Fig. 5

Tetraspanins and EV tumour-related proteins, EpCAM and MICA, can be detected directly in minimal volumes of plasma and ascitic fluid from cancer patients by flow cytometry, with better sensitivity than Western Blot. Plasma (A, B). A 12 µl of PBS 1% casein containing 3000 beads conjugated with anti-CD63, anti-EpCAM or anti-MICA were incubated for 16 h with 12 µl of plasma (EDTA-tubes) from cancer patients (ovarian cancer Ov1-Ov3 and breast cancer Br1-Br5). Samples with addition of 8 µg/ml of polybrene were analysed in parallel (dark symbols). The final volume of the assay was 24 µl. EVs captured in each assay were detected with anti-CD81-PE, anti-CD9-PE or IgG-PE as a negative control to calculate the Relative Fluorescence Intensity (RFI). Mean and Standard Deviation of the RFI from 3 independent experiments (circles, squares and triangles) are represented. B After ultracentrifugation of 200 µl of each plasma, the EV enriched preparation was resuspended in 15 µl. 5 µl of this EV preparation were loaded in a 12% SDS-PAGE gel and transferred to nitrocellulose. The membrane was immunoblotted for detection of EpCAM and MICA and the tetraspanins CD9 and CD81, as general EV markers. In parallel, 1.5 µl of the same EV preparation were incubated for 16 h with 24 µl of PBS 1% casein containing 3000 beads conjugated with anti-CD63, anti-EpCAM or anti-MICA. EVs captured in each assay were detected by incubation with anti-CD81-PE or anti-CD9-PE. RFI values are represented as heatmaps (the number obtained is superimposed). EVs from the H3122 cell line were used as a control for EpCAM positive EVs (5.86 × 10⁸ particles/well for WB and 1.76 × 10⁸ particles/test for Flow Cytometry). Ascites (C, D). Ovarian cancer patients’ ascites [1,2,3,4,5,6,7,8] were subjected to the same protocols as in A, B. Patient samples were obtained at Clínica Universidad de Navarra