CANCER STEM CELLS Enhanced Unique Pattern of Hematopoietic Cell Mobilization Induced by the CXCR4 Antagonist 4F-Benzoyl-TN14003 MICHAL ABRAHAM,a KATIA BIYDER,a MICHAL BEGIN,a HANNA WALD,a IDO D. WEISS,a EITHAN GALUN,a Downloaded from ARNON NAGLER,b AMNON PELEDa 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 www.StemCells.com ABSTRACT An increase in the number of stem cells in blood following cells (WBC) in blood, including monocytes, B cells, and T mobilization is required to enhance engraftment after high- cells, it had no effect on mobilizing natural killer cells. dose chemotherapy and improve transplantation outcome. T-140 was found to efficiently synergize with granulocyte Therefore, an approach that improves stem cell mobilization colony-stimulating factor (G-CSF) in its ability to mobi- is essential. The interaction between CXCL12 and its recep- lize WBC and progenitors, as well as to induce a 660-fold tor, CXCR4, is involved in the retention of stem cells in the increase in the number of erythroblasts in peripheral at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009 bone marrow. Therefore, blocking CXCR4 may result in blood. Comparison between the CXCR4 antagonists mobilization of hematopoietic progenitor and stem cells. We T-140 and AMD3100 showed that T-140 with or without have found that the CXCR4 antagonist known as 4F-benzo- G-CSF was significantly more potent in its ability to yl-TN14003 (T-140) can induce mobilization of hematopoi- mobilize hematopoietic stem cells and progenitors into etic stem cells and progenitors within a few hours post- blood. These results demonstrate that different CXCR4 treatment in a dose-dependent manner. Furthermore, antagonists may have different therapeutic potentials. although T-140 can also increase the number of white blood STEM CELLS 2007;25:2158–2166 Disclosure of potential conflicts of interest is found at the end of this article. Granulocyte colony-stimulating factor (G-CSF)-mobilized INTRODUCTION peripheral-blood mononuclear cells are routinely used as a source of HSCs for transplantation. Despite the potency of All mature blood cells are derived from hematopoietic stem G-CSF in mobilizing stem cells, it results in broad interindi- cells (HSCs) through intermediates that are termed hematopoi- vidual variations in circulating progenitor and stem cell numbers etic progenitor cells (HPCs) [1]. Hematopoietic cells at various [4], requiring repeated dosing, and is frequently associated with stages of differentiation are localized within the bone marrow side effects. Thus, optimal and improved methods to mobilize (BM), their main site of production. Their trafficking between and collect peripheral-blood progenitor and stem cells for he- BM and blood is a physiological process [2, 3], but under matopoietic rescue are warranted. steady-state conditions, HPCs and HSCs circulate in peripheral Over recent years, it has become apparent that the interac- blood at very low frequency. Stem cell frequencies in peripheral tion between CXCL12 and its receptor, CXCR4, plays a pivotal blood can be considerably increased both in response to various role in hematopoietic cell mobilization and engraftment [5–7]. growth factors and during the recovery phase following myelo- The CXCR4 receptor is widely expressed on many cell types, suppressive chemotherapy, a process termed mobilization. Cir- including HSCs and HPCs, and the interaction with its ligand culating stem cells are capable of homing to the BM and was found to be involved in chemotaxis, homing, and survival. regenerating normal hematopoiesis following myeloablative The CXCL12/CXCR4 axis is also involved in the retention of conditioning with high doses of chemotherapy or radiation. hematopoietic cells within the BM microenvironment [8]; con- Recently, the use of peripheral blood as a source of stem cells sequently, the disruption of CXCL12/CXCR4 interactions re- for patients undergoing autologous and allogeneic transplanta- sults in mobilization of hematopoietic cells. Indeed, blocking tion has replaced BM as the preferred source of hematopoietic the CXCR4 receptor with an antagonist, such as AMD3100, rescue. Under steady-state conditions, HPCs and HSCs circulate results in the mobilization of HPCs. Moreover, combining in blood at frequencies too low to allow for efficient collection AMD3100 with G-CSF has produced an additive effect [9]. of numbers sufficient for transplantation. Therefore, an increase These approaches suggest that antagonizing the interactions of in the number of HSCs in blood following mobilization im- BM-produced CXCL12 with CXCR4 that is expressed on HSCs proves transplantation efficiency by shortening the duration of could be an effective HSC mobilizing strategy. cytopenia, enhances immune reconstitution, and reduces mor- Today, there are several known CXCR4 antagonists that bidity. have been described as having different levels of efficiency [10, 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: [email protected] Received March 6, 2007; accepted for publication May 15, 2007; first published online in STEM CELLS EXPRESS May 24, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0161 STEM CELLS 2007;25:2158–2166 www.StemCells.com Abraham, Biyder, Begin et al. 2159 11]. 