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Keywords:

  • Myeloma;
  • Mobilization;
  • SDF-1/CXCR4;
  • VLA4

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Adhesion molecules and stromal cell-derived factor-1 (SDF-1)/CXCR4 signaling play key roles in homing and mobilization of hematopoietic stem cells (HSC). Active signaling through SDF-1/CXCR4 and upregulation of adhesion molecules are required for homing, whereas downregulation of adhesion molecules and disruption of SDF-1/CXCR4 signaling are required for mobilization of HSC. We studied the surface expression of CXCR4 very late activation antigen (VLA)-4 and VLA-5 on myeloma cells mobilized with cyclophosphamide and GM-CSF in 12 multiple myeloma patients undergoing HSC mobilization for autologous transplantation. We also studied the plasma levels of SDF-1 in apheresis collection of these patients. We observed a statistically significant decrease in the levels of SDF-1 and surface expression of CXCR4 on myeloma cells in four consecutive apheresis collections compared with premobilization bone marrow specimens. We also observed a statistically significant decrease in surface expression of VLA-4 in myeloma cells in the apheresis collections compared with premobilization bone marrow samples. Furthermore, myeloma cells derived from apheresis collections had decreased adhesion and trans-stromal migration in response to SDF-1, which could be reversed by short incubation with interleukin-6. Hence, mobilization of myeloma cells involves SDF-1/CXCR4 signaling and downregulation of VLA-4.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mobilization of hematopoietic stem cells (HSC) can be achieved by the administration of chemotherapy, by administration of hematopoietic growth factors such as G-CSF or GM-CSF, or by chemotherapy plus hematopoietic growth factor [18]. The molecular events and signaling molecules involved in HSC mobilization have been extensively studied. Thus, downregulation of adhesion molecules and desensitization of the CXCR4/stromal cell-derived factor (SDF-1) chemotaxis are critical events in growth factor-induced and/or chemotherapy-induced mobilization of HSC [914]. Comobilization of tumor cells following chemotherapy or growth factor has also been documented for hematological malignancies [1521] and for solid tumors [2224].

Recent studies revealed that adhesion molecules are similar to HSC in their homing and release of cancer cells. In particular, the involvement of very late activation antigen (VLA)-4 and VLA-5 in adhesion of myeloma cells to stromal cells and the resulting drug resistance has been extensively studied [2529]. SDF-1/CXCR4 signaling is also active in all cancer cells studied [3047], in solid tumors [3033], and in hematological malignancies, including chronic lymphocytic leukemia [3436], non-Hodgkin's lymphoma [3738], chronic myelogenous leukemia [39], and acute leukemias [40, 41]. The role of SDF-1/CXCR4 signaling in the adhesion of myeloma cells to fibronectin and stromal cells has also been studied [29, 42, 43]. Hideshima et al. [42] have shown that SDF-1α promotes proliferation, induces migration, and protects against dexamethasone-induced apoptosis through the mitogen-activated protein (MAP)/Akt pathway in myeloma cells, but these effects were only modest. Also, SDF-1α upregulated the secretion of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in bone marrow (BM) stromal cells, resulting in proliferation and partial resistance to dexamethasone-induced apoptosis [42]. In addition, the expression and role of various chemokines and chemokine receptors in myeloma cells was studied recently by Moller et al. [43]. They reported that cells derived from myeloma cell lines and primary myeloma cells express functional chemokine receptors CCR1, CXCR3, and CXCR4 and migrate in response to the CCR1 ligands RANTES, macrophage inflammatory protein-1 (MIP-1), and SDF-1α [43]. We have studied the role of MIP-1α in the regulation of myeloma cell adhesion in vitro and myeloma cell growth in vivo in a xenograft mouse model [28]. Blocking of MIP-1α resulted in decreased survival of myeloma cells and decreased bone disease in mice engrafted with ARH-77 myeloma cells transfected with anti-sense MIP-1α-expressing vector [28].

Further support of the general role of SDF-1/CXCR4 signaling in the survival, homing, and mobilization of cancer cells was obtained from several recent studies demonstrating that blocking of CXCR4 by specific antibodies or by the CXCR4-blocking peptides, such as T140, results in mobilization of tumor cells and blocking of human lymphoma, and leukemia cell growth in xenograft mouse models [4447].

