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a Hadassah University Hospital, Gene Therapy Institute. Jerusalem, Israel;
b Hematology Institute, Sheba Medical Center, Tel Hashomer, Israel;
c The Plastic Surgery Department, The Tel-Aviv Sourasky Medical Center, Israel;
d Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
Key Words. Chronic myeloid leukemia • SDF-1 • CXCR4 • Integrins
Correspondence:
Amnon Peled, Ph.D., Hadassah University Hospital, Gene Therapy Institute, P.O. Box 12000 Jerusalem, Israel. Telephone: 972-2-6778780; Fax: 972-2-6430982; e-mail: peled{at}hadassah.org.il
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ABSTRACT |
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INTRODUCTION |
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is not caused by a lack or complete uncoupling of CXCR4, but may rather be due to an intracellular signaling defect downstream of the receptor [10]. It has also been shown that the chemokine SDF-1 and its receptor CXCR4 are essential for homing and repopulation of the murine bone marrow (BM) by human SCID repopulating stem cells (SRCs) [11]. In addition, the major integrins leukocyte function-associated antigen-1, very late activation antigen-4 (VLA-4), and VLA-5 are activated by SDF-1 and are essential for SRC homing and engraftment [12, 13]. These findings suggest a functional role for SDF-1-mediated, integrin-dependent retention of immature CD34+ stem and progenitor cells in the BM of healthy individuals and CML patients. In the present study, we investigated the potential role of SDF-1 activation of integrins in the retention and homing of Ph+CD34+ cells within and to the BM microenvironment. Our findings could be used for developing procedures of purging of malignant cells while maintaining normal stem cells for clinical, autologous transplantation.
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MATERIALS AND METHODS |
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was purchased from R&D Systems (Minneapolis, MN; http://www.rndsystems.com). Bovine serum albumin (BSA; fraction V), Ca2+- and Mg2+-free Hank's balanced salt solution, EGTA, and HEPES were obtained from Sigma Chemical Co. (St. Louis, MO; http://www.sigmaaldrich.com). Human serum albumin (HSA; fraction V) was obtained from Calbiochem (La Jolla, CA; http://www.calbiochem.com). Human FN was obtained from Chemicon International Inc. (Temecula, CA; http://www.chemicon.com). The anti-VLA-4 (MCA697) and VLA-5 (MCA1187) antibodies were purchased from Serotec (Oxford, UK). These antibodies were detected using secondary fluorescein isothiocyanate-conjugated F(ab’)2 fragment goat anti-mouse IgG (H+L) (Jackson; West Grove, PA; http://www.jacksonimmuno.com). The anti-CXCR4 monoclonal antibody 12g5 (conjugated to phycoerythrin) was purchased from Pharmingen, (San Diego, CA; http://www.bdbiosciences.com/pharmingen). Purified mouse IgG (Zymed Lab; South San Francisco, CA; http://www.zymed.com) was used as a control antibody.
Human Cells and Enrichment of CD34+ Cells
Human cord blood (CB) cells were obtained from full-term deliveries. Human peripheral blood lymphocyte cells were obtained from newly diagnosed (ND) CML patients and from CML patients treated with hydroxyurea (HU) plus G-CSF before autologous transplantation [15, 16]. Human cells were obtained after informed consent and approval by the Weizmann Institute ethics committee. The blood samples were diluted 1:1 in phosphate-buffered saline (PBS), supplemented with 1% fetal bovine serum (Bet Haemek; Israel). Low-density MNCs were collected after standard separation on Ficoll-Paque (Pharmacia Biotech; Uppsala, Sweden; http://www.pnu.com) and washed in RPMI with 1% fetal calf serum (FCS). Enrichment of human CD34+ cells was performed with a magnetic bead separation kit (mini MACS; Miltenyi Biotec; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com) according to the manufacturer's instructions. The purity of the enriched CD34+ cells was higher than 90%, as confirmed by fluorescence-activated cell sorting (FACS).
Flow Cytometry Analysis and Calcium Flux
Flow cytometry analysis was done as previously described [11]. Cells were suspended in staining buffer (PBS, 0.1% BSA, 0.02% sodium azide) after lysis of erythrocytes by exposure to ammonium chloride. 105 cells were incubated with 10 µg/ml of purified anti-mouse CD16/CD32 antibody (FcR) (Pharmingen) and 1% human plasma for 20 minutes at 4°C. Cells were next stained with human-specific antibodies and incubated for 30 minutes on ice. Isotype control antibodies (Becton Dickinson [BD]; Lincoln Park, NJ; http://www.bd.com) were used to exclude false positive cells. Dead cells were eliminated by staining with propidium iodide (Sigma). After staining, cells were washed twice in the same buffer and analyzed on a FACSsort cell sorter (BD), using CellQuest software (BD). CD34+-enriched cells from both ND and treated CML patients were sorted on a FACStar cell sorter (BD) based on CXCR4 expression. The purity was found to be >97%. Intracellular free Ca2+ was measured in Fura-3-labeled cells as previously described [17].
