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. 2012 May 1;130(9):2033-43.
doi: 10.1002/ijc.26217. Epub 2011 Aug 8.

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis

Simona Taverna  1 , Anna Flugy, Laura Saieva, Elise C Kohn, Alessandra Santoro, Serena Meraviglia, Giacomo De Leo, Riccardo Alessandro
Affiliations

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis

Simona Taverna et al. Int J Cancer. .

Abstract

Our study is designed to assess if exosomes released from chronic myelogenous leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM-1 and VCAM-1 cell adhesion molecules and interleukin-8 expression. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE-cadherin and β-catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization.

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Figures

Figure 1
Figure 1. LAMA84 exosome characterization
a Exosomes released by LAMA84 cells observed by scanning electron microscopy. b: Coomassie Blue staining of 30 µg of cell lysate (lane 1) and exosome lysate (lane 2) separated on 8% SDS gel. c: Exosomes released by LAMA84 cells and purified on a 30% sucrose/D2O cushion observed by scanning electron microscopy d: Detection of Hsc 70 and CD63 in 30 µg of exosomes purified after ultracentrifugation on 30% sucrose/D2O gradient (lane 1) and 30 µg of cell lysate (lane 2). e: Acetylcholinesterase assay. The activity of acetylcholinesterase, an exosome-specific protein marker, was determined in exosomes (10 µg) (▪), total cell lysates (10 µg) (♦), exosome-deprived Fbs (▲) and exosome-deprived conditioned medium (CM) (■) as negative control.
Figure 2
Figure 2. Exosome treatment modulates IL8, VCAM1 and ICAM1 mRNA expression in HUVEC cells
IL8 (a), VCAM1 (b) and ICAM1 (c) mRNA expression increased in a time- and dose-dependent (10, 20, 50 µg/ml) manner after addition of exosomes to endothelial cell monolayer. Exosome-deprived conditioned medium (CM-Ex) and low-serum medium were used as negative controls d: Immunoprecipitation assay with anti-VCAM1 antibody; top panel: HUVEC were incubated for 6h with low-serum medium (lane 1); 50 µg/ml LAMA84 exosomes (lane 2) or 10 ng/ml TNFα (lane 3); results indicate an increased amount of VCAM-1 in exosome-treated cells. Bottom panel: western blot anti-actin performed, as a loading control, on cell lysates before the immunoprecipitation step (starting material, St) e: Representative overlay histogram showing an increase of surface expression of VCAM 1 on HUVEC treated with 50 µg/ml of LAMA84 exosomes (solid line) compared untreated HUVEC, as control (dot line). f: VCAM1, ICAM1 and IL8 mRNA expression in HUVEC treated for 12h with low serum medium (Ctrl), Ctrl plus 10 µg/ml of a neutralizing anti-IL8 antibody, 50 µg/ml exosomes or 50 µg/ml exosomes plus 10 µg/ml of a neutralizing anti-IL8 antibody. Values are representative of three independent experiments. *p ≤ 0.05; **p ≤ 0.01.
Figure 3
Figure 3. Adhesion of LAMA84 cells to exosome-treated HUVEC monolayer
a: Adhesion of LAMA84 cells to endothelial cell monolayer treated for 6h with different amount of LAMA84 exosomes or with EGM, used as positive control. b: Cells (arrows) treated as in a and observed at contrast phase microscopy. c: Adhesion of LAMA84 cells to HUVEC treated with 50 µg/ml of LAMA84 exosomes, 50 µg/ml of exosomes plus antibodies anti actin (5 µg/ml), 10 ng/ml of recombinant IL8, 50 µg/ml of CML patients exosomes, EGM (as positive control), 50 µg/ml of exosomes plus neutralizing antibodies anti IL8 (5 µg/ml), 50 µg/ml of PBMC-exosomes in low serum medium and low serum medium (as negative control). Values are the mean ± SD of 5 fields in three independent experiments CTRL: control. *p ≤ 0.05; **p ≤ 0.01.
Figure 4
