Eph-B2/Ephrin-B2 Interaction Plays a Major Role in the Adhesion and Proliferation of Waldenstrom's Macroglobulinemia

Receptor tyrosine kinases (RTK) are high affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones (1). RTKs are key regulators of normal cellular processes and in the development and progression of many types of cancer (2). The binding of growth factor to the extracellular domain of RTKs activates the receptor and induces a cell signaling cascade that promotes cell survival and proliferation (1, 2). Ephrin receptors (Eph) represent the largest family of RTKs, including 16 members divided into 2 classes; Eph-A and Eph-B. The classification is based on the affinity of the receptor to different ligands, ephrin-A and ephrin-B (3–5). A-type ephrins are tethered to the cell membrane by a glycosylphosphatidylinositol anchor, whereas B-type receptors have a transmembrane domain that is followed by a short cytoplasmic region (3).

Although used extensively throughout embryogenesis and development, Eph receptors are rarely detected in adult tissues. Eph receptors and ephrin ligand serve as a guidance system to position cells and modulate cell morphology, especially in embryogenesis (6, 7). In adult organisms, Eph/ephrin interactions can also trigger a wide array of cellular responses, including cell boundary formation, motility, adhesion, and egress (8, 9). Elevated levels of expression of both Eph A and B receptors were found in a wide range of cancers, including melanoma, breast, prostate, pancreatic, gastric, and esophageal and colon cancer (10). Moreover, increased Eph expression correlates with more malignant and metastatic tumors, consistent with a role of ephrin in governing cell movement (11).

Waldenstrom's macroglobulinemia (WM) is a low-grade non-Hodgkin lymphoma, characterized by the presence of abnormal lymphoplasmacytic cells producing high levels of immunoglobulin M (IgM) macroglobulins (12). WM is characterized by widespread involvement of the bone marrow, which provides a protective environment for the survival and proliferation of these cells (13). The specific tropism of these cells to the bone marrow niches indicates their dependence on adhesion and critical interaction with the bone marrow microenvironment (14). Inhibiting the adhesion of WM cells to the bone marrow microenvironment reduces survival and proliferation of the tumor cells (15). Direct cell–cell interaction or stimulation through cytokines secreted by endothelial cells has been shown to regulate tumor proliferation in multiple myeloma (16–18). However, the survival effect of these interactions is poorly understood in WM.

In this study, we aimed to characterize the role of RTKs in the interaction of WM cells with the bone marrow microenvironment. We focused on the role of Eph-B2 receptor, which was found to be overexpressed in WM cells, and its interaction with ephrin in the bone marrow microenvironment, including on endothelial cells and bone marrow stromal cells (BMSC). Moreover, we tested the effect of inhibiting the Eph/ephrin interaction in WM cells and endothelial cells through loss of function experiments and blocking antibodies. We showed that Eph/ephrin interaction regulates cell adhesion, proliferation, and cell-cycle progression and tumor progression in WM.

Recombinant ephrin-B2, recombinant SDF1α, Human phospho-RTK Array Kit and anti-ephrin-B2 antibody were purchased from R&D. Anti–Eph-B2 antibody was purchased from Santa-Cruz Biotechnology, Inc. All monoclonal antibodies for Western blotting were purchased from Cell Signaling Technologies. Short interfering RNA (siRNA) for the knockdown of Eph-B2 was obtained from Dermacon.

BCWM1, an IgM secreting lymphoplasmacytic cell line obtained from a patient with WM, and other IgM-secreting cell lines (MEC1, RL) were used in this study. The BCWM1 was a kind gift from Dr. Treon (Dana-Farber Cancer Institute, Boston, MA). MEC1 was a kind gift from Dr. Neil Kay (Mayo Clinic, Rochester, MN). RL was purchased from the American Tissue Culture Collection. All cell lines were cultured in RPMI-1640 containing 10% FBS (Sigma Chemical), 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). The human umbilical vein endothelial cell (HUVEC) lines were purchased from Cambrex, and cultured in EGM-2 MV media (Cambrex), reconstituted according to the manufacturer's instructions.

