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CANCER STEM CELLS |
aGoldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel;
bBone Marrow Transplantation Department, Chaim Sheba Medical Center, Tel-Hashomer, Israel
Key Words. CXCR4 • Mobilization • Hematopoietic stem cells • Hematopoietic progenitors
Correspondence: Amnon Peled, Ph.D., Hadassah Hebrew University Hospital, Gene Therapy Institute, P.O. Box 12000, Jerusalem, 91120 Israel. Telephone: 972-2-677-8780; Fax: 972-2-643-0982; e-mail: peled{at}hadassah.org.il
Received on March 6, 2007;
accepted for publication on May 15, 2007.
First published online in STEM CELLS EXPRESS May 24, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral-blood mononuclear cells are routinely used as a source of HSCs for transplantation. Despite the potency of G-CSF in mobilizing stem cells, it results in broad interindividual variations in circulating progenitor and stem cell numbers [4], requiring repeated dosing, and is frequently associated with side effects. Thus, optimal and improved methods to mobilize and collect peripheral-blood progenitor and stem cells for hematopoietic rescue are warranted.
Over recent years, it has become apparent that the interaction between CXCL12 and its receptor, CXCR4, plays a pivotal role in hematopoietic cell mobilization and engraftment [5–7]. The CXCR4 receptor is widely expressed on many cell types, including HSCs and HPCs, and the interaction with its ligand was found to be involved in chemotaxis, homing, and survival. The CXCL12/CXCR4 axis is also involved in the retention of hematopoietic cells within the BM microenvironment [8]; consequently, the disruption of CXCL12/CXCR4 interactions results in mobilization of hematopoietic cells. Indeed, blocking the CXCR4 receptor with an antagonist, such as AMD3100, results in the mobilization of HPCs. Moreover, combining AMD3100 with G-CSF has produced an additive effect [9]. These approaches suggest that antagonizing the interactions of BM-produced CXCL12 with CXCR4 that is expressed on HSCs could be an effective HSC mobilizing strategy.
Today, there are several known CXCR4 antagonists that have been described as having different levels of efficiency [10, 11]. 4F-benzoyl-TN14003 (T-140) is a short modified peptide (supplemental online Fig. 1), a highly selective CXCR4 antagonist, originally designed as a human immunodeficiency virus (HIV) entry inhibitor through specific binding to CXCR4. T-140 is a competitive inhibitor that hinders CXCL12-mediated cell proliferation and migration [12].
To determine the therapeutic potential of T-140, we evaluated the capacity of T-140 to mobilize hematopoietic cells, alone and in combination with G-CSF. In addition, we compared the effect of T-140 to that of another CXCR4 antagonist, AMD3100.
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MATERIALS AND METHODS |
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Mice and Experimental Protocol
Female C57BL/6 mice (7–8 weeks old) were purchased from Harlan Israel (Rehovot, Israel, http://www.harlanisrael.com) and maintained under specific pathogen-free conditions at the Hebrew University Animal Facility (Jerusalem, Israel). All experiments were approved by the Animal Care and Use Committee of the Hebrew University (Jerusalem, Israel). Mice were injected subcutaneously with various doses of T-140 or AMD3100 (1, 2.5, 5, and 10 mg/kg) in a total volume of 200 µl, 2 hours before sacrifice. In some experiments, mice were sacrificed at different time points: 
, 1, 2, 4, and 24 hours postinjection. G-CSF was subcutaneously injected at a dose of 2.5 µg per mouse twice a day for 4 days. In the combination experiments, 18 hours after the last injection of G-CSF, mice were injected with either T-140 or AMD3100. Control mice were injected with phosphate-buffered saline (PBS) at the appropriate volume.
Cell Isolation and Differential Counts
Peripheral blood cells were collected from mice by cardiac puncture into tubes with heparin followed by lysis of erythrocyte population using a red blood cell lysis solution (0.155 M NH4Cl, 0.01 M KHCO3, 0.01 mM EDTA; pH 7.4). Cells were counted using a hemocytometer, and total number of cells per 1 ml of blood was calculated. In some experiments, blood samples were resuspended and processed to cytospin slides, centrifuged, air-dried, and stained with Giemsa. The percentage and the morphology of mobilized cells were determined by differential counts of Giemsa-stained cytospin slides.
