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Stem Cells 2004;22:65-73 www.StemCells.com
© 2004 AlphaMed Press

Mobilization of Myeloma Cells Involves SDF-1/CXCR4 Signaling and Downregulation of VLA-4

Yair Gazitt, Cagla Akay

University of Texas Health Science Center, San Antonio, Texas, USA

Key Words. Myeloma • Mobilization • SDF-1/CXCR4 • VLA4

Yair Gazitt, Ph.D., Department of Medicine/Hematology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284, USA. Telephone: 210-567-4848; Fax: 210-567-1956; e-mail: e-mail:gazitt{at}uthscsa.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 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 [1–8]. 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 [9–14]. Comobilization of tumor cells following chemotherapy or growth factor has also been documented for hematological malignancies [15–21] and for solid tumors [22–24].

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 [25–29]. SDF-1/CXCR4 signaling is also active in all cancer cells studied [30–47], in solid tumors [30–33], and in hematological malignancies, including chronic lymphocytic leukemia [34–36], non-Hodgkin’s lymphoma [37–38], 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-1{alpha} 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-1{alpha} 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-1{alpha} [43]. We have studied the role of MIP-1{alpha} in the regulation of myeloma cell adhesion in vitro and myeloma cell growth in vivo in a xenograft mouse model [28]. Blocking of MIP-1{alpha} resulted in decreased survival of myeloma cells and decreased bone disease in mice engrafted with ARH-77 myeloma cells transfected with anti-sense MIP-1{alpha}-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 [44–47].

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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 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 x 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 x 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 x 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-1{alpha}, 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-1{alpha} 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 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 1Go. 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. 1Go).



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  		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 the Materials and Methods section. 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.

 
The cumulative results in mobilized blood obtained from 12 myeloma patients are depicted in Figure 2Go. 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. 2AGo). 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 2BGo.



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  		Figure 2. Decreased expression of CXCR4 and VLA4 in mobilized myeloma cells. Cell staining and analysis of stained cells were performed as described in the Materials and Methods section. 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.

 
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 3Go.



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  		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 the Material and Methods section. 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.

 
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 4Go. 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 ({alpha}V4) or CXCR4, but not with anti-VLA-5 ({alpha}c), 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 4Go 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 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); {alpha}V4 and {alpha}V5 = same + 2 µg/ml of anti-VLA-4 or anti-VLA-5 antibodies, respectively; {alpha}C = 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.

 
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 5AGo (percent positive cells) and 5B (MFI).



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  		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 the Materials and Methods section. 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.

 
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. 5AGo). Concomitantly, a substantial (> threefold) increase in the fluorescence intensity of CXCR4 and VLA-4 was also observed following treatment with IL-6 (Fig. 5BGo). 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has been shown that downregulation of adhesion molecules and disruption of CXCR4/SDF-1 signaling occurs during the process of HSC mobilization [9–14]. 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 [26–36, 39–42, 44–46]. 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 [53–56]. 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-1{alpha} 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-1{alpha} [42]. In this regard, we have previously shown that MIP-1{alpha} upregulates the expression of VLA-5, and antisense to MIP-1{alpha} 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-1{alpha}). 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 [57–60] and other B-cell malignancies [61–62] have been reported. Therefore, new strategies for treatment of MM that consider myeloma cells in their microenvironment should be developed, as was previously suggested [63–64].


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vora AJ, Toh CH, Peel J et al. Use of granulocyte colony-stimulating factor (G-CSF) for mobilizing peripheral blood stem cells: risk of mobilizing clonal myeloma cells in patients with bone marrow infiltration. Br J Haematol 1994;86:180–182.[Medline]

  2. Bensinger W, Singer J, Appelbaum F et al. Autologous transplantation with peripheral blood mononuclear cells after administration of G-CSF. Blood 1993;81:3158–3163.[Abstract/Free Full Text]

  3. Chao NJ, Schriber JR, Grimes K et al. Granulocyte colony-stimulating factor "mobilized" peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy. Blood 1993;81:2031–2038.[Abstract/Free Full Text]

  4. Lane TA, Law P, Maruyama M et al. Harvesting and enrichment of HPC mobilized into the peripheral blood of normal donors by GM-CSF or G-CSF: potential role in allogeneic transplantation. Blood 1995;85:275–282.[Abstract/Free Full Text]