WM patient samples were obtained after approval from the Dana-Farber Cancer Institute Institutional Review Board. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. Primary WM cells were obtained from bone marrow samples using CD19⁺ micro-bead selection (Miltenyi Biotec) with more than 90% purity as confirmed by flow cytometric analysis.

For isolation of BMSCs, bone marrow specimens were obtained from patients with WM; mononuclear cells were separated by Ficoll-Hypaque density sedimentation and were used to establish long-term BMSCs cultures. Mononuclear cells were suspended in 10 mL of growth medium containing Dulbecco's Modified Eagle's Medium, 20% fetal calf serum, Pen (100 U/mL), and Strep (100 pg/mL) in 25-cm² flasks. Cells were incubated at 37°C, for 3 to 4 weeks, and when an adherent cell monolayer had developed with predominantly fibroblast morphology, cells were harvested using trypsin/EDTA and used as BMSCs.

Forty-two RTKs were screened using phosphor-RTK array kit following the manufacturer's recommendations (R&D). Briefly, cell lysates were diluted with the assay diluents and applied to capture antibodies against the different RTKs spotted in duplicate on nitrocellulose membranes. After binding the extracellular domain of both phosphorylated and unphosphorylated RTKs, unbound material was washed, and a pan anti–phospho-tyrosine antibody conjugated to horseradish peroxidase was then used to detect phosphorylated tyrosine on activated receptors by chemiluminescence. Quantification of signal intensity was done with ImageJ software (NIH, Bethesda, MD).

The expression of Eph-B2 was detected in specimens from bone marrow aspirates from 6 normal subjects and 6 WM patients. Specimens were rinsed with PBS, fixed with 4% formaldehyde in PBS, dehydrated with ethanol, embedded in paraffin blocks, and sectioned. Sections were stained with rabbit antihuman Eph-B2.

To test the effect of ephrin-B2 on cytoskeletal signaling in WM, BCWM cells (5 × 10⁶) were serum starved for 3 hours and then stimulated with ephrin-B2 (100 ng/mL) for 0, 5, 10, 15, 30, and 60 minutes. In addition, cells were activated with different concentrations of ephrin-B2 (0, 50, 100, 250, 500, and 1,000 ng/mL) for 15 minutes.

We tested the effect of treating BCWM.1 cells with anti–Eph-B2 (5 μg/mL, 1 hour for cytoskeletal signaling or 12 hours for cell-cycle signaling). HUVECs were treated with anti–ephrin-B2 (5 μg/mL, 1 hour for cytoskeletal signaling or 12 hours for cell-cycle signaling) or the combination of both anti-Eph B2 and anti–ephrin-B2. In the experiments involving coculture, HUVECs were cultured for 24 hours before the experiment in 6-well plates at 5 × 10⁴ cells per well, washed with PBS, and treated with the anti–ephrin-B2, and WM cells were then applied for 1 hour. After coculture, WM cells were separated from HUVECs using gentle pipetting. Nontreated WM cells cultured with or without HUVECs served as control for WM cells in the coculture; similarly nontreated HUVECs were used as control for HUVECs in the coculture.

BCWM1 or HUVECs were washed with ice-cold PBS, lysed, and protein concentration was normalized. Proteins were blotted using 8% to 12% acrylamide gels, transferred to a nitrocellulose membrane; membranes were blocked with 5% nonfat dry milk in TBS/T buffer and incubated with primary antibodies for p-FAK (focal adhesion kinase), p-Src, p-Paxillin, pP130, p-cofilin, p-Akt, cyclin D3, cyclin E, p-Rb, or α-tubulin overnight at 4°C. The membranes were then washed, incubated with appropriate horseradish peroxidase-conjugated secondary antibody, washed, and developed using luminol base assay. Luminescence was measured using X-ray films.

