EVs are internalized in vivo and modify the bone marrow microenvironment. (A) HEK293 cells were transfected with luciferase under NF-κB promotor for 24 hours. A total of 2 × 106 cells per mouse were injected s.c. after 30 minutes, EVCONT or EVL265P (150 μg per mouse) were injected IV and incubated for 24 hours. Luciferin was added and, after 10 to 15 minutes, luminescence was measured. PKH67-labeled EVs (100 μg) were injected into bone marrow. After 16 hours, uptake of EVs by bone marrow cells was measured (B). EVCONT or EVMWCL (150 μg) were injected into bone marrow. After 8 days, blood was taken to measure albumin, total protein, and globulin (C-D) concentrations and blood smears (E) for detection of rouleaux formation (red arrow) were prepared. (F) Tissue sections from femurs were prepared and stained for the presence of mast cells (CD40L) (blue). Original magnification ×630 for panels E-F. MVs and exosomes were isolated from bone marrow aspirates of untreated WM patients. MVs, exosomes, serum, and BCWM.1 cell lysate as positive control were used for MyD88 (G) and IRAK4 (H) detection by WB. GAPDH was used as a positive control for EVs and cell lysate.
