Multiplexed exosome analyses with DNA barcoding

 描述(由申请人提供)：循环外切体已经成为一种新的生物标志物，能够实现非侵入性、实时的疾病监测。大多数癌细胞会主动释放大量外切体进入血液循环，这些外切体携带原始细胞的分子成分。因此，捕捉这些信息可以代表一种新的途径来探测和连续监测肿瘤分子状态。我们之前已经开发了便于外切体分析的微型化平台，并通过后续的临床研究建立了外切体用于癌症诊断和监测的临床用途。然而，预测和检测耐药性的出现仍然是具有挑战性的，因为它需要多方面地检测外体蛋白、它们的翻译后Modifi阳离子和mRNAs的变化。本应用的总体目标是提出一种新的综合外体蛋白/信使核糖核酸fi的筛选技术。我们将专门探索fi条形码标记系统，将蛋白质和信使核糖核酸的检测统一为一种分析形式：抗体将被标记的脱氧核糖核酸标签，其序列对于不同的蛋白质靶标是唯一的；连接依赖的脱氧核糖核酸标签将用于检测信使核糖核酸靶标。在目标1中，我们将开发和验证所提出的检测方法。我们将实施一种新的、集成的fluidic系统，以在芯片上进行癌症特定的fic外显体浓缩和DNA条形码。该装置将在热塑性塑料中制造(通过注塑)，以提高系统的健壮性和扩大生产。在目的2中，我们将探讨胞外体蛋白/信使核糖核酸蛋白fi在体内外预测和监测耐药方面的应用。开发的平台将用于分析从卵巢细胞系和接受治疗的卵巢癌患者中收集的外切体。将在基线和纵向监测外体蛋白和信使核糖核酸的靶点，以区分治疗反应和监测耐药性的出现。我们预计，新的检测技术将通过加速外切体的翻译而具有重要的临床意义，不仅作为癌症诊断的生物标志物，而且作为药物疗效的指示物和作为治疗决策的潜在的分子策略。

 DESCRIPTION (provided by applicant): Circulating exosomes have emerged as a new class of biomarker which enables non- invasive, real-time disease monitoring. Most cancer cells actively release large numbers of exosomes into the circulation, that carry molecular constituents of the originating cells. Capturing such information can thus represent a new avenue to probe and serially monitor the tumor molecular status. We have previously developed miniaturized platforms to facilitate exosome analyses and established the clinical utility of exosomes for cancer diagnosis and monitoring through subsequent clinical studies. Predicting and detecting the emergence of drug resistance, however, is still challenging, as it requires multifaceted profiling of exosomal proteins, their post-translational modifications, and mRNA changes. The overall goal of this application is to advance a new screening technology for comprehensive exosomal protein/mRNA profiling. We will specifically explore the DNA-barcode labeling system to unify protein and mRNA detection into a single assay format: antibodies will be labeled with DNA tags whose sequences are unique for different protein targets; and ligation-dependent DNA tags will be used to detect mRNA targets. In Aim 1, we will develop and validate the proposed assay. We will implement a new, integrated fluidic system to perform cancer-specific exosome enrichment and DNA-barcoding on-chip. The device will be fabricated in thermoplastics (via injection molding) to promote system robustness and scaled-up production. In Aim 2, we will investigate the utility of exosomal protein/mRNA profiling in predicting and monitoring drug resistance in vitro and in vivo. The developed platform will be applied to analyze exosomes collected from ovarian cell lines and ovarian cancer patients undergoing therapies. Exosomal protein and mRNA targets will be monitored at baseline and longitudinally to differentiate treatment response and monitor the emergence of drug resistance. We envision that the new assay technology would have significant clinical implications by accelerating the translation of exosomes, not only as a cancer diagnostic biomarker but also as an indicator of drug efficacy and as a potential molecular stratifier for treatment decision.