BCWM1 cells were cultured in the presence of increasing concentrations (0, 50, 100, 500, 1,000 ng/mL) of ephrin-B2 in for 15 minutes, lysed, and lysates were precleared by incubating with protein G-agarose beads slurry for 30 minutes at 4°C, and beads were removed by centrifugation. Supernatant were incubated with anti–Eph-B2 antibody with gentle rocking overnight at 4°C. Protein G agarose beads slurry was added and incubated for 3 hours with gentle rocking for 1 to 3 hours at 4°C. Beads were then collected by centrifugation, washed, and resuspended in 20 μL 3× SDS. The samples were heated to 95°C to 100°C for 5 minutes and samples were then loaded to SDS-PAGE gel (8%) and immunoblotted as described above with pan-anti-p-Tyrosine antibody.

Nontissue culture 96-well plates were incubated over night at 4°C with 50 μL per well of increasing concentrations (0, 50, 100, 500, 1,000 ng/mL) of ephrin-B2. Then wells were washed with PBS, blocked with bovine serum albumin (BSA; 2 μg/mL) for 1 hour at room temperature and washed with PBS. BCWM1, MEC1, RL, or CD19⁺ cells were starved for 3 hours, labeled with calcein-AM (1 μg/mL), washed and added to the plates (10⁵ cells per well) for 1 hour at 37°C. Nonadherent cells were then washed, and adherent cells were detected by measuring fluorescence intensity in the wells using a fluorometer (Ex/Em = 485/520 nm).

To test the role of ephrin-B2/Eph-B2 in adhesion to fibronectin, WM cells were stained with calcein-AM, washed, treated with increasing doses of ephrin-B2 (0, 50, 100, 500, and 1,000 nmol/L) for 15 minutes, and applied on a fibronectin-coated plates (10⁵ cells per well) for 1 hour at 37°C. Nonadherent cells were then washed, and adherent cells were detected by measuring fluorescence intensity in the wells using a fluorometer (Ex/Em = 485/520 nm).

BCWM1 cells were starved 3 hours before migration, applied to the upper chamber of an 8-micron pore filters for trans-well migration assay (Costar), and left to migrate in the absence or presence of increasing concentrations (0, 50, 100, 500, 1,000 ng/mL) of ephrin-B2, in presence or absence of 30 nmol/L SDF1 in the lower chamber for 4 hours at 37°C. Cells that migrated to the lower chambers were counted using flow cytometry.

BCWM1 cells were treated with increasing concentrations of ephrin-B2, fixed in 2% formaldehyde for 15 minutes at room temperature, permeabilized with 0.2% saponin, and stained with (5 μg/mL) phalloidin tagged with either Alexa Fuor 488. Cells were analyzed by flow cytometry, or were spun onto slides, mounted, and analyzed by confocal microscopy.

Negative fractions of bone marrow aspirates from WM patients and HUVECs were treated with mouse antihuman eprine-B2 antibody or isotype control, followed by a secondary fluorescein isothiocyanate (FITC)–goat–antimouse antibody. Bone marrow aspirates cells were then treated with PE–anti-CD31 antibody to gate the endothelial cells population. The expression of ephrin-B2 was measured by flow cytometry as fluorescence intensity of FITC. BMSCs were trypsinized, washed, and stained with mouse antihuman ephrin-B2 antibody or isotype control, followed by a secondary FITC–goat–antimouse antibody.

A confluent monolayer of HUVECs or BMSCs was generated by plating 1 × 10⁴ cells per well in 96-well plates overnight. The next day, HUVECs or BMSCs were treated with 0 or 5 μg/mL anti–ephrin-B2 for 1 hour. BCWM1 or MEC1 cells (1 × 10⁶ cells/mL) were serum starved for 3 hours, prelabeled with calcein-AM, treated with anti–Eph-B2 (5 μg/mL) or with ephrin-B2 (100 ng/mL), and then added to the HUVECs or BMSCs. Cells were cocultured for 1 hour at 37°C and nonadherent cells were washed. Adherent cells were detected by measuring the fluorescence intensity in the wells using a fluorometer (Ex/Em = 485/520 nm).

