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：
9266748
负责人：
Hakho Lee
金额：
$ 19.99万
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 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9266748
关键词：
70-kDa Ribosomal Protein S6 Kinases Antibodies Benchmarking 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 Monitoring Drug resistance ERCC1 gene Enrollment Evolution Filtration Goals Gold Heterogeneity Human Hybrids In Vitro Injection of therapeutic agent Institutional Review Boards Integral Membrane Protein Label Ligation Liquid substance 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 Phosphoproteins 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 Efficacy Treatment outcome Tumor Biology Vesicle analog anticancer research base cancer cell cancer diagnosis cancer therapy clinical practice design diagnostic biomarker drug efficacy exosome high throughput screening in vivo insight mRNA Expression miniaturize novel personalized cancer therapy photolysis pressure protein activation public health relevance resistance mechanism scale up screening treatment response tumor

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（由申请人提供）：循环外泌体已成为一类新型生物标志物，可实现非侵入性实时疾病监测。大多数癌细胞主动将大量外泌体释放到循环中，这些外泌体携带原始细胞的分子成分。因此，捕获此类信息可以代表探测和连续监测肿瘤分子状态的新途径，我们之前开发了微型平台来促进外泌体分析，并通过外泌体建立了用于癌症诊断和监测的临床应用。然而，预测和检测耐药性的出现仍然具有挑战性，因为它需要外泌体蛋白、其翻译后修饰和 mRNA 变化的多方面分析。该应用的总体目标是推进新的筛选。我们将专门探索 DNA 条形码标记系统，将蛋白质和 mRNA 抗体检测统一为单一测定格式：将用其序列的 DNA 标签进行标记。对于不同的蛋白质靶点来说是独一无二的；并且连接依赖的 DNA 标签将用于检测 mRNA 靶点。该设备将采用热塑性塑料（通过注塑）制造，以提高系统的稳健性和扩大生产规模。在目标 2 中，我们将研究外泌体的实用性。预测和监测体外和体内耐药性的蛋白质/mRNA 分析将应用于分析从接受治疗的卵巢细胞系和卵巢癌患者中收集的外泌体，并在基线和纵向上进行监测。我们预计，这项新技术将通过加速外泌体的翻译而产生重大的临床意义，不仅可以作为癌症诊断生物标志物，还可以作为药物的指标。效率并作为治疗决策的潜在分子分层器。