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Exosome profiling with CF® dye tetraspanin panel shows single tetraspanin immunocapture methods may limit cancer biomarker detection

Clinical analysis of extracellular vesicles (EVs) is a promising route for unbiased diagnostic and therapeutic applications. The current method for identifying EVs is the detection of the EV-enriched tetraspanins CD9, CD63, and CD81. These markers are also used for “total” EV capture for a variety of applications including ELISA, flow cytometry, and lab-on-a-chip assays. However, studies that assess the relative expression of these markers on EVs derived from various sources are lacking. In addition, methods for EV enrichment such as size exclusion chromatography (SEC) and ultracentrifugation (UC) yield heterogenous mixtures of EVs with other cellular components. Consequently, using these tetraspanin markers as “EV-specific” biomarkers without considering the variance of tetraspanin expression from EVs obtained through various sources and isolation methods may erroneously bias results.

In a recent preprint in bioRxiv, Mizenko et al. examine tetraspanin profiles of single EVs from a variety of sources using the ExoView® R100 platform developed by NanoView Biosciences. This method entails capture of exosomes by tetraspanin antibodies on a microarray chip for single particle interferometric reflectance imaging sensing (SP-IRIS) coupled with CF®488-anti-CD9, CF®647-anti-CD63, and CF®555-anti-CD81 fluorescent detection antibodies. This platform is capable of measuring captured particles by immunofluorescence with up to 3 colors, as well as particle size with a practical detection limit as low as ~50 nm.

The authors analyzed EVs derived from ovarian cancer cells SK-OV-3 isolated by UC and SEC and demonstrated similar tetraspanin expression across both isolation methods. The authors then analyzed EVs derived from other sources, including pMSCs and human serum, demonstrating unique tetraspanin profiles for each EV source. For clinical relevance, the authors studied whether single tetraspanin capture methods bias downstream detection of known cancer markers such as CD24, EpCAM, and Her2. As a control, the authors utilized a non-specific EV capture method using biotinylated SK-OV-3 EVs and anti-biotin coated chips. Results show significant variance in tetraspanin profiles between single tetraspanin capture and non-specific capture methods. Cumulatively, these results suggest that to avoid bias, EV capture using a combination of tetraspanins or non-specific method such as biotinylation should be used.

Learn more about Biotium’s bright and photostable CF® dyes as well as Biotium’s antibody and membrane stains validated for exosome and EV detection.


Full Citation

Mizenko, R. R., Brostoff, T., Rojalin, T., Koster, H. J., Swindell, H. S., Leiserowitz, G. S., … Carney, R. P. (2021). Tetraspanin immunocapture phenotypes extracellular vesicles according to biofluid source but may limit identification of multiplexed cancer biomarkers. BioRxiv, 2021.03.02.433595.