The effect of ephrin-B2 on the proliferation of WM cells was tested by treating BCWM cells with increasing concentrations of ephrin-B2 for 24 hours, and cell proliferation was detected using ³H-thymidine uptake or bromodeoxyuridine assay as previously described (19). To test the role of ephrin-B2 and Eph-B2 on WM cells and HUVECs proliferation, when cultured alone or with endothelial cells, a confluent monolayer of HUVECs in 96-well plate wells was obtained by seeding 1 × 10⁴ cells per well overnight. The next day, BCWM cells (3 × 10³) were cultured with or without HUVECs for 24 hours. In some cases HUVECs were treated with anti–ephrin-B2, BCWM cells were treated with anti–Eph-B2 or the combination of both.

WM cells were fixed with 70% ethanol, washed, RNA was degraded by RNAase, DNA was stained with 5 μg/mL propidium iodide (Sigma-Aldrich), and cells were analyzed by flow cytometry. For studies using coculture of WM cells with HUVECs, a confluent monolayer of HUVECs in 24-well plate wells was obtained by seeding 5 × 10⁴ cells per well overnight. The next day, BCWM cells (1 × 10⁶) were cultured with or without HUVECs for 24 hours. In some experiments, HUVECs were treated with anti–ephrin-B2, BCWM cells were treated with anti–Eph-B2 or the combination of both.

BCWM1 cells were cultured in OPTI-MEM media overnight in 6-well plates, washed, and a mixture of Lipofectamine 2000 (5 μL) with scramble-siRNA or anti–Eph-B2 siRNA (100 pmol) in a final volume of 2 mL of OPTI-MEM was added to each well. Transfection solution was replaced with complete media after 24 hours and cells were used after 48 hours.

Quantitative reverse transcriptase PCR (qRT-PCR) for expression of Eph-B2 was done on an Applied Biosystems AB7500 Real Time PCR system as described previously (20). All PCR reactions were run in triplicate, and mRNA expression was expressed relative to glyceraldehyde-3-phosphate dehydrogenase.

BCWM1 cells transfected with scramble siRNA or Eph-B2 siRNA were injected to severe combined immunodeficient (SCID)/bg mice (age 7–9 weeks; n = 3 per group); 3 × 10⁶ cells per mouse, by intravenous tail-vein injection. To determine early differences in tumor growth patterns and to confirm that the cells did not loose siRNA inhibition, the mice were sacrificed after 1 week (21, 22). The bone marrow from 2 femurs of each mouse was extracted. Red blood cells were lysed, and the mononuclear cells were used for flow cytometry analysis. Cells were resuspended in blocking solution (2% BSA in PBS) for 30 minutes on ice, cells were then incubated with mouse-antihuman-CD19 antibody conjugated to Alexa Fluor 488 (5 μg/mL), and rabbit anti–Eph-B2 followed by incubation in Alexa 633 conjugate of secondary antirabbit antibody. Cells were then washed 2 times with blocking solution, and the CD19⁺ cells were analyzed for the expression of Alexa-633-Eph-B2 using BD Canto II flow cytometer.

The role of Eph-B2/ephrin-B2 interaction in the angiogenesis was determined using an In Vitro Angiogenesis Assay Kit (Chemicon). HUVECs were cultured in the presence or absence of BCWM1 cells, and presence or absence of anti–EpH-B2 and anti-ephrin blocking antibodies on polymerized matrix gel at 37°C. After 8 hours, any tube formation by endothelial cells was evaluated using Nikon inverted TE2000 microscope, and a 20× Plan-fluor DIC NA0.5 objective. Images were processed with Adobe Photoshop 7.0 software (Adobe). Tube formation was calculated as the total length of the tubes per frame and normalized to nontreated HUVECs. Moreover, the number of WM cells adherent to the tubes was calculated, divided by the total length of tubes per frame, and normalized to nontreated coculture of BCWM1 cells and HUVECs.

