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Two-Dimensional Multi-Stage Isotachophoretic Technology for Multiplex Analysis of Cancer Exosomes and Proteins Marker Panel

用于癌症外泌体和蛋白质标记物组多重分析的二维多级等速电泳技术

基本信息

批准号：
10322022
负责人：
WEN-JI DONG
金额：
$ 16.88万
依托单位：
WASHINGTON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10322022
关键词：
3D Print Address Adopted Antibodies Antigens Area Behavior Binding Biological Blood Blood Circulation CD44 gene Cancer Patient Cancer Prognosis Cancerous Cations Cell secretion Cells Cellular Phone Clinical Communication Consumption Death Rate Detection Devices Diagnosis Disease Distal Early Diagnosis Epidermal Growth Factor Receptor Exhibits Hematopoietic Neoplasms Immune Immune Targeting Lead Liquid substance Malignant Neoplasms Membrane Methods MicroRNAs Microfluidics Modeling Neoplasm Metastasis Normal Cell Paper Pathologic Processes Patients Plasma Proteins Play Point of Care Technology Population Primary Health Care Process Prognosis Proteins Quality of life Research Role Sampling Scheme Screening for cancer Sensitivity and Specificity Serum Serum Proteins Signaling Molecule Survival Rate Techniques Technology Testing Time Tissues Translating Translational Research Tumor Markers Tumor-Derived base biomarker panel cancer biomarkers cancer cell cancer diagnosis cell type clinical diagnosis clinical prognosis clinically significant cost cost effective design early screening exosome extracellular extracellular vesicles improved interdisciplinary approach liquid biopsy malignant breast neoplasm miniaturize multiplex assay neoplastic cell noninvasive diagnosis novel novel strategies point of care protein biomarkers rapid technique success survivin tool translational potential tumor tumor growth tumor progression two-dimensional

项目摘要

Abstract Exosomes are small-sized (30–120 nm) extracellular vesicles. They are secreted by most cell types and play important roles in extracellular communication in normal and pathological processes. The exosomes derived from cancers shuttle signaling molecules (e.g. proteins and miRNAs) from parental cancer cells and tissue to distal recipient cells to reprogram the recipient cells and promote tumor growth and metastasis. Therefore, circulating tumor-derived exosomes carrying signature protein markers of the tumor hold great potential as invaluable liquid biopsy tools for the noninvasive diagnosis of early-stage cancers. Despite their potential clinical significance, translating disease-derived exosomes into point-of-care (POC) applications for the early diagnosis of cancers is hampered by a critical technical barrier: lack of a cost-effective POC approach capable of the simultaneous analysis of specific exosomes and their content markers in clinical samples. Although a number of methods for exosome isolation and characterization have been developed, either they are singly functionalized, nonspecific, laborious, or time-consuming, or they lack the robustness to be adopted as a cost-effective POC technique. Therefore, there is an urgent need for an effective, precise, easy to use, low-cost approach to multiplex POC sample analysis to detect trace levels of specific populations of exosomes released by specific cancer cells at an early stage and comprehensively profile the cancer markers carried by the exosomes. This application aims to fill the gap by taking a multidisciplinary approach to developing a novel, disposable two- dimensional paper-based multistage isotachophoresis (ITP) technology platform capable of the simultaneous analysis of specific target exosomes and exosomal proteins in a cost-effective way. Our objective is to develop an integrated paper-based isotachophoretic platform on which: 1) anionic cascade ITP is used to deplete high- abundance plasma proteins and enrich target exosomes before their capture and analysis; 2) a second ITP process simultaneously analyzes multiplex exosomal proteins released by lysing the exosomes captured in the first ITP process; and 3) a miniaturized smartphone-based detection module quantifies the target exosomes and protein markers captured by novel graded-binding test lines. We expect that integrating effective isolation/identification of specific exosomes with multiplex analysis of their contents in a cost-effective modular platform will provide a robust POC approach to advancing basic and clinical translational research on disease- derived exosomes. Exosomes derived from breast cancer will be used as model targets to validate our technology. The success of this project will not only provide a clinically compatible POC tool for tracking specific exosomes and markers for early screening for cancers, but also prompt research on the profile analysis of exosomal markers for the precision diagnosis of other diseases. Therefore, the technique may have broad translational potential in managing patients with malicious diseases and improving their quality of life.

抽象的外泌体是小尺寸（30-120 nm）的细胞外囊泡。它们由大多数细胞类型分泌并发挥作用在正常和病理过程中细胞外通讯中发挥重要作用。衍生的外泌体癌症将信号分子（例如蛋白质和 miRNA）从亲本癌细胞和组织传递到远端受体细胞对受体细胞进行重新编程并促进肿瘤生长和转移。所以，携带肿瘤特征蛋白标记物的循环肿瘤来源的外泌体具有巨大的潜力用于早期癌症无创诊断的宝贵液体活检工具。尽管它们具有潜在的临床意义重大，将疾病来源的外泌体转化为护理点（POC）应用以进行早期诊断癌症的治疗受到关键技术障碍的阻碍：缺乏具有成本效益的 POC 方法同时分析临床样本中的特定外泌体及其含量标记物。尽管有一些外泌体分离和表征的方法已经开发出来，它们要么是单一功能化的，非特异性、费力或耗时，或者缺乏作为具有成本效益的 POC 的稳健性技术。因此，迫切需要一种有效、精准、易用、低成本的方法多重 POC 样本分析可检测特定外泌体释放的痕量水平早期阶段的癌细胞并全面分析外泌体携带的癌症标志物。这该应用旨在通过采取多学科方法来开发一种新颖的一次性双三维纸基多级等速电泳 (ITP) 技术平台，能够同时进行以经济有效的方式分析特定目标外泌体和外泌体蛋白。我们的目标是发展一个集成的纸基等速电泳平台，其上：1) 阴离子级联 ITP 用于耗尽高在捕获和分析之前丰富血浆蛋白并富集目标外泌体； 2) 第二个 ITP 该过程同时分析通过裂解捕获的外泌体释放的多重外泌体蛋白第一个 ITP 流程； 3) 基于智能手机的小型化检测模块可量化目标外泌体并由新型分级结合测试线捕获的蛋白质标记。我们期望整合有效的通过经济高效的模块对其内容物进行多重分析来分离/鉴定特定的外泌体平台将提供强大的 POC 方法来推进疾病的基础和临床转化研究衍生的外泌体。来自乳腺癌的外泌体将用作模型靶标来验证我们的研究技术。该项目的成功不仅将提供一种临床兼容的 POC 工具，用于跟踪特定的用于癌症早期筛查的外泌体和标记物，同时也促进了对癌症特征分析的研究用于其他疾病精确诊断的外泌体标记物。因此，该技术可能具有广泛的在管理恶性疾病患者和改善他们的生活质量方面具有转化潜力。