Multiplexed exosome analyses with DNA barcoding

使用 DNA 条形码进行多重外泌体分析

基本信息

批准号：
9099367
负责人：
Hakho Lee
金额：
$ 23.98万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9099367
关键词：
70-kDa Ribosomal Protein S6 Kinases Antibodies Benchmarking Binding Biological Biological Assay Biological Markers Biopsy Biosensor Blood Circulation Caliber Cancer Diagnostics Cancer Patient Carboplatin Cell Line Cells Cisplatin Clinical Clinical Research Complex DNA DNA Microarray Chip DNA Repair Detection Devices Disease Drug Efflux Drug resistance ERCC1 gene Enrollment Evolution Filtration Goals Gold Human In Vitro Injection of therapeutic agent Institutional Review Boards Integral Membrane Protein Label Ligation MLH1 gene Malignant Neoplasms Malignant neoplasm of ovary Membrane Messenger RNA Methods MicroRNAs Microfluidics Molds Molecular Molecular Profiling Molecular Target Monitor Oligonucleotides Ovarian Pathway interactions Pattern Performance Pharmaceutical Preparations Pharmacotherapy Phosphatidylinositide 3-Kinase Inhibitor Phospholipids Platinum Post-Translational Protein Processing Production Proteins Proto-Oncogene Proteins c-akt Protocols documentation Regimen Scientific Advances and Accomplishments Screening for cancer Signal Pathway Signal Transduction System Technology Testing Therapeutic Therapeutic Trials Time Tissues Translations Treatment outcome Tumor Biology Vesicle analog anticancer research base cancer cell cancer diagnosis cancer therapy clinical practice design diagnostic biomarker exosome high throughput screening in vivo insight mRNA Expression miniaturize novel personalized cancer therapy photolysis pressure protein activation protein profiling public health relevance resistance mechanism scale up screening treatment response tumor tumor heterogeneity

项目摘要

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.

描述（由适用提供）：循环外泌体已成为一种新的生物标志物，可实现无创，实时疾病监测。大多数癌细胞都会积极地将大量外泌体释放到循环中，这些外泌体构成了原始细胞的分子。因此，捕获此类信息可以代表探测和串行监测肿瘤分子状态的新途径。我们以前已经开发了微型平台，以促进外泌体分析，并通过随后的临床研究建立了外泌体进行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受到挑战，因为它需要多方面的外泌体蛋白质，其翻译后修饰和mRNA变化。该应用程序的总体目标是推进一项新的筛选技术，以供全面的外泌体蛋白/mRNA培养。我们将专门探索DNA-Barcode标记系统，以将蛋白质和mRNA检测统一为单个测定格式：抗体将用DNA标签标记，其序列对于不同蛋白质靶标的序列是独特的；并依赖于连接的DNA标签来检测mRNA靶标。在AIM 1中，我们将开发并验证拟议的测定法。我们将实施一个新的，集成的流体系统，以在片上执行特定于癌症的外泌体富集和DNA-Barcoding。该设备将在热塑性塑料（通过注入成型）中制造，以促进系统的鲁棒性和扩展生产。在AIM 2中，我们将研究外泌体蛋白/mRNA培养在预测和监测体外和体内耐药性方面的效用。开发的平台将用于分析接受热感应的外泌体细胞系和卵巢癌患者。外泌体蛋白和mRNA靶标将在基线和纵向上进行监测，以区分治疗反应并监测耐药性的出现。我们设想，新测定技术不仅可以作为癌症诊断生物标志物，而且作为药物效率的指标以及作为治疗决策的潜在分子层面层的指标，还可以加速外泌体的翻译来具有显着的临床意义。