Cell Adhesion Assay
24-well plates (Falcon; BD; Plymount, UK), were incubated overnight at 4°C with 500 µl PBS containing 20 µg/ml human FN or 2.5% BSA as a control. Wells were washed with PBS, blocked with 1,000 µl of 2.5% BSA in PBS, and incubated for 1 hour at room temperature with or without neutralizing antibodies to VLA-4 (MCA697) and VLA-5 (MCA1187) (10 µg/ml). Plates were then washed three times with adhesion medium (RPMI-1640 supplemented with 0.2% BSA). CD34+-enriched cells were added to the precoated wells, 6 x 104 cells in 200 µl adhesion medium. The cells were allowed to adhere for 30 minutes at 37°C in a humidified atmosphere containing 5% CO2. Next, the cells were washed four times with prewarmed adhesion medium to remove nonadherent cells. The adherent cells were collected with medium containing 0.01% EDTA and by gentle shaking with vortex. Finally, the cells were counted.
Controlled Detachment Adhesion Assays
Laminar flow assays were performed as previously described [18]. sVCAM-1 was coated at 10 µg/ml in the presence of 2 µg/ml HSA carrier on polystyrene plates (BD). The plates were washed three times with PBS, then blocked with HSA (20 µg/ml in PBS) for 2 hours at room temperature. Alternatively, washed plates were coated with 10 µg/ml SDF-1 in PBS for 30 minutes at room temperature before being blocked with HSA. The plates were then placed so as to form the lower wall of a parallel wall flow chamber and mounted on the stage of an inverted microscope. CB or CML CD34+-enriched cells (2 x 106/ml, purity >98%) were suspended in binding buffer, perfused into the chamber, and allowed to settle on the substrate-coated chamber wall for 1 minute at 37°C. Flow was initiated, then increased by two to two and one-half-fold increments every 5 seconds, generating controlled shear stress on the wall. Cells were visualized in the 20 x objective of an inverted phase-contrast Diaphot Microscope (Nikon; Tokyo, Japan; http://www.nikon-image.com/eng) and photographed with a long-integration LIS-700 change-coupled device video camera (Applitech; Holon, Israel), connected to a video recorder (AG-6730 S-VHS; Panasonic; Osaka, Japan; http://www.panasonic.com). The number of adherent cells resisting detachment by the elevated shear forces was determined after each interval by analysis of videotaped cell images, and was expressed as a percentage of originally settled cells. All experiments were performed at a temperature of 37°C maintained by warming the microscope stage with heating lamps in a humidified atmosphere.
Real-Time Tracking of CD34+ Cell Migration in 3-D ECM-Like Gels
Migration assays in three-dimensional extracellular matrix-like (3-D ECM-like) gels were performed as previously described [19]. Purified (>98%) CB or CML CD34+-enriched cells were suspended in a 10-µl drop consisting of type I collagen (1.8 µg/ml), laminin (6 µg/ml), and FN (2.5 µg/ml) in RPMI. A second drop without cells was placed 1.5 mm from drop I. A SDF-1 depot was created in a third drop supplemented with SDF-1 (250-500 ng/ml) and placed 1.5 mm downstream of drop II and 3-5 mm from drop I. Once the drops began to polymerize, they were gently connected with a fine needle to form a continuous 3-D gel, and cell migration within this gel was tracked by time-lapse video microscopy. Cell images were visualized and videotaped on a time-lapse video recorder (AG-6730 S-VHS; Panasonic) at 25 frames per minute. The proportions of polarized, nonmotile, randomly migrating, and directionally migrating cells within the entire population of cells in the field were determined within 60-90 minutes of tracking.
Chemokines and Chemotaxis Assay
Chemotaxis experiments were conducted using Costar; (Cambridge, MA; 6.5-mm diameter, 5-µm pore) transwells, as previously described [11]. One hundred microliters of chemotaxis buffer (RPMI 1640, 1% FCS) containing 2 x 105 CD34+ cells were added to the upper chamber, and 0.6 ml of chemotaxis buffer with or without SDF-1 or microphage inhibitory protein-1 (125 ng/ml) was added to the bottom chamber. After 4 hours, migrating (bottom chamber) and nonmigrating (upper chamber) cells were counted for 30 seconds using a FACSort cell sorter (BD).