Results were reported as the mean ± SD for experiments done in 3 replicates samples and were compared by the Student t test. Results were considered significantly different for P values less than 0.05.

We first examined the expression and phosphorylation of different RTKs in primary WM samples and cell lines using an antibody-based RTK-array. As shown in Fig. 1A and B, there was activation of RTKs in CD19⁺ cells from healthy donors and from WM patient samples. Eph-B2 was phosphorylated in WM cell lines, BCWM.1, and IgM secreting cell lines RL and MEC1. Figure 1A shows quantification of the expression of Eph-B2 in the different groups, showing that Eph-B2 receptor was activated in all patient samples and cell lines compared with control, with a 5-fold increase in CD19⁺ primary WM cells. Figure 1B shows representative images of the membranes. These results were confirmed by immunohistochemical detection on primary normal subjects and WM patient bone marrow biopsies that showed expression of Eph-B2 in WM cells (Fig. 1C). The RTK array included other members of the Eph family, including Eph-A1, Eph-A2, Eph-A3, Eph-A4, Eph-A6, Eph-A7, Eph-B1, Eph-B2, Eph-B4, and Eph-B6. The only member that was highly expressed on WM cells and BCWM1 cell line compared with normal control was Eph-B2.

We next examined the activity of Ephrin-B2/Eph-B2 on adhesion WM cells. To test the effect of ephrin-B2 on adhesion, we coated adhesion plates with increasing concentrations of ephrin-B2 and tested the adhesion of starved WM cell lines and IgM secreting cell lines to these adhesion plates. We found that ephrin-B2 increased adhesion of WM cell lines (Fig. 1D) and primary cells (Fig. 1E), with a maximal increase of 150% to 170% compared with control in plates coated with 100 ng/mL of ephrin-B2.

Furthermore, the treatment of starved WM cells with recombinant ephrin-B2 (the ligand of Eph-B2) activated cell adhesion signaling. Activation of WM cells with ephrin-B2 100 ng/mL showed that peak activation of Eph and downstream signaling was achieved at 15 minutes. This was evidenced by increased phosphorylation of FAK and Src (Fig. 1F). We further examined different concentrations of ephrin-B2 on activation of adhesion-related proteins at the 15-minute time point and showed that ephrin-B2 induced maximum activation at 100 ng/mL, evidenced by increased phosphorylation of Eph-B2, FAK, Src, P130, paxillin, and cofilin, without changing the total Eph-B2 (Fig. 1G).

When we tested the effect of activation with ephrin-B2 on adhesion to fibronectin, we found that activation of WM cells with increasing concentrations of ephrin-B2 (0, 50, 100, 500, 1,000 nmol/L) did not change the adhesion of WM cells to fibronectin (Fig. 1H).

We next determined the functional effects of Eph-B2 on migration of WM cells. First, we tested the effect of ephrin-B2 on the chemotaxis of BCWM1 cells in response to SDF1. Figure 2A shows that ephrin-B2 did not change the chemotactic response of BCWM1 cells to SDF1. However, Fig. 2B shows that increasing concentrations of ephrin-B2 significantly decreased spontaneous chemotaxis of WM cells. This is in agreement with the decrease in phosphorylation of myosin-light-chain (pMLC), which is critical for cell movement (Fig. 2C). In addition, we have tested actin polymerization in BCWM cells after treatment with increasing concentration of ephrin-B2. Figure 2D shows that Ephrin-B2 significantly decreased actin polymerization as quantified by flow cytometry analysis. Figure 2E shows a confirmation of the decrease of actin polymerization in response to ephrin-B2 using confocal imaging.

