Abstract
Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30–150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids.
Highlights
Exosomes from breast cancer MDA-MB-231 cells and human serum samples were mainly isolated by differential ultracentrifugation[27] (Fig. 1A) unless specified in this report, which remains the most widely used and unbiased purification method[28]
According to the guidelines of the International Society of Extracellular Vesicles (ISEV) for the characterization of exosomes[29], multiple approaches were used to characterize the physical features and molecular markers of the isolated extracellular vesicles in order to identify these as exosomes
Due to improved ability to rapidly analyze small particles at an unprecedented sensitivity for fluorescent signals and extreme light scatter performance using three distinct angle ranges, the Micro flow cytometer (MFC) is capable of measuring surface protein profiles of single exosomes isolated from cell culture or human blood
Summary
Latex beads in micrometer sizes have been used to bind to multiple exosomes to enhance the ability to detect exosomes stained with fluorophore-conjugated antibodies by conventional flow cytometry[10]. This bead-based approach does not provide single exosome profiling and fails to discriminate between different subsets of exosomes, which may result in the loss of distinctive signatures with potential diagnostic importance. We report a new, automated analytic approach utilizing a micro flow cytometer[13], and present data on its use to profile protein expressions of individual exosomes isolated from cell lines and human blood of breast cancer patients and healthy controls, as a proof of principle. The expression of CD47 on the surface of the cancer cells prevents recognition by macrophages and natural killers, thereby inhibiting their ability to engulf and destroy those cancer cells[25,26]
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