Flow Cytometry
Flow cytometry was used to assess the number of cells in blood and distinguish between the different populations. Cells were gated according to forward scatter and side scatter to exclude dead cells and to determine granulocytes, mononuclear cells, and mature macrophage populations (Fig. 2E). The number of each cell population was counted. Cells were also stained in 0.1 ml of fluorescence-activated cell sorting (FACS) buffer with fluorescence antibodies directed against Gr-1, CD3, mac-1, F4/80, natural killer 1.1 (NK1.1), B220, and Ter-119 molecules or matched isotype controls (all from eBioscience, San Diego, CA, http://www.ebioscience.com) for 30 minutes and washed with FACS buffer. Immunostained cells were analyzed by flow cytometry using the FACSCaliber flow cytometer (BD Biosciences, San Diego, http://www.bdbiosciences.com); the data were analyzed using software from CellQuest (version 3.3; BD Biosciences).
Migration Assay
A migration assay with human Jurkat T cells was assayed by using Costar migration buffer (6.5 mm/diameter, 5 µm/pore; Corning Costar, Cambridge, MA, http://www.corning.com/lifesciences; RPMI 1640, 1% FCS) containing 2 x 105 Jurkat cells added to the upper chamber, and 0.6 ml of chemotaxis buffer with or without CXCL12 (50 ng/ml) and with or without T-140 or T-140 pretreated with proteinase K was added to the bottom chamber. Cells migrating within 4 hours to the bottom chamber of the Transwell were counted using FACSCalibur (BD Biosciences, San Jose, CA, http://www.bdbioscience.com).
HPC Assay
To evaluate the number of progenitor cells, a colony-forming cell assay was used. Burst-forming units-erythrocyte (BFU-E), colony-forming units granulocyte-macrophage (CFU-GM), colony-forming units megakaryocytes (CFU-M), and colony-forming units granulocyte-erythroblasts-macrophages-megakaryocyte (CFU-GEMM) were assayed by plating the cells in Iscove's modified Dulbecco's medium containing 1% methylcellulose, 15% fetal bovine serum, 1% bovine serum albumin, 3 U/ml recombinant human (rh) EPO, 104 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombinant mouse (rm)SCF, 10 ng/ml rmIL-3, 10 µg/ml rh insulin, 10 ng/ml rh interleukin-6 (IL-6), and 200 µg/ml human transferrin (Methocult GF M3434; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. Seven days later, typical colonies were visually scored by morphologic criteria using a light microscope, and the frequency of CFU was calculated. Staining colonies with benzidine dihydrochloride (Sigma-Aldrich) was used to localize hemoglobin-containing cells.
HSC Assay
C57BL/6 mice, serving as donors, were injected with 5 mg/kg of either T-140 or AMD3100. Two hours later, peripheral blood cells were collected, followed by erythrocyte lysis (as described above), and i.v. transferred into C57BL/6 recipient mice that had been pretreated with a lethal dose of irradiation (900 cGy) 24 hours before. Total cells obtained from 900 or 225 µl of blood were inoculated into a single recipient mouse in a total volume of 200 µl of PBS. Recipient mice that were inoculated with cells obtained from blood of untreated mice or with normal BM cells (5 x 106 cells per mouse) served as controls. The survival of mice was monitored for 4 months. Four months after the first transplantation, BM cells recovered from the first recipient mice repopulated by donor cells were injected i.v. into lethally irradiated secondary mice.
Statistical Analysis
Results are expressed as average ± SD. Statistical differences were determined by an analysis of two-tailed Student's t test. Values of p < .05 were considered to be statistically significant.
To evaluate whether the effect we observed for the combined treatments was synergistic, we used Student's t-test and compared the sum of values obtained for each different treatment (estimated additive effect) and the values obtained for the combined treatment. Synergistic effect was defined as an effect that was significantly greater (p < .05) than the sum of the individual effects.
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RESULTS |
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, 1, 2, 4, and 24 hours after injection of T-140, mice were sacrificed and cells were collected from blood. A single-dose administration of 5 mg/kg T-140 resulted in a rapid elevation in the total number of WBC in blood, a threefold change from the control after 30 minutes, slowly decreasing until full return to the baseline level after 24 hours (Fig. 1A). The characterization of the mobilized WBC, using a flow cytometer, showed alteration in the absolute number of granulocytes (stained positive with Gr-1), mononuclear cells (MNCs), and mature macrophages (stained positive with both mac-1 and F4/80 [13]). Further characterization using cytospin assay and Giemsa staining defined the granulocyte population as neutrophils. When we looked at the number of neutrophils in blood, there was an elevation starting 30 minutes postinjection, reaching a peak after 1–2 hours (more than a 12-fold change from control), and slowly decreasing to the baseline level after 24 hours (Fig. 1B). The same pattern was observed for mature macrophages; these cells reached a peak 1 hours postinjection of T-140 and returned to the baseline level after 24 hours (Fig. 1D). When we tested the effect of T-140 on the mobilization of MNCs, we observed a twofold rapid elevation change from control after 30 minutes, which rapidly decreased until full return to the baseline level after 4 hours (Fig. 1C).
