CLL cells secrete exosomes that rapidly enter stromal cells in culture. (A) A 24-hour coculture assay of primary CLL cells (1 × 10⁶ in upper compartment) and BM-MSCs (2 × 10⁴) was established in 6-well plates containing 0.4-µm pore inserts. BM-MSCs were cultured in the absence (Ctrl) or presence (+CLL) of primary PKH67-labeled CLL cells (green). Images were captured by fluorescence confocal microscopy. Nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI) (blue). Representative image of n = 6 experiments. Scale bar, 20 µm. (B) Electron microscopy image of purified CLL exosomes. Scale bar, 100 nm. (C) Western blot analysis of the fractions collected after sucrose density gradient of the 110 000g pellets obtained using ultracentrifugation of CLL cell supernatants. Positive control was CLL cell lysate. (D) Size analysis of CLL exosomes using tunable resistive pulse sensing (TRPS)-based analysis (qNano). (E) The BM-derived stromal cell line HS-5 and the endothelial cell line HMEC-1 were incubated with 50 µg/mL PKH67-labeled CLL exosomes (MEC-1) for the indicated times before 4 washes and fixation. (Upper) Exosome uptake was followed by fluorescence confocal microscopy. Scale bar, 20 µm. (Lower) Quantification of exosome uptake by ImageJ software. Data are presented as fold change relative to 0 hours (n = 3). (F) HS-5 and HMEC-1 cells were incubated for 4 hours in the absence (Ctrl) or presence of 20 µg/mL PKH67-labeled CLL exosomes (MEC-1) untreated (Exo) or pretreated for 30 minutes with 10 ng/mL heparin (Exo + H). (Left) Images were captured by fluorescence confocal microscopy. Scale bar, 50 µm. (Right) Quantification of exosome uptake by ImageJ software. Data are presented as fold change relative to Ctrl (n = 3). **P < .01, ***P < .001.