In this communication, we report the involvement of adhesion molecules and SDF-1/CXCR4 signaling in the mobilization of myeloma cells. We report a decrease in plasma levels of SDF-1 and a decrease in surface expression of VLA-4 and CXCR4 in mobilized myeloma cells compared with premobilization BM myeloma cells. We also observed a concomitant decrease in adhesion and trans-stromal migration of mobilized myeloma cells. Preliminary results from this work were presented at the 94th Annual Meeting of the American Association of Cancer Research [48].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Patient Population

Samples were obtained with consent from 12 multiple myeloma (MM) patients undergoing peripheral blood stem cell (PBSC) mobilization for autologous transplantation with cyclophosphamide (6 g/m2) and GM-CSF (250 μg/m2) as previously described [19]. All patients underwent four apheresis collections and were transplanted with ≥ 2 × 106 CD34+ cells/kg.

Staining of Myeloma Cells for VLA-4, VLA-5, and CXCR4

BM and peripheral blood samples from apheresis collections were obtained from consented myeloma patients as mentioned above. The mononuclear cell fraction was obtained following Ficoll-Hypaque separation. Myeloma cells were defined as CD38+CD138+ cells. Tubes containing 1 × 106 mononuclear cells were placed in 100 μl phosphate-buffered saline (PBS) plus 1% bovine serum albumin (BSA) and samples were stained for 20 minutes at 4°C. Staining for VLA-4 (CD49d), VLA-5 (CD49e), and CXCR4 was performed as described before [49] by three-color immunofluorescence staining using phycoerythrin (PE)-conjugated anti-CD38 and APC-conjugated anti-CD138 (syndican-1) to mark myeloma cells. Antibodies to CXCR4, VLA-4, and VLA-5 were fluorescein isothiocyanate (FITC) conjugated. Isotype immunoglobulin controls conjugated to FITC, PE, and APC were used for each test. All antibodies and immunoglobulin isotype controls were from Becton Dickinson (Mountain View, CA; http://www.bd.com). Region was set on CD38+CD138+ cells (R1+R2), and the fluorescence in the FITC channel was determined in R3 for the three FITC-conjugated antibodies. Stained cells were analyzed using the FACSCalibur flow cytometer (Becton Dickinson). Results were quantitated by CellQuest software. At least 50,000 cells were analyzed.

Flow Sorting and Culture of Freshly Isolated CD38+CD138+ Myeloma Cells

Sample from the BM or peripheral blood apheresis collections were stained as above for myeloma cell markers (CD38+/CD138+ cells), and flow sorting was performed by the FACSstarPlus Turbo-Fast Sorter (Becton Dickinson; 10,000 event/second) as previously described [50]. Peripheral blood and BM myeloma cells with >95% purity were routinely obtained and were stored at −80°C until used. For studies of adhesion and trans-stromal migration, purified myeloma cells were cultured in RPMI1640 medium containing 10% autologous plasma.

Isolation of Stromal Cells

Isolation of autologous stromal cells was performed by first depleting monocytes from peripheral blood or BM mononuclear cells by passing them through CD14-conjugated immunomagnetic beads (Miltenyi Biotech; Auburn, CA; http://www.miltenyibiotec.com) as recommended by the manufacturer. Unbound cells were collected and cultured in 35-mm petri dishes in RPMI1640 containing 10% autologous plasma for 7 days in a humidified CO2 incubator. Nonadherent cells were discarded and the medium was changed every other day. Homogeneous populations of clean fibroblast-looking cells were obtained. Purified stromal cells were stored at −80°C until used.

Cell Adhesion Assay

Briefly, stromal cell suspension (200,000 cells/500 μl medium/well) was placed in a 24-well plate (Falcon, Becton Dickinson) and incubated overnight at 37°C in a humidified CO2 incubator followed by washing three times with adhesion medium (RPMI1640 supplemented with 0.2% BSA). Prior to adhesion assays, myeloma cells were fluorescently labeled by incubating with 20 μM tetramethylrhodamine ethyl ester (TMRE; Molecular Probes; Eugene, OR; http://www.probes.com) according to the manufacturer's instructions [51]. Purified fluorescently labeled myeloma cells (100,000 cells/200 μl adhesion medium) were added to wells precoated with stromal cell layer, with or without 50 ng/ml SDF-1. The cells were allowed to adhere for 1 hour at 37°C in a humidified CO2 incubator and were then washed three times with prewarmed adhesion medium to remove nonadherent cells. Adherent cells were dislodged by addition of 1 mM EDTA for 5 minutes at room temperature following gentle shaking on a vortex plate. Nonadherent and adherent cells were collected and counted by flow cytometry. Controls for nonspecific adhesion were performed in the same way but with 1 mM EDTA. All adhesion experiments were performed in triplicate wells.

