Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease.
Researchers at the University of Sydney have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence.
DotScan analysis of LIM1215 cells (b) and their EV (c).
The key (a) shows antibody locations, with shaded antibodies indicating cell capture. Duplicate antibody arrays (outlined) are surrounded by a frame of alignment dots consisting of a mixture of CD44/CD29 antibodies. Detection of captured cells was by optical scanning (b). EV (7.8×108 particles derived from 335 µl of LIM1215-conditioned medium) were detected by ECL using biotinylated EpCAM (CD326) antibody, with a 10 min exposure on ECL film (c). NanoSight analysis shows the size distribution of LIM1215 EV (d). The number above the peak represents mode size in nm. A Venn diagram compares surface profiles of LIM1215 cells with their EV (e).
This approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.