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

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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.