Chemotaxis Assays

Chemotaxis experiments were performed using transwell plates (Costar; Cambridge, MA; diameter, 6.5 mm; pore, 5 μm). One hundred microliters of chemotaxis buffer (RPMI1640 with 1% fetal calf serum [FCS]) containing 200,000 fluorescently labeled myeloma cells (described above) were added to the upper chamber, and 0.6 ml chemotaxis buffer with or without 50 ng/ml SDF-1 (hu-recombinant [rh], R&D Systems; Minneapolis, MN; http://www.rndsystems.com) added to the bottom chamber. After 4 hours at 37°C, migrating (bottom chamber) and nonmigrating (upper chamber) cells were counted by flow cytometry. Prior to the chemotaxis assay, stromal cells were seeded (400,000 cells/well) onto the upper chamber and further cultured for 48 hours at 37°C in a CO2 incubator. For blocking of cell adhesion and trans-stromal migration experiments, 1 × 106 myeloma cells/ml were preincubated for 30 minutes at 4°C with 2 μg anti-VLA-4, anti-CXCR4, or with 2 μg isotype-matching immunoglobulin control (Becton Dickinson). Cells were washed before onset of adhesion and trans-stromal migration assays. For restoration of cell adhesion and trans-stromal migration, myeloma cells were incubated for 24 hours in RPMI1640 medium containing 10% autologous plasma and 50 ng/ml IL-6 (R&D Systems). The cells were then washed and incubated with TMRE for fluorescence labeling as described above. All assays were performed in triplicate wells.

Determination of SDF-1 by Enzyme-Linked Immunosorbent Assay

Plasma levels of SDF-1 were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) as described before [52]. Briefly, aliquots of 100 μl/well of anti SDF-1 (5–10 μg/ml; monoclonal antibody, R&D Systems) were used to coat 96-well plates (MaxiSorp Immunoplate; Nalge Nunc; Rochester, NY; http://www.nalgenunc.com) and incubated overnight at 4°C. Plates were blocked with PBS containing 1% BSA for 1 hour at room temperature, followed by washing with buffer containing 0.1% BSA plus 0.05% Tween-20 in PBS. Plasma samples were diluted 1:2 and 1:4 with PBS and were dispensed into the wells. To generate a standard curve, rh-SDF-1 (SDF-1α, R&D Systems) was used at a concentration range of 25 ng/ml to 100 pg/ml in 10 serial dilutions in PBS plus 1% BSA. After a 2-hour incubation at room temperature, plates were washed and 100 μl of a 0.5-μg/ml biotin-anti-SDF-1α antibody (clone 79018-111, R&D Systems) were added to each well. After a second 2-hour incubation at room temperature, plates were thoroughly washed and 100 μl of a 1:10,000 dilution of peroxidase--streptavidin conjugate (Jackson Immunoresearch Laboratory; West Grove, PA; http://www.jacksonimmuno.com) were added into each well, and plates were incubated at room temperature for 1 hour. After washing of unbound antibody, 100 μl of tetramethylbenzidine-peroxidase substrate/chromogen solution (Kirkegaard & Perry; Gaithersburg, MD; http://www.kpl.com) were added to each well and incubated at room temperature for 10–20 minutes. The reaction was stopped with 100 μl of 1M H3PO4 and absorbence was monitored at 450 nm by an automated ELISA reader (THERMOmax; Molecular Devices; Menlo Park, CA; http://www.moleculardevices.com). Patient samples were run in triplicate wells. Negative controls for assay background were also run for each plate.

Statistical Analyses

Paired Student's t-test was performed to compare BM and apheresis samples. Analysis of variance between multiple groups was performed by Tukey's Multiple Comparison Test or by the Newman-Keuls Multiple Comparison Test. The statistical package of the GraphPad Prism graphics software (San Diego, CA; http://www.graphpad.com) was used.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Adhesion molecules and CXCR4 signaling were implicated in mobilization of HSC and various cancer cells. However, the expression of adhesion molecules and CXCR4 on mobilized myeloma cells was not studied. We first studied the expression of VLA-4, VLA-5, and CXCR4 on CD38+CD138+ myeloma cells in the blood of mobilized myeloma patients compared with premobilization BM myeloma cells. An example is shown in Figure 1. A marked decrease in the expression of CXCR4 and VLA-5 was observed in the apheresis collections obtained from all patients studied, relative to steady-state BM (Fig. 1).

