Extracellular vesicles (EVs) are of considerable interest as
tumor biomarkers because
tumor-derived EVs contain a broad array of information about
tumor pathophysiology. However, current EV assays cannot distinguish between EV
biomarker differences resulting from altered abundance of a target EV population with stable
biomarker expression, altered
biomarker expression in a stable target EV population, or effects arising from changes in both parameters. We now describe a rapid nanoparticle- and
dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV
biomarker levels to EV abundance. In this approach, EVs are captured from complex samples (e.g., serum), stained with a lipophilic
dye, and hybridized with antibody-conjugated
quantum dot probes for specific EV surface
biomarkers. EV
dye signal is used to quantify EV abundance and normalize EV surface
biomarker expression levels. EVs from malignant and nonmalignant pancreatic cell lines exhibited similar staining, and probe-to-
dye ratios did not change with EV abundance, allowing direct analysis of normalized EV
biomarker expression without a separate EV quantification step. This EV
biomarker normalization approach markedly improved the ability of serum levels of two
pancreatic cancer biomarkers, EV
EpCAM and EV EphA2, to discriminate
pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its modular design permits it to be rapidly adapted to quantitate other EV
biomarkers by the simple expedient of swapping the antibody-conjugated
quantum dot probes for those that recognize a different disease-specific EV
biomarker.