  5. Watts MJ, Sullivan AM, Jamieson E et al. Progenitor-cell mobilization after low-dose cyclophosphamide and granulocyte colony-stimulating factor: an analysis of progenitor-cell quantity and quality and factors predicting for these parameters in 101 pretreated patients with malignant lymphoma. J Clin Oncol 1997;15:535–546.[Abstract/Free Full Text]

  6. Gazitt Y, Freytes CO, Callander N et al. Successful PBSC mobilization with high dose G-CSF for patients failing a first round of mobilization. J Hematother 1999;8:173–183.[CrossRef][Medline]

  7. Peters WP, Rosner G, Ross M et al. Comparative effect of GM-CSF and G-CSF on priming of peripheral blood progenitor cells for use with autologous bone marrow after high dose chemotherapy. Blood 1993;81:1709–1719.[Abstract/Free Full Text]

  8. Lane TA, Ho AD, Bashey A et al. Mobilization of blood-derived stem and progenitor cells in normal subjects by granulocyte-macrophage- and granulocyte-colony-stimulating factors. Transfusion 1999;39:39–47[CrossRef][Medline]

  9. Gazitt Y. Immunological profiles of effector cells and peripheral blood stem cells mobilized with different growth factors. STEM CELLS 2001;18:390–398.

  10. Gazitt Y. Recent developments in the regulation of peripheral blood stem cell mobilization. J Hematother Stem Cell Res 2001;10:229–236[CrossRef][Medline]

  11. Gazitt Y. Comparison between granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the mobilization of peripheral blood stem cells. Curr Opin Hematol 2002;9:190–198.[CrossRef][Medline]

  12. Petit I, Szyper-Kravitz M, Nagler A et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol 2002;3:687–694.[CrossRef][Medline]

  13. Lapidot T, Kollet O. The essential roles of the chemokine SDF-1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune-deficient NOD/SCID and NOD/SCID/B2m (null) mice. Leukemia 2002;16:1992–2003.[CrossRef][Medline]

  14. Lapidot T, Petit I. Current understanding of stem cell mobilization. The roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 2002;30:973–981.[CrossRef][Medline]

  15. Sharp JG, Joshi SS, Armitage JO et al. Significance of detection of occult non-Hodgkin’s lymphoma in histologically uninvolved bone marrow by aculture technique. Blood 1992;79:1074–1080.[Abstract/Free Full Text]

  16. Dreyfus F, Ribrag V, Leblond V et al. Detection of malignant B cells in peripheral blood stem cell collections after chemotherapy in patients with multiple myeloma. Bone Marrow Transplant 1995;15:707–712.[Medline]

  17. Gazitt Y, Shaughnessy P, Liu Q. Differential mobilization of CD34+ stem cells and lymphoma cells in non-Hodgkin’s lymphoma patients mobilized with cyclophosphamide with different growth factors. J. Hematother Stem Cell Res 2001;10:167–176.[CrossRef]

  18. Gazitt Y, Reading C, Hoffman R et al. Purified CD34+ Lin-Thy+ stem cells do not contain clonal myeloma cells. Blood 1995;86:381–389.[Abstract/Free Full Text]

  19. Gazitt Y, Tian E, Barlogie B et al. Differential mobilization of myeloma cells and normal hematopoietic stem cells in multiple myeloma after treatment with cyclophosphamide and GM-CSF. Blood 1996;87:805–811.[Abstract/Free Full Text]

  20. Gazitt Y, Reading C. Autologous transplantation with tumor-free graft: a model for multiple myeloma patients. Leuk Lymphoma 1996;27:202–212.

