Fig. 1

Workflow of sample processing and experimental design. Conditioned media were collected over a 24 h incubation period from two human cell lines, RT4 and J82, grown in monolayer culture. Urine samples were collected from patients with bladder urothelial carcinoma. Extracellular vesicles (EVs) were isolated and characterized using scanning electron microscopy (SEM), nanoparticle tracking analysis (NTA), and high-resolution flow cytometry. The homotypic effects of EVs were evaluated via cell-based functional assays with different readouts of cellular phenotypes (A). Cell and EV morphology were visualized using light microscopy and SEM, respectively (B). Cells (C) and cell- and urine-derived Evs (D) were analyzed for surface markers. The particle number and size distribution of the EVs were analyzed in unfractionated conditioned medium, centrifugation supernatant containing small EVs (sEVs), and centrifugation pellet containing large, microvesicle-like EVs (lEVs) by NTA (E), and the presence of tetraspanin EV markers was analyzed by flow cytometry (F) of both the EV pellet and centrifuge supernatant derived from muscle-invasive J82 cells. The parameters of the urine-derived EVs are presented as medians with 10th and 90th percentiles and were compared using the non-parametric Mann–Whitney U test. MFI, mean fluorescence intensity