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T-140 Synergizes with G-CSF to Mobilize WBC
Recently, the CXCR4 antagonist AMD3100 was reported to promote mobilization of stem cells and was found to synergistically augment G-CSF-induced mobilization of HPCs [9, 14]. Thus, we evaluated the mobilizing capability of T-140 in comparison with G-CSF, AMD3100, and the combination of both CXCR4 antagonists with G-CSF. Similarly to T-140, injection of AMD3100 (s.c.) induced the mobilization of WBC after 2 hours at the optimal concentration of 5 mg/kg. Both T-140 and AMD3100 induced a 2.5-fold increase in the number of WBC in blood compared with control. When G-CSF was injected subcutaneously at a dosage of 2.5 µg per mouse twice a day for 4 days, there was a 1.9-fold increase in the number of WBC in blood. To study the combination of G-CSF and either T-140 or AMD3100, the CXCR4 antagonists were injected at a concentration of 5 mg/ml, 18 hours after the fourth injection of G-CSF; 2 hours later, mice were sacrificed, and the number and type of cells mobilized in blood were tested.
The combination of T-140 with G-CSF induced a 5.1-fold increase in the number of WBC in blood (Fig. 3A). This induction was significantly higher than the increase seen in WBC stimulated by a combination of AMD3100 and G-CSF (3.7-fold increase over control; p = .006; Fig. 3A).
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T-140 and AMD3100 induced a significant and similar fold change in the number of MNCs in blood compared with control (2.3- and 3.4-fold increase over control, respectively; p < .005). Comparison with both CXCR4 antagonists showed that G-CSF, in our experimental model, is an inferior mobilizer of MNCs (Fig. 3B, 3D). The combination of G-CSF with T-140 induced a synergistic increase in the number of MNCs (4-fold increase over control), whereas the combination of G-CSF with AMD3100 did not induce such an effect (Fig. 3D). T-140 and AMD3100 also induced a significant change in the number of mature macrophages in blood compared with control (4.2- and 6.1-fold increase over control, respectively; p < .05). Comparison with both CXCR4 antagonists showed that G-CSF is also a poor mobilizer of mature macrophages (Fig. 3B, 3E). Similarly to the effect on MNCs, the combination of G-CSF with T-140 induced a synergistic increase in the number of MNCs (8.3-fold increase over control; p < .005), whereas the combination of G-CSF with AMD3100 did not induce such an effect (Fig. 3E). These results show that the CXCR4 antagonists are capable of mobilizing different subtypes of cells compared with G-CSF and that T-140, but not AMD3100, synergized with G-CSF to further stimulate the mobilization of MNCs and mature macrophages.
Effects of T-140 Administration on Progenitor Cell Mobilization
To further study the effect of T-140 on mobilization of HPCs, we collected cells from the blood of treated and untreated mice and tested for the presence of colony-forming cells. Injection of T-140 at a concentration between 1 and 10 mg/kg induced a dose-response increase (up to 10-fold over control) in the number of progenitor cells in blood (Fig. 4A; p < .005). Treatment of mice with T-140 induced a robust dose-response mobilization of myeloid CFU-GM colonies and the mobilization of multipotent progenitors CFU-M, CFU-GEMM and the erythroid progenitors BFU-E. Interestingly, we found that the most dramatic increase in progenitors was observed in the number of BFU-E, reaching a significant increase of more than 110-fold over control (p < .01; Fig. 4B).
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T-140 Synergizes with G-CSF to Mobilize HPCs
Blocking the CXCR4 receptor with AMD3100 resulted in the mobilization of HPCs [9, 15, 16]. When we compared the effect of T-140 (5 mg/kg) on the mobilization of progenitor cells to that of AMD3100 (5 mg/kg), we found that T-140 was significantly more potent in its ability to mobilize progenitor cells into blood. T-140 increased the number of progenitor cells in blood by 7.1-fold over control (p < .05), whereas AMD3100 induced the mobilization of progenitor cells by 4.2-fold over control (p = .001). G-CSF induced a 13.6-fold increase in the number of progenitors in blood over control, whereas in combination with T-140, it induced a 76.8-fold elevation in the number of progenitors over control. This progenitor increase induced by a combination of G-CSF and T-140 was significantly higher than the synergistic induction in the number of progenitors by the combination of G-CSF with AMD3100 (76.8-compared with 46.4-fold over control; p = .001; Fig. 5A).