Prior studies have shown that ephrin-B2 is highly expressed on endothelial cells (23). In addition, angiogenesis and the interaction of WM cells with endothelial cells in the bone marrow is critical for tumor proliferation (14, 24). We therefore sought to examine the interaction of endothelial cells and WM cells and the role of ephrin-B2/Eph-B2 in this interaction. We first tested the expression of ephrin-B2 on endothelial cells and BMSCs from bone marrow aspirates of WM patient. We found that ephrin-B2 was expressed on endothelial cells and BMSCs in the bone marrow of WM patients, and also on the endothelial cell line (HUVEC; Fig. 3A).

We further confirmed that the interaction between ephrin-B2 on the endothelial cells and the Eph-B2 receptor on WM cells is critical for cell–cell adhesion. Figure 3B shows that inhibition of Eph-B2 on WM cells or inhibition of ephrin-B2 on endothelial cells reduced the adhesion of WM cells to endothelial cells. Pretreatment of WM cells with exogenous recombinant ephrib-B2 compensated the adhesion inhibition induced by blocking the endogenous ephrin-B2 on endothelial cells.

These results were further confirmed by testing the role of ephrin-B2 and Eph-B2 in the induction of cytoskeletal signaling and cell adhesion–related proteins in WM cells by interaction with endothelial cells. Figure 3C shows that the coculture of WM cells with endothelial cells activates cell adhesion–related proteins, including phosphorylation of FAK and Src. Inhibition of either ephrin-B2 on endothelial cells or Eph-B2 on WM cells or both reduced the activation of cell adhesion pathways in WM cells.

Similar result were obtained when BCWM1 were tested, indicating that the interaction between ephrin-B2 on the BMSCs and the Eph-B2 receptor on BCWM1 cells is critical for cell–cell adhesion (Fig. 3D).

When WM cells were cultured alone, inhibition of Eph-B2 with a blocking antibody induced a mild reduction of WM cell proliferation. The use of anti–ephrin-B2 antibody did not alter the proliferation of WM cells and did not change the effect of the anti–Eph-B2 antibody (Fig. 4A). However, coculture of WM cells with endothelial cells induced an increase in WM cells proliferation compared with WM cells cultured alone. This effect was abolished by blocking antibody for the Eph-B2 receptor on WM cells and significantly decreased by a blocking antibody for the ephrin-B2 ligand on endothelial cells (Fig. 4B).

To investigate the proliferative effect induced by ephrin-B2/Eph-B2 axis in WM cells when cultured with or without endothelial cells, we tested its role on cell cycle of WM cells. Treatment of WM cells with anti–Eph-B2 or with anti–ephrin-B2 or the combination did not alter the cell-cycle regulation of these cells (Fig. 4C). Coculture of endothelial cells with starved WM cells induced transition of cells from G₁ to S and G₂/M phases, an effect that was reversed with either inhibition of Eph-B2 on the WM cells or inhibition of ephrin-B2 on the endothelial cells by blocking antibodies (Fig. 4D). These results were confirmed by immunoblotting showing activation of AKT, cyclin D3, cyclin E, and pRb in WM cells cocultured with endothelial cells compared with WM cells alone (Fig. 4E). These effects were reversed by inhibition of Eph-B2, ephrin-B2, or the combination of both.

To confirm the selective role of Eph-B2, we knocked down the expression of the receptor using siRNA. Figure 5A shows that the expression of Eph-B2 gene was downregulated to 27% compared with transfection with scramble siRNA. Similarly, the downregulation of Eph-B2 protein expression was confirmed using flow cytometry (Fig. 5B). Loss of function of Eph-B2 induced a significant reduction of WM cell adhesion to endothelial cells (Fig. 5C) and BMSCs (Fig. 5D). Moreover, knockdown of Eph-B2 in WM cells prevented the proliferative effect (Fig. 5E) and cell-cycle regulation (Fig. 5F) in WM cells induced by coculture with endothelial cells. Mechanistically, these results were confirmed by immunoblotting showing that coculture with endothelial cells increased proliferative pathways (such as AKT), and cell-cycle proteins (such as cyclin-D, cyclin-E and pRb); however, these effects were not observed in WM cell that were transfected with Eph-B2 siRNA (Fig. 5G).

