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. 2010 Mar 4;115(9):1755-64.
doi: 10.1182/blood-2009-09-242719. Epub 2009 Dec 17.

Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression

Asish K Ghosh  1 , Charla R Secreto, Traci R Knox, Wei Ding, Debabrata Mukhopadhyay, Neil E Kay
Affiliations

Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression

Asish K Ghosh et al. Blood. .

Abstract

Microvesicles (MVs) released by malignant cancer cells constitute an important part of the tumor microenvironment. They can transfer various messages to target cells and may be critical to disease progression. Here, we demonstrate that MVs circulating in plasma of B-cell chronic lymphocytic leukemia (CLL) patients exhibit a phenotypic shift from predominantly platelet derived in early stage to leukemic B-cell derived at advanced stage. Furthermore, the total MV level in CLL was significantly greater compared with healthy subjects. To understand the functional implication, we examined whether MVs can interact and modulate CLL bone marrow stromal cells (BMSCs) known to provide a "homing and nurturing" environment for CLL B cells. We found that CLL-MV can activate the AKT/mammalian target of rapamycin/p70S6K/hypoxia-inducible factor-1alpha axis in CLL-BMSCs with production of vascular endothelial growth factor, a survival factor for CLL B cells. Moreover, MV-mediated AKT activation led to modulation of the beta-catenin pathway and increased expression of cyclin D1 and c-myc in BMSCs. We found MV delivered phospho-receptor tyrosine kinase Axl directly to the BMSCs in association with AKT activation. This study demonstrates the existence of separate MV phenotypes during leukemic disease progression and underscores the important role of MVs in activation of the tumor microenvironment.

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Figures

Figure 1
Figure 1
Identification and characterization of MVs. (A) MVs were identified by evaluating their ability to bind annexin V by flow cytometry. Annexin positivity is shown by histogram from 2 representative MV preparations from CLL plasma. (B) Heterogeneity and sizes of the MVs were determined by electron microscopy after negative membrane staining with phosphotungstic acid (magnification level is indicated by the horizontal bar). (C) Plasma levels of MVs were determined by measuring the total protein content and presented as micrograms of MV per milliliter of plasma (isolated from CLL patients [n = 58] or healthy human subjects [n = 5]). (D-E) MVs isolated from CLL patients at various disease stages as indicated were phenotypically characterized by the use of CD61 for platelet-derived or CD19 for B lymphocyte–derived cell-surface marker by flow cytometry. Results from individual patients are presented with mean values.
Figure 2
Figure 2
MVs can integrate into various target cells. (A) Freshly isolated primary CLL B cells were incubated with MVs isolated from different CLL patients for 48 hours. Transfer of CD61 marker from MV to CLL B cells was analyzed by flow cytometry. (B) Purified MVs were labeled with the membrane dye PKH67 (green) and incubated with CLL-BMSCs for the indicated periods. Binding and internalization of MVs into BMSCs was visualized after staining with DAPI (nuclear stain) under confocal microscope (magnification level is indicated by the horizontal bar). (C) MV-mediated transfer of message into BMSCs was examined by Western blot after a 24-hour incubation by the use of an antibody to CD31. Expression of CD31 on MVs was verified by flow cytometry.
Figure 3
Figure 3
MVs activate human bone marrow stromal cells. (A) MVs isolated from different CLL patients' plasma exhibiting variable degrees of AKT activation in HS-5 cell line as determined by densitometric analysis of the results from Western blot. (B-D) MVs activate primary BMSCs isolated from CLL or healthy human subjects as analyzed by Western blot. A sustained level of AKT activation is detected at least up to 24 hours after incubation with MVs.
Figure 4
Figure 4
MVs activate the AKT/p70S6K/HIF-1α signaling axis in BMSCs. CLL or healthy BMSCs (“normal”) were exposed to MVs with or without presence of wortmannin or rapamycin for 24 hours. BMSC lysates were analyzed for the activation of the AKT/p70S6K signaling pathway as shown by Western blots by the use of specific antibodies. Expression of HIF-1α, a downstream target of AKT/p70S6K axis, also is shown. Actin was used as loading control. MV-mediated modulation of the AKT/p70S6K/HIF-1α axis is shown for better understanding (right).
Figure 5
Figure 5
MVs induce expression of VEGF in CLL-BMSCs. (A) Production of total VEGF in the conditioned media of BMSCs stimulated with MVs as described previously was measured by ELISA and mean values are presented with SDs (pg/mL per 105 cells). *Statistical significance (P < .001) compared with the unstimulated controls. (B) CLL-BMSCs were exposed to MVs with or without presence of wortmannin or rapamycin for 24 hours. Cell lysates were analyzed for the expression of different isoforms of VEGF by Western blot by the use of an antibody to human VEGF. Results indicate that MVs induce expression predominantly of the VEGF165 isoform in CLL-BMSC. Actin was used as loading control. (C) Production of the antiangiogenic isoform VEGF165b in the aforementioned (B) conditioned media of BMSCs stimulated with MVs was measured by ELISA and mean values are presented (pg/mL per 105 cells).
Figure 6
Figure 6
MVs modulate GSK3β/β-catenin signaling in BMSCs. (A) The BMSC lysates stimulated with MVs described in Figure 4A were analyzed for AKT-mediated phosphorylation of GSK3β and β-catenin at Ser552 by Western blot by the use of specific antibodies. Phosphorylation of AKT is also shown for comparison. (B) Translocation of β-catenin was visualized in BMSCs stimulated with MVs from CLL patients (MV1 and MV2) by the use of a specific antibody to β-catenin and nuclear stain, DAPI under confocal microscope (magnification level is indicated by the horizontal bar). (C) AKT-mediated phosphorylation of β-catenin at Ser552 and GSK3β was further examined in BMSCs stimulated with MVs for 24 or 48 hours by Western blotting. Expression of cyclin D1 and c-myc, downstream targets of GSK3β/β-catenin, was shown in BMSCs after stimulation with MVs (MV22 and MV23) compared with the unstimulated controls in Western blot analysis.
Figure 7
Figure 7
MVs activate RTKs in BMSCs. (A) Activation of RTKs in BMSCs stimulated with MVs obtained from CLL patients at Rai stages 0 (MV1), I (MV2), or II (MV3) was analyzed on human phospho-RTK antibody array blots and the level of activation compared with the unstimulated control BMSCs. Activation of various RTKs was analyzed and presented as fold-activation by bar diagrams. (B) Lysates from BMSCs stimulated with MVs after treatment with the Axl-inhibitor, SKI-606 (SKI) or anti-VEGF neutralizing antibody (NAB) or left unstimulated or untreated were examined for the phosphorylation of AKT by Western blot. Treatment with SKI-606 showed substantial inhibition of MV-mediated activation of AKT; however, anti-VEGF antibody did not show any effect. Phosphorylation of Axl is also shown for comparison. Actin was used as loading control. (C) MVs obtained from CLL patients at various Rai stages (0, I, II) were analyzed for the presence of constitutively active Axl by Western blot with the use of a phospho-specific antibody to Axl. Actin was used as loading control.
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