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, researchers at Case Western Reserve University report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Their 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.
Micro flow cytometer detection of the ApogeeMix Beads and blood exosomes
(A) Schematic representation of Apogee A50 MFC denoting the mode of action of nanoparticle detection. The sample flows from top to bottom and is surrounded by sheath fluid. The laser intersects with the sample stream, generating 3 different light scatters (LALS, MALS, SALS) and fluorescence signals. LALS: Large angle light scatter; MALS: Middle angle light scatter; SALS: Small angle light scatter. (B) Cytogram of the reference beads, an aqueous mixture of the green fluorescent latex and non-fluorescent silica (Si) spheres in 110–1300 nm plotted at green fluorescence (L488-FITC) and LALS signals with minimized background noise. The red rectangle and circled box represent the 110 nm and 500 fluorescence beads respectively. (C) Cytogram of the reference ApogeeMix Beads in 110–1300 nm with similar MALS and LALS signals at the modified high threshold setting. (D) Cytogram of the unlabeled exosomes from human blood at green fluorescence and LALS signals. (E) Fluorescent signal comparison between the ApogeeMix beads from B and the unlabeled exosomes from D shown at the L488–FITC channel. (F) Histogram comparing the size of the gated exosomes from D with that of the gated 110 nm fluorescence beads in B (red rectangles). (G) Flow analysis of CD44 expression on exosomes derived from MDA-MB-231 and MCF-12A cells using A50 MFC. (H) Immunoblot of CD44 expression in the exosome lysates derived from MDA-MB-231 and MCF-12A cells. β-actin (~42 kDa) serves as a loading control.