Biologically Active Tissue Factor-Bearing Larger Ectosome-Like Extracellular Vesicles in Malignant Effusions from Ovarian Cancer Patients: Correlation with Incidence of Thrombosis

Figures

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  Figure 1. Ascites from patients with ovarian cancer contains high amounts of larger EVs. (A) Purification of EV subsets using sequential centrifugation. (B) Gating strategy for the identification of intact high-speed EVs (hsEVs) by high-resolution flow cytometry. A gate representing the size range of interest (0.1–0.9 µm) was set according to known diameters of standard beads (upper plots). Within the estimated size range of 0.1–0.9 µm, carboxyfluorescein diacetate succinimidyl ester (CFSE+) events were identified as intact hsEVs (lower plots). For setting the CFSE gate, an unstained PBS control (black histogram) and a stained hsEV sample (red histogram) were overlaid. Of note, the intermediate signal occurring in the stained hsEV sample can be assigned to CFSE noise, as it has been recently shown to be EV independent [12]. (C) Concentration of CFSE+ hsEVs/mL isolated from ascites of ovarian carcinoma patients expressed as a box plot including median (line), upper and lower quartiles (box), and extreme values (whiskers). (D) Flow cytometry analysis of tetraspanin markers CD9, CD63, and CD81 on hsEVs (upper panel), fluorescence minus one (FMO) control (lower panel left), and isotype control…

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  Figure 2. Elevated levels of TF-bearing EVs correlate with the incidence of thrombosis in ovarian cancer patients. (A) Tissue-factor (TF) activity on ascites-derived hsEVs isolated from ovarian carcinoma patients. The median of 20 individual samples (80 pg/mL) is shown. The four response categories of EV-TF activity proposed by Hisada and coworkers served as reference [14] (right, scale in red). (B) Quantification of ascites-derived hsEVs isolated from ovarian carcinoma patients in the size range of 0.1–0.9 μm. The median of 6 × 106 hsEVs/mL ascites (depicted by the red dotted line) is implemented. The correlation between the absolute amount of hsEVs/mL ascites and the incidence of thrombosis was analyzed using the chi-square test (* p = 0.019) (see table on the right).

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  Figure 3. Ascites-derived EVs are biologically active and induce intracellular signaling. 10 min of co-cultivation with ascites-derived hsEVs (left box-plot, *** p = 0.0002) and induction of tumor cell migration after 48 h of co-cultivation with ascites-derived hsEVs (right box-plot * p = 0.013). For these experiments, we used human pancreas carcinoma COLO 357 cells, as these cells have a stable high level of PAR2 expression, and we used them in our previous experiments in which we demonstrated the signal chain PAR2/ERK/migration [3].