Tumor cell derived extracellular vesicles (EV) are being explored as circulating biomarkers for cancer detection. Up to now however, clinical results have been mixed for a number of reasons including the predominant use of bulk measurements, the inability to differentiate tumor from host cell derived vesicles, the general absence of uniquely identifying biomarkers and the unknown frequency of stochastically distributed biomarkers into single circulating vesicles.
Researchers at Massachusetts General Hospital hypothesized that a single EV analysis (sEVA) technique could potentially improve diagnostic accuracy necessary to detect early cancers but the actual biomarker frequency and practical detection limits are currently unknown. Using pancreatic cancer, the researchers carefully analyzed the composition of putative cancer markers in 11 established and new patient derived models. In parental PDAC cells positive for KRASmut and/or P53mut proteins only ~40% of EVs were also positive (range: 30-64%). This rate of positivity increased to 57% when additional PDAC biomarkers were considered (MUC1, EGFR, αFG-P4OH) in cell lines. In a blinded study involving 16 patients with surgically proven stage 1 PDAC, KRASmut and P53mut protein was detectable at much lower levels, generally in < 0.1% of vesicles. With the analytical capabilities of sEVA however, 15 of the 16 patients with stage 1 PDAC expressed low levels of biomarker positive EV. Using a modeling approach, the researchers estimate that the current PDAC detection limit is at ~0.1 cm3 tumor volume, below clinical imaging capabilities. These findings establish the potential for single-EV analysis for early cancer detection.
Workflow of single EV analysis
A) In an initial screen, EV are labeled for pooled biomarkers as shown. For example, onco/tumor suppressor gene labeling contains 3 antibodies labeled with the same fluorochrome (KRASG12D, KRASG12V, P53mut; red channel); additional pancreatic cancer markers (EGFR, MUC1, and ⍺FGP4OH) are shown in the blue channel. Note the high signal-to-noise ratio of labeling individual EV as shown in the dashed lines (e.g. 1, 2 or 3). B) In case of positivity, any of the channels can be sub-speciated where each antibody contains a separate fluorochrome as shown.