BACKGROUND: Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface.
METHODS: We investigated the potential of a 48- multiplex surface plasmon resonance imaging (SPRi) system to perform EV phenotyping. Antigen surface density of 11 antigens was measured on the human breast cancer cell lines HS578T, MCF7, and SKBR3 and their EVs by use of both SPRi and the widely used flow cytometry (FCM).
RESULTS: For cells, the SPRi andFCMsignals for antigen exposure correlated (R2 HS578T cells = 0.66, R2 MCF7 cells = 0.78, R2 SKBR3 cells = 0.60). With regard to EVs, SPRi detected 31 out of 33 tested antibody-EV pairs, whereas our flow cytometer detected 5 antibody-EV pairs because of high blank and isotype control signals. For HS578T-derived EVs, the SPRi and FCM signals correlated (R2 HS578T EVs = 0.98). However, on MCF7- and SKBR3-derived EVs, insufficient antigens were detected by our flow cytometer. To confirm that the SPRi responses correlated with mean antigen density on EVs, the SPRi responses of EVs were correlated with antigen density on parental cells as measured by FCM (R2 HS578T = 0.77, R2 MCF7 = 0.49, R2 SKBR3 = 0.52).
CONCLUSIONS: SPRi responses correlate with mean antigen density. Moreover, SPRi detects lower antigenexposure levels than FCM because SPRi measures an ensemble of EVs binding to the sensor surface, whereas FCM detects antigens of single EV.