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To determine whether the effects we observed were specifically related to the activity of T-140, we used the neutralized T-140 treated with proteinase K and compared its effect with the natural T-140. We found that treatment with proteinase K digests the peptide into smaller fragments, as demonstrated in supplemental online Figure 3A and 3B. These fragments did not inhibit the migration of Jurkat T cells toward CXCL12, the ligand for CXCR4, and could not induce the mobilization of WBC or progenitor cells (supplemental online Fig. 3C–3E, respectively). These results demonstrate that the mobilizing effect we observed was specifically related to presence of the intact T-140 peptide capable of interacting with CXCR4.
T-140 Mobilizes HSCs with Long-Term Repopulating Capacity
Although mobilization of HPCs may be of use for short-term repopulation in a transplant setting, HSCs are required for long-term repopulation. To assess the T-140 effect on mobilization of murine HSCs, we used a transplantation model in which donor C57Bl/6 mice were treated with T-140 or AMD3100. After 2 hours, blood was collected, and cells that were isolated from an equal blood volume (900 or 225 µl) were transferred into lethally irradiated recipients C57Bl/6 mice. Survival of recipient mice, as indicated by long-term repopulating activity and presence of HSCs, was monitored.
As shown in Figure 6A, 100% of mice transplanted with cells mobilized by T-140 (900 µl of blood) survived the treatment. In contrast, only 73% of mice transplanted with cells that were mobilized by AMD3100 survived the treatment (p = .06; Fig. 6A). Transplantation of cells mobilized by T-140 using a blood volume of 225 µl resulted in the survival of 70.6% of the treated mice, compared with the survival of only 37.5% of the treated mice when mobilization was performed by AMD3100 (p < .05). Although all untreated mice died after irradiation, 100% of mice survived irradiation when normal BM cells had been transplanted (Fig. 6A, 6B). To test for self-renewal and long-term repopulating activity, the transplanted mice were maintained for more than 4 months, and then their BM cells were injected into a second recipient. Stem cells mobilized by T-140 maintained 100% of the transplanted mice for longer than 4 months and were capable of producing long-term rescue in lethally irradiated secondary transplanted mice. These results indicate that T-140 is a powerful mobilizer of long-term repopulating stem cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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G-CSF was first identified as a colony-stimulating factor for granulocyte precursors and as a stimulator of granulocyte differentiation and activation [20]. Due to its activity, G-CSF is used to reduce neutropenia and increase the production of neutrophils [21, 22]. In addition to inducing the production and mobilization of neutrophils, G-CSF is also a powerful mobilizer of HSCs. Although several cytokines, such as GM-CSF, IL-3, stem cell factor, and flt-3 ligand, and chemokines, such as IL-8, were also shown to mobilize HPCs and HSCs [23–25], the cytokine G-CSF remains the major growth factor used for mobilization of stem cells for transplantation. The current protocols for stem cell mobilization necessitate 4–5 days of 1–2 s.c. injections of G-CSF; moreover, one or more pheresis procedures are required because of failure to collect the number of CD34+ cells necessary for successful transplantation. It is suggested that G-CSF induces the mobilization of HSCs through an indirect mechanism by activating neutrophils within the BM. Indeed, it was recently demonstrated that G-CSF stimulates neutrophils to secrete a variety of proteolytic enzymes, including elastase, cathepsin G, matrix metalloproteinase (MMP)-2, and MMP-9, that are capable of degrading CXCL12, the ligand for the chemokine receptor CXCR4, a key molecule that is expressed by the BM stroma and plays an important role in the retention of stem cells expressing CXCR4 within the BM [7, 26].
An important role for the interaction between CXCL12 and its receptor CXCR4 has been described for the mobilization of hematopoietic progenitors and stem cells [5–7]. More specifically, AMD3100, a selective antagonist of CXCR4, was recently shown to rapidly mobilize CD34+ HPCs from the human marrow into the peripheral blood. Furthermore, AMD3100 significantly increased both G-CSF-stimulated (10 µg/kg per day) mobilization of CD34+ cells (3.8-fold) and leukapheresis yield of CD34+ cells. Moreover, AMD3100-mobilized leukapheresis products contained significantly greater numbers of T and B cells compared with G-CSF-stimulated leukapheresis products [27]. AMD3100 was also reported to mobilize HSCs with long-term repopulating capacity in humans [28] and nonhuman primates [29]. In addition, AMD3100 was reported to be a safe and effective agent for the mobilization of CD34+ cells in patients with multiple myeloma or lymphoma receiving prior chemotherapy [30, 31]. On the other hand, a clinical trial with AMD3100 in HIV-infected individuals produced premature ventricular contraction side effects, resulting in the discontinuation of this trial [27].
