Multiplexed exosome analyses with DNA barcoding

 描述（由申请人提供）：循环外泌体已成为一类新的生物标志物，其能够进行非侵入性实时疾病监测。大多数癌细胞主动释放大量的外泌体进入循环，这些外泌体携带起源细胞的分子成分。因此，捕获这些信息可以代表探测和连续监测肿瘤分子状态的新途径。我们之前已经开发了小型化平台以促进外泌体分析，并通过后续临床研究建立了外泌体用于癌症诊断和监测的临床实用性。然而，预测和检测耐药性的出现仍然具有挑战性，因为它需要对外泌体蛋白、其翻译后修饰和mRNA变化进行多方面的分析。这项申请的总体目标是推进一种新的筛选技术，用于全面的外泌体蛋白/mRNA表达。我们将专门探索DNA条形码标记系统，以将蛋白质和mRNA检测统一为单一检测格式：抗体将使用DNA标签标记，其序列对于不同的蛋白质靶标是唯一的;连接依赖性DNA标签将用于检测mRNA靶标。在目标1中，我们将开发和验证所提出的检测方法。我们将实施一个新的集成化微生物系统，以进行癌症特异性外泌体富集和芯片上DNA条形码化。该设备将由热塑性塑料制成（通过注塑成型），以提高系统的鲁棒性和扩大生产。在目标2中，我们将研究外泌体蛋白/mRNA在体外和体内预测和监测耐药性中的效用。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泌体。将在基线和纵向监测外泌体蛋白和mRNA靶标，以区分治疗反应并监测耐药性的出现。我们设想，新的检测技术将通过加速外泌体的翻译而具有重大的临床意义，不仅作为癌症诊断生物标志物，而且作为药物有效性的指标和作为治疗决策的潜在分子分层器。

 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.