Moreover, the downregulation of Eph-B2 in WM cell decreased tumor progression in vivo (Fig. 5H). One week after injecting 3 × 10⁶ BCWM1 cells to SCID mice, the bone marrow contained 25% of CD19⁺ cells in mice injected with BCWM1 cells transfected with scramble siRNA, whereas significant tumor progression was observed in mice injected with BCWM1 cells transfected with Eph-B2 siRNA, with only 10% of CD19⁺ cells in the bone marrow. Downregulation of Eph-B2 expression in the recovered CD19⁺ cells was confirmed by flow cytometry (Fig. 5I).

We investigated the role of ephrin-B2 receptor on endothelial cells and their regulation of the tumor clone. We investigated the effect of ephrin-B2 and Eph-B2 on regulation of tube formation and angiogenesis induced by WM cells. We found that coculture of WM cells with endothelial cells induces vessel formation, which was decreased by either inhibition of Eph-B2 on WM cells or by inhibition of ephrin-B2 on endothelial cells (Fig. 6A and B). Similarly, the adhesion of WM cells to the tubes formed was also decreased by inhibition of either Eph-B2 or ephrin-B2 (Fig. 6C). Mechanistically, it was shown that the interaction between WM cells and endothelial cell induced signaling in the endothelial cells, through phosphorylation of ephrin-B2 and activation of adhesion-related signaling including, P130, FAK, and Src (Fig. 6D). In addition, we investigated the effect of blocking ephrin-B2 and Eph-B2 on regulation of the proliferation of endothelial cells without coculture with WM cells. Figure 1E shows no effect on the proliferation of HUVECs.

Eph receptors and ephrin ligands are both membrane bound; therefore, binding and activation of Eph and ephrin induces cell–cell interactions (3). The Eph system also affects integrin-mediated cell communication with the extracellular environment (25). Ephrin-B2 can interact with other members of Eph receptors, including EphB1, EphB2, EphB3, EphB4, and EphA4 (26, 27). Activation of Eph receptors and ephrins has been shown to affect cell attachment by means of integrin and focal adhesion protein–dependent mechanisms (28). Prior studies have shown that ephrin-A1 induces cell adhesion and actin cytoskeletal changes in fibroblasts in a FAK-dependent and P130 cas-dependent manner through activation of EphA2 receptor (29).

Eph receptors and ephrins are overexpressed in many human cancers and correlate with aggressive, invasive, and metastatic potential of these cancers. In addition, signaling through Eph/ephrins can lead to cell adhesion or cell repulsion, depending on which signaling pathways are activated. The role of Eph/ephrins in low-grade B-cell lymphomas has not been previously investigated.

In this study, we used WM as a model of low-grade B-cell lymphoma that has the potential of dissemination and metastasis to the bone marrow and lymph nodes. We first showed that among all tyrosine kinases, Eph-B2 was highly expressed in primary WM samples and cell lines. The expression of Eph-B2 has been shown in many other cancers, such as colon cancer, and correlated with aggressive behavior (30). Previous reports have shown that ephrin-B2 is a key regulator of this process and, thereby, controls angiogenic, lymphangiogenic, and tumorigenic growth (23, 31); therefore, we tested the role of ephrin-B2 and Eph-B2 on the WM interaction with the bone marrow microenvironment, especially the endothelial cells and BMSCs. The interaction of Eph-B2 with ephrin-B2 itself led to WM cell adhesion. We next showed that, when activated with recombinant ephrine-B2, Eph-B2 signaling leads to activation of adhesion and cytoskeletal reorganization molecules, including FAK, Src, cofilin, and paxillin. These results, therefore, indicate that Ephrin-B2/Eph-B2 interaction in WM regulates cell–cell interaction, which is integrin dependent in our model system through downstream activation of FAK and Src. These results are in agreement with previous studies showing that ephrin-induced cytoskeletal reorganization requires FAK and P130 (29). In agreement with previous reports showing that Eph receptors and ephrins regulate cell migration and tissue assembly (27), we found that ephrin-B2/Eph-B2 interaction downregulated spontaneous migration of WM cells but has no effect on SDF1-induced migration.

