During the past year, we have developed a comprehensive pipeline for EV analysis, which we refer to as the Multiplex-to-Single-EV-Analysis (Mt-SEA) pipeline. This Mt-SEA pipeline includes multiplex high dimensional EV surveys as well as high resolution single EV studies, including nanoFACS protocols for fluorescence detection and calibration, for counting, and for high-throughput sorting, to sort EV subsets from plasma and other biofluids by nanoFACS. Using multiple distinct vesicle populations, we find that nanoFACS sorting is uniquely able to produce high fidelity EV subsets (95-97% pure), with functional biological cargo (RNA and protein). Our current focus is exclusively on application of this method for clinically important studies, for the study of cancer disease burden, metastatic potential, and responses to treatment. Furthermore, we have devised a new class of labels (Molecular NanoTags) that can be used with nanoFACS, not only to improve the detection of EV epitopes, but also to enable single molecule counting by flow cytometry. The specialized instrumentation, expertise, and biospecimen pipeline that we have assembled and are continuing to improve for this nanoFACS program is a unique and distinctive asset for NCI.
