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Figure 2. Selective binding of EV-PSMA^scFv to PSMA-expressing prostate cancer cells. (A) Flow cytometric analysis of surface PSMA expression in prostate cancer cell lines. Representative histograms of anti-PSMA antibody staining in LNCaP, CWR22Rv1, PC3, and DU145 cells. Unstained and IgG isotype controls are shown; (B) Quantification of relative MFI of PSMA expression in prostate cancer cell lines; (C and D) Flow cytometric analysis of cell-associated EV binding at the single-cell level in prostate cancer cells following exposure to labeled EVs. Representative dot plots show gated EV-positive cell populations, with percentages indicating EV-bound cells among total analyzed cells (C). Quantification of EV-associated MFI is shown in (D). Representative flow cytometry plots are shown. Experiments were conducted in three independent biological repeats (n = 3), with each repeat using an independently prepared EV batch and analyzed in triplicate. Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple-comparisons test for the indicated comparisons. ns; ^***P < 0.001. EVs: Extracellular vesicles; PSMA: prostate-specific membrane antigen; MFI: mean fluorescence intensity; IgG: immunoglobulin G; PE: phycoerythrin; ANOVA: analysis of variance; ns: not significant; EV-PSMA^scFv: extracellular vesicles displaying PSMA-targeting single-chain variable fragments; FITC-A: fluorescein isothiocyanate area; PE-A: phycoerythrin area.