SEVEN – Single Extracellular VEsicle Nanoscopy

Extracellular vesicles (EVs) and their cargo constitute novel biomarkers. EV subpopulations have been defined not only by abundant tetraspanins (e.g., CD9, CD63 and CD81) but also by specific markers derived from their source cells. However, it remains a challenge to robustly isolate and characterize EV subpopulations. Researchers at the City of Hope Comprehensive Cancer Center have combined affinity isolation with super-resolution imaging to comprehensively assess EV subpopulations from human plasma. Their Single Extracellular VEsicle Nanoscopy (SEVEN) assay successfully quantified the number of affinity-isolated EVs, their size, shape, molecular tetraspanin content, and heterogeneity. The number of detected tetraspanin-enriched EVs positively correlated with sample dilution in a 64-fold range (for SEC-enriched plasma) and a 50-fold range (for crude plasma). Importantly, SEVEN robustly detected EVs from as little as ∼0.1 μL of crude plasma. The researchers further characterized the size, shape and molecular tetraspanin content (with corresponding heterogeneities) for CD9-, CD63- and CD81-enriched EV subpopulations. Finally, they assessed EVs from the plasma of four pancreatic ductal adenocarcinoma patients with resectable disease. Compared to healthy plasma, CD9-enriched EVs from patients were smaller while IGF1R-enriched EVs from patients were larger, rounder and contained more tetraspanin molecules, suggestive of a unique pancreatic cancer-enriched EV subpopulation. This study provides the method validation and demonstrates that SEVEN could be advanced into a platform for characterizing both disease-associated and organ-associated EV subpopulations.

Validation of EV size and shape measurements using SEVEN

(a) Scheme of an affinity isolated and fluorescently labelled EV. Anti-TSPAN Abs (mixture of unlabelled anti-CD9, anti-CD63 and anti-CD81 Abs) were covalently immobilized on the polymer-coated glass coverslip surface. After affinity capture, EVs were stained using a mixture of fluorescently labelled anti-TSPAN Abs. (b) Raw SMLM image of TSPAN-enriched EVs in red. (c) TSPAN-enriched EVs are imaged in SMLM (red, signal from fluorescent anti-TSPAN Abs) and TIRF (green, signal from GFP). (d) Tessellation polygons (blue lines) are outlining SMLM localization (red dots); EV is outlined in white dashed line using the Voronoi tessellation algorithm described in Methods. (e) TEM image of EVs. (f) Number of detected EVs per ROI for spot coated with either a mixture of anti-TSPAN Abs or anti-rabbit IgG control. (g) EV diameter detected with SMLM imaging and tessellation analysis (SMLM_T); SMLM imaging and EVSCAN analysis (SMLM_E); TEM and segmentation analysis; and NTA. (h) EV circularity detected with SMLM_T, SMLM_E and TEM. Box plots indicate interquartile range (box), median (centre line), mean (cross); the hollow dots (for TEM and SMLM) indicate EVs detected beyond 1.5-times the interquartile range (marked by the Whisker lines under and over the box). Error bars, SEM; *** indicates p < 0.001.