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Figure Figure 1.. Three-color immunofluorescence staining of myeloma cells expressing CXCR4, VLA4, and VLA5.An example staining of BM myeloma cells (BM) and day-1 apheresis collection (PBSC1) for CXCR4, VLA4, and VLA5 is shown. Mononuclear cells were stained with PE-anti-CD38, APC-anti-CD138 (syndican-1), FITC-conjugated anti-CXCR4, anti-VLA4, and anti-VLA5. Region was set on CD38+CD138+cells (R1+R2) and the fluorescence in the FITC channel was determined in R3 for the three FITC-conjugated antibodies. Immunofluorescence staining was performed as described in theMaterials and Methodssection. Dot plots represent immunoglobulin isotype control (upper left) and staining for CD49d (lower right). Results were quantitated by CellQuest software. At least 50,000 cells were analyzed. Dotted lines are isotype-matched controls, and solid lines represent staining with specific antibodies. Note the decrease in expression of CXCR4 and VLA4 and the slight decrease in VLA5 in the apheresis sample versus the BM sample of the same patient. Note also the relatively low expression of VLA5 compared with the high expression of VLA4 and CXCR4 in BM and apheresis samples of myeloma cells.

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The cumulative results in mobilized blood obtained from 12 myeloma patients are depicted in Figure 2. The mean expression of VLA-4 and CXCR4 on mobilized myeloma cells was significantly decreased compared with premobilization myeloma cells in all apheresis collections. In contrast, expression of VLA-5 was low and was not significantly decreased (Fig. 2A). Concomitant with a decrease in the proportion of myeloma cells expressing VLA-4 and CXCR4, we observed a statistically significant decrease in the mean fluorescence intensity (MFI) of VLA-4 and CXCR4 on mobilized myeloma cells compared with premobilization BM myeloma cells. In contrast to VLA-4 and CXCR4, no decrease in MFI was observed for VLA-5. The data obtained from 12 patients are presented in Figure 2B.

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Figure Figure 2.. Decreased expression of CXCR4 and VLA4 in mobilized myeloma cells.Cell staining and analysis of stained cells were performed as described in theMaterials and Methodssection. A) BM myeloma cells were compared with four consecutive apheresis collections (d1 to d4). The results are depicted as percent myeloma cells expressing CXCR4, VLA-4, or VLA-5 of the total number of cells analyzed (absolute percentage). Bars represent mean ± SD of each apheresis collection obtained from 12 myeloma patients. Asterisks represent significant (p < 0.05) differences relative to premobilization BM samples. Note the significant decrease in percent expression of VLA-4 and CXCR4 of mobilized myeloma cells with only a small decrease in VLA-5 expression. B) BM myeloma cells were compared with four consecutive apheresis collections derived from 12 patients. The results are presented as mean fluorescence intensity (MFI) of the expression of CXCR4, VLA-4, and VLA-5. MFI is an indirect estimate of the number of molecules/cell. Bars represent mean ± SD of each apheresis collection obtained from 12 myeloma patients. Asterisks represent significant (p < 0.05) differences relative to premobilization BM samples. Note the significant decrease in MFI of VLA-4 and CXCR4 of mobilized myeloma cells with only a small decrease in VLA-5 expression.

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We also studied the plasma levels of SDF-1 in the apheresis collections and in premobilization BM samples. We observed a significant decrease (∼50%) in plasma SDF-1 in each of the apheresis collections compared with premobilization BM. The results are depicted in Figure 3.

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Figure Figure 3.. Decrease in plasma SDF-1 in the apheresis collections of mobilized myeloma patients.Changes in plasma levels of SDF-1 in mobilized blood (day 1 to day 4) compared with BM plasma before mobilization. Determination of SDF-1 levels by ELISA was carried out as described in theMaterial and Methodssection. BM = bone marrow before mobilization; d1 to d4 = apheresis collections at day 1 to day 4. Results were derived from 12 myeloma patients. Bars represent mean ± SD. Asterisks represent significant differences (p < 0.05) relative to premobilization BM samples.

