fig1

Figure 1. Characterization of EVs derived from HGSOC patients. (A) Spheroids were directly isolated from the ascites of HGSOC patients and subsequently cultured before EV collection. Then, EVs were isolated from the conditioned medium. Representative brightfield and fluorescence microscopy images show spheroid structures with DAPI-stained nuclei (blue) and microtubules (green). Scale bar: 100 µm; (B) Characterization of isolated EVs by TEM. Representative images are shown with scale bars of 1 µm (left) and 100 nm (right); (C) Median size of particles of EVs analyzed by nFC; (D) Single-particle phenotyping by nFC. EVs were fluorescently labeled with FITC-conjugated antibodies specific to CD9 and PE-conjugated CD63. Bivariate dot plots of indicated fluorescence vs. SS-A are shown. In addition, the percentage of CD9 and CD63 positive particles is depicted; (E) A Venn diagram displaying the overlap between EV proteins identified in this study and entries from ExoCarta and Vesiclepedia databases; (F) GO term enrichment analysis of proteins identified in EVs isolated from ovarian cancer spheroids. The bar plot shows the top enriched terms grouped by GO category: Cellular Component, Biological Process, and Molecular Function. Enrichment significance is indicated by -log₁₀-adjusted P-values. Dashed line indicates multiple testing threshold (Bonferroni-corrected P = 0.05). EVs: Extracellular vesicles; HGSOC: high-grade serous ovarian cancer; DAPI: 4’,6-diamidino-2-phenylindole; TEM: transmission electron microscopy; nFC: nanoflow cytometry; FITC: fluorescein isothiocyanate; PE: phycoerythrin; SS-A: side scatter area; GO: gene ontology; UC: ultracentrifugation; SEC: size-exclusion chromatography.