Fig 2: Secretion of extracellular vesicles is elevated in disease-associated stromal cells. (a) Patient-matched normal and tumour-associated stromal cells, at passage 6, were seeded onto imaging chamber slides, and when 80% confluent subject to fixation and immuno-fluorescent labelling for the specified markers (green). DAPI was used to identify the nucleus (blue) (Scale Bar = 100 µm). (b) Secreted EVs were isolated from stromal cell conditioned media by ultracentrifugation and compared to whole cell lysates by western blot. 20 µg of cell or EV lysates were loaded per lane. Membranes were probed using antibodies against ALIX, TSG101, Vimentin, or GAPDH. (c) Stromal cell-derived EVs were immobilised onto plates and immunofluorescently labelled with antibodies against CD9, CD81, and CD63 (bars show mean ± SE, n = 3). (d) EVs isolated from normal or tumour-associated stromal cell-conditioned media were visualised by cryo-electron microscopy (Scale Bar = 100 nm). Panels (a–d) represent contiguous data from patient WCB949 and is representative of six patient samples. (e) Size distribution of particles within preparations of stromal cell-derived EVs, isolated by ultracentrifugation, was assessed by nanoparticle tracking analysis (data from patient WCB955 is shown, and is representative of six patient samples). (f) Subsequent assessment of concentration of particles within stromal EV isolates was assessed by nanoparticle tracking analysis and normalised based on cell number (mean ± SE, n = 6)

Fig 3: EV isolation and characterization. (A) Workflow of the study. (B) Western blot of CD63, ALIX, TSG101 and calnexin in LNCaP prostate cancer cells and pre-operation urinary and plasma EVs from three different patients. (C) Representative TEM image of urinary EVs. (D) Representative TEM image of plasma EVs. (E) Paired dot plots showing the numbers of EVs per ml of urine before (PreOp) and after radical prostatectomy (PostOp). (F) Paired dot plots showing the numbers of EVs per ml of plasma before and after radical prostatectomy. Wilcoxon matched-paired signed rank test was used to assess the statistical significance of the differences between groups.