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In addition we studied cell adhesion and trans-stromal migration of myeloma cells in response to SDF-1 derived from the apheresis collections compared with that of premobilization BM myeloma cells. The results are depicted in Figure 4. We observed about a 50% decrease in SDF-1-dependent adhesion and trans-stromal migration of purified myeloma cells derived from day-1 apheresis collections compared with premobilization BM myeloma cells. Furthermore, preincubation of myeloma cells with antibodies to VLA-4 (αV4) or CXCR4, but not with anti-VLA-5 (αc), resulted in abrogation of cell adhesion and trans-stromal migration of myeloma cells. Most importantly, incubation of myeloma cells for 24 hours with 100 ng/ml of IL-3 and 50 ng/ml SCF resulted in varying degrees of restoration of cell adhesion and trans-stromal migration of myeloma cells, with best results obtained with IL-6. The results for IL-6 presented in column R in Figure 4 demonstrate full restoration of SDF-1-dependent cell adhesion and trans-stromal migration. Very small (<10%) adhesion and migration of myeloma cells was observed in the absence of SDF-1. Thus, the observed decrease in VLA-4, CXCR4, and SDF-1 was functionally relevant and could be reversed by IL-6. Anti-CXCR4 and anti-VLA-4 antibodies blocked adhesion and trans-stromal migration of premobilization BM-derived myeloma cells (results not shown).

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Figure Figure 4.. Decrease in cell adhesion and trans-stromal migration of myeloma cells derived from mobilized blood of MM patients.CD38+CD138+myeloma cells were purified by flow cytometry from premobilization BM and from day-1 apheresis collections (A1) of myeloma patients mobilized with cyclophosphamide and GM-CSF. C = control BM with no SDF-1; BM = bone marrow + 50 ng/ml SDF-1; A = apheresis (day-1 collection + 50 ng/ml SDF-1);αV4 andαV5 = same + 2 μg/ml of anti-VLA-4 or anti-VLA-5 antibodies, respectively;αC = same + 2 μg/ml of anti-CXCR4 antibodies; R = myeloma cells from apheresis collections (A1) incubated for 24 hours with 50 ng/ml IL-6 and assayed for adhesion and trans-stromal migration with 50 ng/ml of SDF-1. Error bars are ± SD derived from 12 patients. Asterisks represent significant (p < 0.05) decrease in adhesion and trans-stromal migration of peripheral blood myeloma cells compared with premobilization BM myeloma cells, or significant blocking by antibodies to VLA4 and CXCR4.

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These results strongly suggest that mobilization of myeloma cells requires downregulation of VLA-4 and disruption of SDF-1/CXCR4 signaling, and thus myeloma cells follow the same requirement for mobilization as was described for HSC.

In order to delineate the mechanism for the observed reversal of cell adhesion and trans-stromal migration of myeloma cells, we tested the effect of a 24-hour incubation of myeloma cells with 100 ng/ml IL-3 or 50 ng/ml IL-6, or 50 ng/ml SCF on the re-expression of CXCR4, VLA-4, and VLA-5 in myeloma cells derived from apheresis collections of three different MM patients. The results are presented in Figure 5A (percent positive cells) and 5B (MFI).

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Figure Figure 5.. Growth factors induce re-expression of CXCR4 and VLA-4 on the surface of myeloma cells derived from mobilized blood of MM patients.CD38+CD138+myeloma cells were purified by flow cytometry from day-1 apheresis collections of 3 patients mobilized with cyclophosphamide and GM-CSF. F = fresh myeloma cells. Myeloma cells were cultured for 24 hours with 50 ng/ml of IL-6 or 50 ng/ml SCF, or 100 ng/ml IL-3. Error bars are ± SD derived from apheresis collections of 3 different patients. Cells were stained for CXCR4 and VLA-4 and analyzed by flow cytometry as described in theMaterials and Methodssection. A) depicts percent positive cells relative to Ig-isotype controls. B) depicts increase in MFI relative to Ig-isotype controls. Asterisks represent significant (p < 0.05) increases in the expression of CXCR4 or VLA-4 in IL-6-treated cells relative to 24 hours incubation in medium lacking IL-6.