Figure 4. LAMA84 exosomes promote HUVEC migration
a: Confluent, scrape-wounded endothelial cell monolayer incubated with low serum medium (negative control), 50 µg/ml of LAMA84 exosomes, and EGM medium (positive control), for 3h. b: Percentage of closure of the wounded area measured after addition of different amount of exosomes. c: Effects of exosomes on endothelial cell migration as measured by Boyden chamber assay. Addition of exosomes (10, 20, 50 µg/ml) for 6h to the bottom wells of the chamber induced a dose-dependent increase of HUVEC migration. Values are the mean ± SD of 3 fields in three independent experiments *p ≤ 0.05; **p ≤ 0.01. d: 50 µg/ml of LAMA84 exosomes, 50 µg/ml of LAMA84 exosomes plus antibodies anti actin (5 µg/ml), 10 ng/ml of recombinant IL8, 50 µg/ml of CML patients exosomes, EGM (as positive control), 50 µg/ml of LAMA84 exosomes plus neutralizing antibodies anti IL8 (5 µg/ml), 50 µg/ml of PBMC-exosomes in low serum medium and low serum medium (as negative control) were added as chemoattractants to the bottom wells. Values are the mean ± SD of 3 fields in three independent experiments *p ≤ 0.05; **p ≤ 0.01.
Figure 5
Figure 5. Alteration of HUVEC monolayer after addition of LAMA84 exosomes
a: Analysis at confocal microscopy of VE Cadherin localization in HUVEC cells treated with LAMA84 exosomes revealed a decrease of immunostaining compared to untreated cells (control). b: Decrease of immunostaining for β catenin in cell membranes was revealed after 6h incubation of HUVEC with 50 µg/ml of LAMA84 exosomes compared to control cells (section at 2 µm from the cell surface). c: figure shows the translocation of β catenin in the cytoplasm and nucleus compared to control (section at 4 µm from the cell surface). d: modification of cytoskeletal structures as observed with actin localization in HUVEC monolayer treated with 50 µg/ml of exosomes compared to control cells. These fields are representative of three independent experiments. Scale bar = 10 µm. e: Semi-quantitative analysis of VE-Cadherin fluorescence intensity in the plasma membrane of HUVEC treated with 50 µg/ml of LAMA84 exosomes compared to control cells. f: Semi-quantitative analysis of β-Catenin fluorescence intensity in the plasma membrane and nuclei of HUVEC treated with 50 µg/ml of LAMA84 exosomes compared to control cells.
Figure 6
Figure 6. LAMA84 exosomes stimulate in vitro and in vivo angiogenesis
Panel a: Phase contrast micrographs showing that exosomes induce an endothelial network formation on matrigel. No tube formation is observed when HUVEC are plated in low-serum medium (upper left panel) or in the presence of 50 µg/ml of exosomes plus neutralizing antibody against IL8 (upper right panel); the addition to HUVEC cells of 50 µg/ml of LAMA84 exosomes (lower right panel) or 50 µg/ml of exosomes plus a non specific antibody against actin (lower left panel) caused the formation of capillary-like structures. Panel b: Measurement of the cables length by ImageJ software. Panel c: Matrigel plug containing LAMA84 exosomes stimulate angiogenesis in nude mice. Ctrl: Negative control (Matrigel plus PBS), Ex + Ab nIL8: LAMA84 exosomes (100 µg) plus 10 µg/ml of an antibody neutralizing anti-IL8, Ex + Ab Actin: LAMA84 exosomes (100 µg) plus 10 µg/ml of a non specific antibody against actin, Ex LAMA84: LAMA84 exosomes (100 µg). Panel d: Western blot analysis of pMAPK and MAPK in HUVEC treated with 50 µg of LAMA 84 exosomes. HUVEC were starvated for 3h with serum free medium and then treated with 50 µg of LAMA 84 exosomes for 15 min (lane 3) and 30 min (lane 4), or with low serum medium alone for 15 min (lane 1) and 30 min (lane 2) as control.
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