On the basis of this knowledge, we evaluated the ability of a novel specific CXCR4 antagonist, known as T-140, to mobilize hematopoietic cells. T-140 and its analogues were first developed initially as anti-HIV agents that inhibit HIV-1 infection through their specific binding to CXCR4 [32, 33]. Unlike AMD3100, T-140 is a short, modified peptide. Comparative studies between T-140 and AMD3100 found that each of these agents inhibited CXCR4 via different mechanisms [34, 35]. Whereas T-140 binds residues in extracellular domains of the CXCR4 receptor and regions of the hydrophobic core proximal to the cell surface, amino acids in the central hydrophobic core are critical to binding of AMD3100 [34]. Analysis of antagonists revealed that exposure to AMD3100 induced G-protein activation by CXCR4, whereas T-140 decreased autonomous signaling [35]. Therefore, AMD3100 was defined to have a weak partial agonist activity, whereas T-140 functions as an inverse agonist.
Our results indicate that T-140 can efficiently induce mobilization of mature white blood cells, progenitors, and stem cells into blood within a few hours after treatment in a dose-dependent manner and can efficiently synergize with G-CSF for this effect. A variety of cell types express the CXCR4 receptor, including monocytes/macrophages, B and T lymphocytes, progenitors, and stem cells. Indeed, we found that T-140 can induce mobilization of all of these cell subsets. Although CXCR4 is also expressed by NK cells [36] and platelets [37], we could not demonstrate the effect of T-140 on their mobilization. Similarly, CXCR4/CXCL12 interaction was not found to be involved in platelet activation, as demonstrated by the inability of CXCL12 to augment aggregation or stimulate calcium mobilization of platelets [37].
Administration of T-140, AMD3100, and G-CSF can induce an effective mobilization of WBC into blood. However, whereas G-CSF induces mainly the exit of neutrophils, both CXCR4 antagonists induced a robust mobilization of MNCs and mature macrophages.
T-140 or AMD3100 alone induced the mobilization of a similar number of MNCs and activated macrophages. However, the combination of T-140 with G-CSF induced a significant increase in the mobilization of MNCs and mature macrophages into blood compared with AMD3100.
Even though mobilization of WBC is an important factor for transplantation, the major factor contributing to transplantation efficiency is the number of HPCs and HSCs in blood. When we looked at the effect of T-140 on mobilization of progenitor cells, we observed a significant elevation in the number of both myeloid pluripotent and erythroid progenitors, that is, CFU-GM, CFU-M, CFU-GEMM, and BFU-E. A comparative study demonstrated that administration of a single dose of T-140 resulted in a significant increase of progenitor cell mobilization compared with AMD3100. Moreover, the combination of T-140 with G-CSF resulted in a dramatic elevation in the number of progenitor cells in blood that was superior to G-CSF alone or to a combination of G-CSF with AMD3100. This superiority was highly significant when we witnessed the number of CFU-GEMM and BFU-E in the blood of treated mice.
Although improved mobilization of HPCs in response to T-140 is of interest, proof that T-140 also mobilizes HSCs is of paramount importance. A comparison of the abilities of T-140 and AMD3100 to mobilize HSCs into blood was made using a transplantation model. We found that T-140 is more potent in its ability to mobilize HSCs with long-term repopulating activity compared with AMD3100.
Mobilization of WBC and progenitor cells was synergistically enhanced when T-140 was used in combination with G-CSF, and this effect was significantly superior to that observed for the combination of G-CSF with AMD3100. Future studies should address the questions about the mechanism(s) by which T-140 induces cell mobilization, including studies regarding the blocking of the CXCR4 receptor and the immunogenicity properties of T-140.
In summary, our results indicate that the ability of the CXCR4 antagonist T-140 to mobilize WBC, as well as progenitor and stem cells, differs both qualitatively and quantitatively from that of the well-characterized CXCR4 antagonist AMD3100. Furthermore, T-140 differs from AMD3100 in its ability to efficiently synergize with G-CSF. It is therefore possible that an improved CXCR4 antagonist will provide better methods of collecting mobilized stem cells for transplantation. Future studies should address these issues and define the potential clinical advantage and usefulness of T-140 in clinical stem cell mobilization and transplantation.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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, p < .05 of T-140 compared with AMD3100. The data are the average ± SD of four mice per group from a total of four separate experiments performed. Abbreviations: AMD, AMD3100; FSC, side scatter; GCSF, granulocyte colony-stimulating factor; MNC, mononuclear cell; T140, 4F-benzoyl-TN14003; WBC, white blood cells.