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Hence, myeloma cells incubated for 24 hours with various growth factors exhibited a marked increase in surface expression of both CXCR4 and VLA-4, with best results obtained with IL-6 (70%–80% positive cells, Fig. 5A). Concomitantly, a substantial (> threefold) increase in the fluorescence intensity of CXCR4 and VLA-4 was also observed following treatment with IL-6 (Fig. 5B). No similar increase in the expression of VLA-5 was observed with any of the growth factors used (results not shown). Interestingly, a slight increase in the expression of CXCR4 and VLA-4 was observed following 24-hour incubation in medium (RPMI1640 plus 15% FCS) without growth factors added, compared with fresh, uncultured myeloma cells.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

It has been shown that downregulation of adhesion molecules and disruption of CXCR4/SDF-1 signaling occurs during the process of HSC mobilization [914]. Similarly, it has been shown that circulating cancer cells exhibit reduced expression of adhesion molecules and CXCR4, and upregulation of CXCR4 and adhesion molecules were detected in metastatic cancer cells [2636, 3942, 4446]. Hence it is widely accepted that mobilization of HSC and cancer cells are parallel processes and occur concurrently in the same patient during the course of treatment with anticancer drugs and/or growth factors.

In earlier studies we demonstrated that downregulation of VLA-4 and disruption of the CXCR4/SDF-1 signaling pathway predicts successful mobilization of HSC in non-Hodgkin's lymphoma patients undergoing PBSC mobilization for autologous transplantation [49, 52]. Here we studied the expression of VLA-4, VLA-5, and CXCR4 on myeloma cells in 12 MM patients undergoing PBSC mobilization for autologous transplantation. We also studied the plasma levels of plasma SDF-1 in apheresis collections of these patients. We observed downregulation of VLA-4, but not VLA-5, in mobilized myeloma cells. Similarly, we observed downregulation of SDF-1 and its receptor CXCR4 in mobilized myeloma cells. Most importantly, we observed that myeloma cells from mobilized peripheral blood have impaired response to SDF-1-dependent adhesion and trans-stromal migration. However, this impairment was reversible upon brief incubation of myeloma cells with physiological concentrations of IL-6, which is widely reported for myeloma patients. IL-6 upregulates the expression of CXCR4 and VLA-4 as reported previously for HSC [5356]. Given the similarity in the function of SDF-1/CXCR4 signaling in HSC and in myeloma cells, we tested the hypothesis that IL-6 upregulates the expression of CXCR4 and VLA-4, resulting in restoration of cell adhesion and trans-stromal migration of myeloma cells. The results presented in this paper further support the general hypothesis that myeloma cells and HSC are mobilized by the same mechanism. Hence, the observed decrease in the expression of VLA and the observed disruption of CXCR4/SDF-1 signaling occurring in the course of mobilization of myeloma cells and the reversal by short incubation with IL-6 could both occur in vivo and could probably be relevant to the process of release and spread of myeloma cells to distal sites in the BM and to extramedullary sites.

In agreement with the results presented here are observations reported by Moller et al. [43] demonstrating that the CXCR4 chemokine receptor is expressed on myeloma cells and that myeloma cells migrate in response to SDF-1. Other studies by Hideshima et al. [42] confirmed the involvement of SDF-1α in promoting proliferation, migration, and protection against dexamethasone-induced apoptosis of myeloma cells through the MAP/Akt pathway. They also observed upregulation of IL-6 and VEGF by SDF-1α [42]. In this regard, we have previously shown that MIP-1α upregulates the expression of VLA-5, and antisense to MIP-1α downregulates the expression of VLA-5 in vitro and blocks human myeloma cell growth and bone disease in a xenograft severe combined immunodeficient mouse model [28].

We therefore propose that, following each cycle of HSC mobilization or chemotherapy, myeloma cells lose adhesion molecules and CXCR4 signaling and surviving myeloma cells are released from the primary or secondary sites (eg, BM, lymph node) into circulation. Myeloma cells circulate until re-expression of adhesion molecules and CXCR4 receptors occurs. Subsequently, myeloma cells migrate and home to stromal cells in the marrow or to extracellular matrix in extramedullary sites using SDF-1/CXCR4 signaling or other chemokines (eg, MIP-1α). Hence repeated cycles of chemotherapy could result in increased drug resistance and in the spread of the disease. Numerous examples of the role of the microenvironment and IL-6 in the survival and drug resistance of myeloma cells [5760] and other B-cell malignancies [6162] have been reported. Therefore, new strategies for treatment of MM that consider myeloma cells in their microenvironment should be developed, as was previously suggested [6364].

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank Dr. Cesar Freytes and Dr. Natalie Callander for enrolling patients on this mobilization protocol. We also would like to thank Mr. Charles Thomas and the Institutional Flow Cytometry Laboratory for performing the cytofluorometric analyses.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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