The poor survival rate of ovarian cancer is attributed to the lack of effective biomarkers and screening methods for early disease. Extracellular vesicles (EVs) of exosomal size (30-150 nm) and their molecular cargo, are emerging as promising biomarker candidates. We hypothesise that ovarian-specific EVs can be isolated from vaginal swabs for use in early cancer screening. However, optimising isolation methods remains essential to ensure sufficient EV yield, purity and reproducibility. Here, we evaluate EV isolation methods in preparation for downstream application to vaginal swab samples.
Using patient serum for optimisation, three isolation strategies were evaluated: (a) combined ultracentrifugation (UC) and size-exclusion chromatography (SEC) (UC + SEC; qEV1 single 35nm, Izon); (b) UC alone; and (c) SEC (qEV1 35nm, Izon) alone. Particle size and concentration were assessed using tuneable resistive pulse sensing (qNano Gold, Izon), and morphology using cryo-transmission electron microscopy. EV protein markers (CD63, CD81, CD9) and contamination markers (GM130) were evaluated via dot blot and EV-derived miRNA marker expression (miR-16-5p, miR-21-5p) was measured by quantitative PCR.
Preliminary results (n=3, 2 technical replicates) showed inconsistent recovery with UC+SEC, likely due to EV fragility post UC, supported by CD63 expression in the protein wash-through qEV fractions. Ultracentrifugation and SEC alone yielded distinctly sized populations (110-188nm and 62-67nm, respectively) and concentrations (3x1010-1x1011 particles/mL and 8x1010-2.5x1011 particles/mL, respectively), but exhibited similar levels of intercellular (GM130) contamination.
This work informs method selection for EV isolation from vaginal swabs and is foundational for biomarker discovery in non-invasive ovarian cancer screening.