Fluorescence In Situ Hybridization (FISH) Probes and Procedures
BM cells from transplanted NOD/SCID mice or sorted CD34+ populations (1-2 x 105 cells) were concentrated by cytospin, then fixed with methanol:acetic acid (3:1). The LSI BCR/ABL and LSI BCR/ABL extra signal dual-color DNA probe kits were used (Vysis; Downers Grove, IL; http://www.vysis.com). FISH was performed according to the standard FISH protocol developed by Esa et al. [20]. A DAPI/Antifade (Oncor, Inc.; Gaithersburg, MD) mixture was used as a counterstaining blue color of the nuclei while preventing the signals from bleaching. Slides were analyzed using an Olympus BH2 fluorescence light microscope equipped with PlanApo objective 100 x 1.4 oil, an appropriate spectral filter (BH-TFC1 Triple band filter), and a 100-W mercury arc lamp. The number of human cells from in vitro migration assay and CD34+-sorted samples counted in each experiment was between 200 and 600. The limit of detection for this assay is approximately 0.1 µg of DNA [8].
Statistical Analyses
Data are expressed as the mean ± range or standard deviation (SD), or standard error (SE). Statistical comparisons of means were performed by a two-tailed unpaired Student's t test.
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RESULTS |
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) and migration to SDF-1 (Fig. 1, IIA, B). Ph+CD34+ cells from 6 out of 11 patients, however, expressed normal levels of CXCR4 (Fig. 1, IC), and their migration levels to SDF-1 were similar to those of CB and mobilized CD34+ cells expressing CXCR4 (Fig. 1, IIC). Migrating Ph+CD34+ cells also responded to SDF-1 by elevating their intracellular Ca2+ levels (Fig. 1, ID). Although immature Ph+CD34+CXCR4+ cells migrated well in response to SDF-1, we found that the migrating population contained two- to threefold higher levels of normal CD34+ cells than the nonmigrating fraction (Table 1). These results suggest that normal cells have a migratory advantage over Ph+CD34+ cells.
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; Table 1). Interestingly, Ph+CD34+ cells present in the blood of treated patients (2/2) lost their ability to migrate in response to SDF-1 and were successfully purged in vitro from normal CD34+ cells, based on migration to SDF-1 (Table 1). For example, more than 97% of the SDF-1-migrating CD34+ cells from two of these patients were normal, whereas the cells in the nonmigrating fraction were 13% and 45% Ph+ (Table 1). We further sorted enriched CD34+ cells from treated patients into CD34+CXCR4+ and CD34+CXCR4- cells, then tested the cells for the presence of bcr-abl. We found that Ph+CD34+ cells were equally distributed between the CD34+CXCR4+ and CD34+CXCR4- fractions (data not shown). These results may suggest that CML cells resistant to chemotherapy and expressing the chemokine receptor CXCR4 have a defect in the signaling pathway of this receptor.
Immature Ph+CD34+ cells have lower ß1-integrin-mediated adhesion to FN [5, 6]. We therefore tested the ability of Ph+CD34+CXCR4+ cells, which migrate in response to SDF-1 in a transwell migration assay, to migrate in response to SDF-1 through a 3-D ECM-like gel. In all three samples of leukemic Ph+CD34+CXCR4+ cells tested, we found lower SDF-1-mediated, integrin-dependent polarization and migration (Fig. 2, I, II). Furthermore, we found that Ph+CD34+ cells had a higher random polarization than normal CB CD34+ cells, and that their polarization was only slightly increased upon SDF-1 activation (Fig. 2, I). This result is in agreement with a previous report by Salgia et al. [9]. Previously, it was shown that VLA-4 and VLA-5 are crucial for SDF-1-dependent adhesion of normal CD34+ cells to FN, migration through 3-D ECM-like gels, and homing and engraftment of primitive SRCs [12, 13]. In the present study, we show that the ability of SDF-1-responsive Ph+CD34+ cells to adhere to FN in the presence of SDF-1 is lower (Fig. 3, I). Furthermore, the binding of Ph+CD34+ cells to FN in response to SDF-1 was found to be dependent on the integrins VLA-4 and VLA-5 (Fig. 3, I). The binding of leukemic CD34+ cells to VCAM-1 through VLA-4, in the presence or absence of SDF-1, was also substantially lower (Fig. 3, I). Interestingly, the malignant Ph+CD34+ cells were found to express normal levels of VLA-4 and VLA-5 (Fig. 3, II).
