Figure 6
CLL exosomes promote cell proliferation, remodeling of the actin cytoskeleton, cell migration, and angiogenesis in vitro and in vivo. (A) Proliferation index of stromal cells after 96 hours of incubation with increasing concentrations of CLL exosomes (MEC-1) assessed using CCK8 assay. Data are reported as fold change (FC) of Ctrl (n = 4). *P < .05, **P < .01, ***P < .001. (B) Microscopy images of wound healing assay showing closure of the scratch when HS-5 or HMEC-1 cells were cultured in the absence (Ctrl) or presence (Exo) of 50 or 100 µg/mL CLL exosomes (MEC-1) in serum-free medium for 18 hours (left). Scale bar, 100 µm. Wound closure (×104 µm2) was quantified from images using WimScratch (Wimasis; n = 4) (right). **P < .01. (C) Representative images of immunofluorescence staining of α-SMA (green) in primary BM-MSCs untreated (Ctrl) or treated (Exo) for 15 days with exosomes produced by healthy donor B cells (B), lymphoblastoid cell line (ST-EAH), or primary CLL cells, captured by confocal microscopy (nucleus stained with DAPI, blue). Scale bar, 50 µm. (D) Representative images of immunohistochemistry staining of α-SMA in paraffin-embedded sections of human lymph nodes from 2 healthy individuals or 2 CLL patients (representative of n = 5). Scale bar, 50 µm. (E) (Upper) C57BL/6 mouse aorta pieces were incubated in vitro in the absence (Ctrl) or presence (Exo) of 100 µg/mL CLL exosomes (MEC-1) for 7 days. Representative microscopy images are shown. Scale bar, 100 µm. (Lower) Quantification of sprouts area and length using WimSprout (Wimasis; n = 4 replicates). **P < .01. (F) (Left) HMEC-1 cells untreated (Ctrl) or treated for 30 minutes with 50 or 100 µg/mL CLL exosomes (MEC-1; Exo) and then seeded on Matrigel for 3 hours. Scale bar, 100 µm. (Right) Quantification of several parameters of the tube formation assay using WimTube (Wimasis; n = 4). *P < .05, **P < .01, ***P < .001. (G) Matrigel plug assay performed by subcutaneous injection of Matrigel mixed with PBS (Ctrl) or 100 µg of CLL exosomes (MEC-1; Exo) in 5 NSG mice. rhIL-8 was used as positive control. (Left) Images depict surgically removed Matrigel plugs after 14 days. Scale bar, 5 mm. (Right) Quantification of hemoglobin content in Matrigel plugs using Drabkin reagent. Data are reported as FC of Ctrl (n = 5). ***P < .001.

CLL exosomes promote cell proliferation, remodeling of the actin cytoskeleton, cell migration, and angiogenesis in vitro and in vivo. (A) Proliferation index of stromal cells after 96 hours of incubation with increasing concentrations of CLL exosomes (MEC-1) assessed using CCK8 assay. Data are reported as fold change (FC) of Ctrl (n = 4). *P < .05, **P < .01, ***P < .001. (B) Microscopy images of wound healing assay showing closure of the scratch when HS-5 or HMEC-1 cells were cultured in the absence (Ctrl) or presence (Exo) of 50 or 100 µg/mL CLL exosomes (MEC-1) in serum-free medium for 18 hours (left). Scale bar, 100 µm. Wound closure (×104 µm2) was quantified from images using WimScratch (Wimasis; n = 4) (right). **P < .01. (C) Representative images of immunofluorescence staining of α-SMA (green) in primary BM-MSCs untreated (Ctrl) or treated (Exo) for 15 days with exosomes produced by healthy donor B cells (B), lymphoblastoid cell line (ST-EAH), or primary CLL cells, captured by confocal microscopy (nucleus stained with DAPI, blue). Scale bar, 50 µm. (D) Representative images of immunohistochemistry staining of α-SMA in paraffin-embedded sections of human lymph nodes from 2 healthy individuals or 2 CLL patients (representative of n = 5). Scale bar, 50 µm. (E) (Upper) C57BL/6 mouse aorta pieces were incubated in vitro in the absence (Ctrl) or presence (Exo) of 100 µg/mL CLL exosomes (MEC-1) for 7 days. Representative microscopy images are shown. Scale bar, 100 µm. (Lower) Quantification of sprouts area and length using WimSprout (Wimasis; n = 4 replicates). **P < .01. (F) (Left) HMEC-1 cells untreated (Ctrl) or treated for 30 minutes with 50 or 100 µg/mL CLL exosomes (MEC-1; Exo) and then seeded on Matrigel for 3 hours. Scale bar, 100 µm. (Right) Quantification of several parameters of the tube formation assay using WimTube (Wimasis; n = 4). *P < .05, **P < .01, ***P < .001. (G) Matrigel plug assay performed by subcutaneous injection of Matrigel mixed with PBS (Ctrl) or 100 µg of CLL exosomes (MEC-1; Exo) in 5 NSG mice. rhIL-8 was used as positive control. (Left) Images depict surgically removed Matrigel plugs after 14 days. Scale bar, 5 mm. (Right) Quantification of hemoglobin content in Matrigel plugs using Drabkin reagent. Data are reported as FC of Ctrl (n = 5). ***P < .001.

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