  21. Tricot G, Gazitt Y, Leemhuis T et al. Collection, engraftment kinetics and tumor contamination of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma. Blood 1998;91:4489–4495.[Abstract/Free Full Text]

  22. Moss TJ, Cairo M, Santana VM et al. Clonogenic circulating BM cells: implications regarding peripheral stem cell transplantation. Blood 1994;83:3085–3090.[Abstract/Free Full Text]

  23. Brugger W, Bross KJ, Glatt M et al. Mobilization of tumor cells and hematopoietic stem cells into peripheral blood of patients with solid tumors. Blood 1994;83:636–640.[Abstract/Free Full Text]

  24. Shpall EJ, Jones R. Release of tumor cells from bone marrow. Blood 1994;83:623–628.[Free Full Text]

  25. Robledo MM, Sanz-Rodriguez F, Hidalgo A et al. Differential use of VLA-4 and -5 integrins by hematopoietic precursors and myeloma cells to adhere to bone marrow stroma. J Biol Chem 1998;273:12056–12060.[Abstract/Free Full Text]

  26. Okada T, Hawley RG, Kodaka M et al. Significance of VLA-4-VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model. Clin Exp Metastasis 1999;17:623–629.[CrossRef][Medline]

  27. Damiano JS, Cress AE, Hazlehurst LA et al. Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 1999;93:1658–1667.[Abstract/Free Full Text]

  28. Choi SJ, Oba Y, Gazitt Y et al. Antisense inhibition of macrophage inflammatory protein-1-alpha blocks bone destruction in a model of myeloma bone disease. J Clin Invest 2001;108:1833–1841.[CrossRef][Medline]

  29. Sanz-Rodríguez F, Hidalgo A, Teixidó J. Chemokine stromal cell-derived factor-1 modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1/fibronectin and VCAM-1. Blood 2001;97:346–351.[Abstract/Free Full Text]

  30. Libura J, Drukala J, Majka M et al. CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion. Blood 2002;100:2597–2606.[Abstract/Free Full Text]

  31. Taichman RS, Cooper C, Keller ET et al. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res 2002;62:1832–1837.[Abstract/Free Full Text]

  32. Schrader AJ, Lechner O, Templin M et al. CXCR4/CXCL12 expression and signaling in kidney cancer. Br J Cancer 2002;86:1250–1256.[CrossRef][Medline]

  33. Payne AS, Cornelius LA. The role of chemokines in melanoma tumor growth and metastasis. J Invest Dermatol 2002;118:915–922.[CrossRef][Medline]

  34. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath BM stromal cells. Blood 1999;94:3658–3667.[Abstract/Free Full Text]

  35. Burger JA, Kipps TJ. Chemokine receptors and stromal cells in the homing and homeostasis of chronic lymphocytic leukemia B cells. Leuk Lymphoma 2002;43:461–466.[CrossRef][Medline]

  36. Majka M, Pituch-Noworolska A, Drukala J et al. SDF-1/ CXCR4 interactions are crucial in the migration and survival of chronic lymphocytic leukemia cells in the bone marrow microenvironment. Blood 2002;100:381a.

  37. Durig J, Schmucker U, Duhrsen U. Differential expression of chemokine receptors in B cell malignancies. Leukemia 2001;15:752–756.[CrossRef][Medline]

  38. Donnard M, Trimoreau F, Jaccard A et al. Chemokines receptor expression profile in low grade non Hodgkin lymphoma with leukemic phase. Blood 2002;100:349a.

  39. Durig J, Rosenthal C, Elmaagacli A et al. Biological effects of stroma-derived factor-1 alpha on normal and CML CD34+ haemopoietic cells. Leukemia 2000;14:1652–1660.[CrossRef][Medline]

  40. Mohle R, Schittenhelm M, Failenschmid C et al. Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia. Br J Haematol 2000;110:563–572.[CrossRef][Medline]

  41. Shen W, Bendall LJ, Gottlieb DJ et al. The chemokine receptor CXCR4 enhances integrin-mediated in vitro adhesion and facilitates engraftment of leukemic precursor-B cells in the bone marrow. Exp Hematol 2001;29:1439–1447.[CrossRef][Medline]

  42. Hideshima T, Chauhan D, Hayashi T et al. The biological sequelae of stromal cell-derived factor-1alpha in multiple myeloma. Mol Cancer Ther 2002;1:539–544.[Abstract/Free Full Text]

  43. Moller C, Stromberg T, Juremalm M et al. Expression and function of chemokine receptors in human multiple myeloma. Leukemia 2003;17:203–210.[CrossRef][Medline]

  44. Bertolini F, Dell’Agnola C, Mancuso P et al. CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin’s lymphoma. Cancer Res 2002;62:3106–3112.[Abstract/Free Full Text]

  45. Lagneaux L, Delforge A, Dejeneffe M et al. Antagonists of the chemokine receptor CXCR4 block chemotaxis and inhibit stromal dependent proliferation of acute lymphoblastic leukemia cells. Blood 2002;100:762a.

