability in EV profiles are important for enabling basic biology, clinical and personalized medicine applications. Although it is beyond the purpose of this grant, the long-term goal of our team is to establish reproducible methods for blood collection and sample processing that would allow us to identify the specific molecular EV signatures during the day/night cycle, with the aim of pinpointing the ideal, organ-specific times for blood collection, which would increase the reliability and specificity of early cancer detection. We believe that establishing biological and methodological baselines are absolutely vital for correctly comparing proteomics and glycomics data obtained in various studies, as well as interpreting the data obtained from patients’ samples.