4F-benzoyl-TN14003 (T-140) is a short modified peptide Migration Assay (supplemental online Fig. 1), a highly selective CXCR4 antag- A migration assay with human Jurkat T cells was assayed by using onist, originally designed as a human immunodeficiency virus Costar migration buffer (6.5 mm/diameter, 5 ␮m/pore; Corning (HIV) entry inhibitor through specific binding to CXCR4. T-140 Costar, Cambridge, MA, http://www.corning.com/lifesciences; is a competitive inhibitor that hinders CXCL12-mediated cell RPMI 1640, 1% FCS) containing 2 ϫ 105 Jurkat cells added to the proliferation and migration [12]. upper chamber, and 0.6 ml of chemotaxis buffer with or without To determine the therapeutic potential of T-140, we evalu- CXCL12 (50 ng/ml) and with or without T-140 or T-140 pretreated ated the capacity of T-140 to mobilize hematopoietic cells, alone with proteinase K was added to the bottom chamber. Cells migrat- Downloaded from and in combination with G-CSF. In addition, we compared the ing within 4 hours to the bottom chamber of the Transwell were effect of T-140 to that of another CXCR4 antagonist, counted using FACSCalibur (BD Biosciences, San Jose, CA, http:// AMD3100. www.bdbioscience.com). HPC Assay MATERIALS AND METHODS 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 www.StemCells.com Reagents units megakaryocytes (CFU-M), and colony-forming units granu- AMD3100 was purchased from Sigma-Aldrich (Rehovot, Israel, locyte-erythroblasts-macrophages-megakaryocyte (CFU-GEMM) http://www.sigmaaldrich.com). G-CSF (Neupogen [filgrastim], were assayed by plating the cells in Iscove’s modified Dulbecco’s Amgen, Thousand Oaks, CA, http://www.amgen.com) was kindly medium containing 1% methylcellulose, 15% fetal bovine serum, provided by Prof. Arnon Nagler. T-140 was kindly provided by 1% bovine serum albumin, 3 U/ml recombinant human (rh) EPO, Biokine Therapeutics, Ltd. The activity of T-140 was neutralized by 104 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng/ml recombi- incubating with Proteinase K (DAKO, Glostrup, Denmark, http:// nant mouse (rm)SCF, 10 ng/ml rmIL-3, 10 ␮g/ml rh insulin, 10 www.dako.com) for 20 minutes at 37°C followed by 10 minutes of ng/ml rh interleukin-6 (IL-6), and 200 ␮g/ml human transferrin at Bernman National Medical Library, Hebrew University of Jerusalem on April 18, 2009 incubation at 95°C. (Methocult GF M3434; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). The cultures were incubated at Mice and Experimental Protocol 37°C in a humidified atmosphere containing 5% CO2. Seven days later, typical colonies were visually scored by morphologic criteria Female C57BL/6 mice (7–8 weeks old) were purchased from Har- using a light microscope, and the frequency of CFU was calculated. lan Israel (Rehovot, Israel, http://www.harlanisrael.com) and main- Staining colonies with benzidine dihydrochloride (Sigma-Aldrich) tained under specific pathogen-free conditions at the Hebrew Uni- was used to localize hemoglobin-containing cells. versity 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 HSC Assay with various doses of T-140 or AMD3100 (1, 2.5, 5, and 10 mg/kg) C57BL/6 mice, serving as donors, were injected with 5 mg/kg of in a total volume of 200 ␮l, 2 hours before sacrifice. In some either T-140 or AMD3100. Two hours later, peripheral blood cells experiments, mice were sacrificed at different time points: 1⁄2,1,2, were collected, followed by erythrocyte lysis (as described above), 4, and 24 hours postinjection. G-CSF was subcutaneously injected and i.v. transferred into C57BL/6 recipient mice that had been at a dose of 2.5 ␮g per mouse twice a day for 4 days. In the pretreated with a lethal dose of irradiation (900 cGy) 24 hours combination experiments, 18 hours after the last injection of G- before. Total cells obtained from 900 or 225 ␮l of blood were CSF, mice were injected with either T-140 or AMD3100. Control inoculated into a single recipient mouse in a total volume of 200 ␮l mice were injected with phosphate-buffered saline (PBS) at the of PBS. Recipient mice that were inoculated with cells obtained appropriate volume.