  46. Boehmler AM, Kuci S, Seitz G et al. In vitro and preclinical activity of the novel AMD3100 CXCR4 antagonist in lymphoma models. Blood 2002;100:579a.

  47. Schwarz MK, Wells TN. New therapeutics that modulate chemokine networks. Nat Rev Drug Discov 2002;1:347–358.[CrossRef][Medline]

  48. Gazitt Y, Akay C, Thomas C et al. The role of adhesion molecules and CXCR4/SDF-1 axis in mobilization of malignant myeloma cells. Cancer Res 2003,94:524a.

  49. Gazitt Y, Shaughnessy P, Liu Q. Expression of adhesion molecules on CD34+ cells in mobilized peripheral blood of non-Hodgkin’s lymphoma patients. STEM CELLS 2001;19:134–143.[Abstract/Free Full Text]

  50. Gazitt Y. TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma pts and is not cytotoxic to hematopoietic stem cells. Leukemia 1999;13:1817–1824.[CrossRef][Medline]

  51. Liu Q, Hilsenbeck S, Gazitt Y. Arsenic trioxide-induced apoptosis in myeloma cells: p53-dependent G1 or G2/M cell cycle arrest, activation of caspase-8 or caspase-9 and synergy with APO2/TRAIL. Blood 2003;101:4078–4087.[Abstract/Free Full Text]

  52. Gazitt Y, Liu Q. Plasma levels of SDF-1 and expression of SDF-1 receptor on CD34+ cells in mobilized peripheral blood of non-Hodgkin’s lymphoma patients. STEM CELLS 2001;19:37–45.[Abstract/Free Full Text]

  53. Forster R, Kremmer E, Schubel A et al. Intracellular and surface expression of HIV-1 co-receptor CXCR4/fusin on various leukocytes subsets: rapid internalization and recycling upon activation. J Immunol 1998;160:1522–1531.[Abstract/Free Full Text]

  54. Peled A, Petit I, Kollet O et al. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 1999;283:845–848.[Abstract/Free Full Text]

  55. Peled A, Kollet O, Ponomaryov T et al. The chemokine SDF-1 activates the integrin LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000;95:3289–3296.[Abstract/Free Full Text]

  56. Odemis V, Moepps B, Gierschik P et al. Interleukin-6 and cAMP induce stromal cell-derived factor-1 chemotaxis in astroglia by up-regulating CXCR4 cell surface expression. J Biol Chem 2002;277:39801–39808.[Abstract/Free Full Text]

  57. Jourdan M, De Vos J, Mechti N et al. Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1. Cell Death Diff 2000;7:1244–1252.[CrossRef][Medline]

  58. Chatterjee M, Honemann D, Lentzsch S et al. In the presence of bone marrow stromal cells human multiple myeloma cells become independent of the IL-6/gp130/STAT3 pathway. Blood 2002;100:3311–3318.[Abstract/Free Full Text]

  59. Lauta VM. A review of the cytokine network in multiple myeloma. Cancer 2003;97:2440–2452.[CrossRef][Medline]

  60. Bisping G, Leo R, Wenning D et al. Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma. Blood 2003;101:2775–2783.[Abstract/Free Full Text]

  61. Mudry RE, Fortney JE, York T et al. Stromal cells regulate survival of B-lineage leukemic cells during chemotherapy. Blood 2000;96:1926–1932.[Abstract/Free Full Text]

  62. Gibson LF. Survival of B lineage leukemic cells: signals from the bone marrow microenvironment. Leuk Lymphoma 2002;43:19–27.[CrossRef][Medline]

  63. Landowski TH, Olashaw NE, Agrawal D et al. Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-kappaB (RelB/p50) in myeloma cells. Oncogene 2003;22:2417–2421.[CrossRef][Medline]

  64. Dalton WS. The tumor microenvironment: focus on myeloma. Cancer Treat Rev 2003;29(suppl 1):11–19.

Received July 18, 2003; accepted for publication September 16, 2003.



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