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DISCUSSION |
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We have demonstrated that human osteoblasts, as well as stromal and endothelial cells within the human BM, express the chemokine SDF-1 [26]. Recently, it was shown that overexpression of SDF-1 in the peripheral circulation resulted in the mobilization of hematopoietic cells with repopulating capacity, progenitor cells, and precursor cells [27]. Moreover, in a phase I clinical study, AMD-3100, a selective CXCR4 inhibitor, was shown to induce the mobilization of white blood cells [28]. These results strongly suggest a role for CXCR4 and its ligand, SDF-1, in the retention and release of white blood cells. Interestingly, CML, a malignant myeloproliferative disorder originating from a pluripotent stem cell that expresses the bcr-abl oncogene, is characterized by an abnormal release of the expanded, malignant stem cell clone from the BM into the circulation [1, 2]. We have already shown that the migration of mobilized PB CD34+ cells from multiple donors in response to SDF-1 is variable (from 8%-60%), suggesting involvement of SDF-1 in the mobilization process [11]. Similar results were also reported by Voermans et al. [29]. In this study, we have shown that the migration of PB Ph+CD34+ cells from ND CML patients is also variable, suggesting involvement of SDF-1 in the mobilization of Ph+CD34+ cells in CML patients.
Overexpression of bcr-abl in cell lines was shown to inhibit the migration of the cells in response to SDF-1 in vitro [9]. When enriched Ph+CD34+ cells from ND CML patients were tested, we found, in the majority of patients, that the malignant CD34+ cells migrated well in response to SDF-1. Although Ph+CD34+ cells migrated in response to SDF-1, the level of normal CD34+ cells was greater in the fraction of migrating than nonmigrating cells. These results suggest that normal cells have a migratory advantage over Ph+CD34+ cells. CD34+ cells collected from patients after intensive chemotherapy treatment and mobilization with G-CSF usually contain variable levels of immature Ph+CD34+ cells, which are most probably responsible for the recurrence of the malignant clone despite the lack of contamination of the mature MNC population. These CD34+ cells are commonly used in protocols aiming to purge malignant cells while maintaining normal CD34+ cells for autologous transplantation. Normal CD34+ cells collected from such patients exhibited greater migration levels to SDF-1 than Ph+CD34+ cells. Furthermore, in some patients, the leukemic cells were successfully purged from normal cells based on migration to SDF-1. Although 50% of the Ph+CD34+ cells in these samples expressed CXCR4 on their surface, they failed to migrate in response to SDF-1. One possible explanation is that, in cell populations where the percentage of Ph+ cells is lower, the normal CD34+ cells will have a migratory advantage over malignant cells. Another possibility is that chemotherapy, and or G-CSF, select for Ph+CD34+ cells with lower migration potential to SDF-1. This may be the result of overexpressing Ph+ in the remaining Ph+CD34+ cells. This hypothesis is supported by the latest report by Salgia et al., which shows that overexpression of bcr-abl can inhibit the migration of cells in response to SDF-1 in the presence of normal levels of CXCR4 [9].
Malignant Ph+CD34+ cells have lower VLA-4- and VLA-5-dependent adhesion to stromal cells and to FN [3–6]. Therefore, we postulated that migration of leukemic CD34+ cells, in response to SDF-1 through the ECM, could be significantly lower. Indeed, we found that Ph+CD34+ cells have lower integrin-dependent directional migration toward SDF-1. We further found that Ph+CD34+ cells have greater spontaneous, random migration through a 3-D ECM-like gel than normal CD34+ cells. Similar results were obtained by Salgia et al. with the murine BAF-3 cell line, which exhibited a round morphology with little movement on a FN-coated surface [9]. Time-lapse video microscopy showed that SDF-1-stimulated BAF-3 cells underwent a dramatic increase in spontaneous motility. Transformed BAF-3 cells with bcr-abl exhibited a high degree of spontaneous motility; however, upon SDF-1 stimulation, these cells did not further increase their motility [9]. The increase in the spontaneous motility of Ph+CD34+ progenitor cells on FN-coated plates [9] and in ECM-like gels, together with a reduction in their migration to SDF-1 through the ECM, may be the result of inappropriate activation of integrins. Indeed, it was recently reported that Ph+CD34+ cells had impaired ß1-integrin capping [30]. Our results suggest that signaling events controlling the function of integrins via CXCR4 in Ph+ progenitor cells are impaired. Consequently, this may impede the binding of VLA-4 and VLA-5 to their endothelial/stromal and ECM ligands, VCAM-1 and FN, respectively, and may contribute to their lower retention within the BM microenvironment. Our data suggest that Ph+CD34+ cells have impaired abilities to interact with the BM endothelial/stromal cells and ECM in response to SDF-1. These deficiencies could affect their homing to the BM and retention within the stromal microenvironment.
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ACKNOWLEDGMENT |
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REFERENCES |
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) or Ph+CD34+ (CML = 
) cells without SDF-1 or with a gradient of SDF-1 (Normal + SDF-1 = 
, CML + SDF-1 = 
) were measured. The average of three different experiments ± SD are shown.