We further examined cell–cell interaction by examining the interaction of WM cells with endothelial cells. Previous studies in multiple myeloma and WM have shown that endothelial cells and angiogenesis are critical regulators of tumor proliferation and survival in these tumors of the bone marrow. However, the mechanism by which cell–cell interaction of these cells with endothelial cells regulates cell adhesion and survival has not been previously elucidated. Here, we showed that endothelial cells obtained form patients with WM expressed ephrin-B2 and the interaction of WM cells (expressing Eph-B2) with endothelial cells led to significant adhesion and proliferation of the tumor cells. Moreover, we have shown that this interaction induced signaling in endothelial cells and activated adhesion pathways and tube formation. Previous studies have shown that endogenous Eph-B2 and Eph-B4 signaling are required for the formation of capillary-like vascular structures and that Eph-B2 and Eph-B4 activation induced signaling in endothelial cells (32). Other studies have shown that these receptors are also critical for the interaction of multiple myeloma cells with mesenchymal cells. Eph-B4-Fc treatment of myelomatous SCID-hu mice inhibited myeloma growth, osteoclast activation and angiogenesis, and stimulated osteoblastogenesis and bone formation, whereas ephrin-B2-Fc stimulated angiogenesis, osteoblastogenesis, and bone formation but had no effect on osteoclastogenesis and myeloma growth (33). The study in multiple myeloma did not examine interaction of ephrin-B2 with Eph-B2 on tumor cells. We now show that ephrin-B2/Eph-B2 is dysregulated in WM cells and is critical for cell–cell interaction of tumor cells with endothelial cells for adhesion, survival, and proliferation of WM cells.

We showed that activation of the Eph-B2 receptor with ephrin-B2 did not affect WM cell proliferation and cell cycle; however, it induced activation of adhesion cascades that increased adhesion of WM cells to endothelial cell, which in turn, promoted WM cell proliferation through cell-cycle transition. And we showed that downregulation of Eph-B2 in WM cells reduced tumor progression in vivo. The link between adhesion-regulating molecules and proliferative signaling pathways in WM is interesting and may lead to discovery of new therapeutic targets, further investigation in this area is warranted. Here, we showed that these receptors/ligands are critical in the regulation of adhesion, cell-cycle progression, and tumor proliferation in WM cells, as well as reverse signaling in the endothelial cells and activating adhesion and tube formation (Fig. 7).

In summary, this study examines the interaction of Eph-B2 receptor in a low-grade B-cell lymphoma model, WM, and shows that ephrin-B2/Eph-B2 axis regulates adhesion, activation of downstream signaling of integrin-related molecules, survival, cell cycle and proliferation; and tumor progression in vivo through the interaction of tumor cells with the cells in the bone marrow microenvironment, specifically, by altering adhesion and migration properties. This interaction can be abrogated by specific ephrin-B2 or Eph-B2 inhibitors. Further studies to examine the role of Eph-B2 as a novel therapeutic target in WM and in other B-cell malignancies are warranted.

I.M. Ghobrial is a consultant and is on the advisory board of Celgene, Millenium, Novartis, and Onyx.

F. Azab and A.K. Azab carried out research, designed research, analyzed data, and wrote the manuscript. P. Maiso, T. Calimeri, and R.D. Carrasco conducted research and analyzed data. Y. Liu, P. Quang, A.M. Roccaro, A. Sacco, H.T. Ngo, Y. Zhang, and B.L. Morgan analyzed data. I.M. Ghobrial designed research and wrote the manuscript.

The authors thank for the contribution of Jennifer Stedman in editing this work.

The work was supported by the Kirsch Laboratory for Waldenstrom's Macroglobulinemia, the Heje fellowship for WM, and the International Waldenstrom's Macroglobulinemia Foundation (IWMF).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.