ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2137-2137 ◽  
Author(s):  
Linda J. Bendall ◽  
Robert Welschinger ◽  
Florian Liedtke ◽  
Carole Ford ◽  
Aileen Dela Pena ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Progenitors ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cell Mobilization ◽  
Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

Disruption of CXCR4 Utilizing AMD3100 Bypasses Mobilization Deficiency Exhibited by CD26−/− Mice and Results in Efficient Mobilization of Myeloid Progenitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3492-3492
Author(s):  
Laura A. Paganessi ◽  
Andrew L. Walker ◽  
Stephanie A. Gregory ◽  
Henry C. Fung ◽  
Kent W. Christopherson
Keyword(s):  
Bone Marrow ◽  
Blood Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Genetically Engineered Mice ◽  
Colony Forming Units ◽  
Myeloid Progenitors

Abstract The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood 2003 and Christopherson, et al Exp Hematol 2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/− mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2 inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2 and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/− mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/− mice as compared to untreated C57BL/6 and CD26−/− mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/− mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

The Disease-Related Bone Marrow Microenvironment Alters Hematopoietic Stem and Progenitor Function in Multiple Myeloma Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2898-2898
Author(s):  
Ingmar Bruns ◽  
Ron-Patrick Cadeddu ◽  
Ines Brückmann ◽  
Sebastian Buest ◽  
Julia Fröbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Microenvironment ◽  
Tgf Beta ◽  
Hematopoietic Stem ◽  
Healthy Donors ◽  
Stem And Progenitor Cells ◽  
Related Bone Marrow

Abstract Abstract 2898 Multiple myeloma (MM) patients often suffer from hematopoietic impairment already at the time of diagnosis with anemia as the prevailing symptom. Given the overt affection of the bone marrow in MM patients by the invasion of malignant plasma cells, we hypothesized that hematopoietic insufficiency in these patients may originate from a functional impairment of hematopoietic stem and progenitor cells. Quantitative analysis of BM CD34+ HSPC cell subsets from MM patients and age-matched healthy donors showed a significant decline of all HSPC subsets including hematopoietic stem cells, common myeloid and lymphoid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in MM patients. The greatest diminution was observed in megakaryocyte-erythrocyte progenitors (MEP) which were 4.9-fold reduced in comparison to healthy donors. Transcriptional analyses of CD34+ HSPC subsets revealed a significant deregulation of signaling pathways that was particularly striking for TGF beta signaling and suggested increased activation of this signaling pathway. Immunhistochemical staining of phosphorylated smad2, the downstream mediator of TGF receptor I kinase activation, in bone marrow sections and immunoblotting of purified CD34+ HSPC of MM patients confirmed the overactivation of TGF beta signaling. On a functional level, we observed significantly reduced long-term self-renewal and clonogenic growth, particularly of the erythroid precursors BFU-E and CFU-E, in CD34+ HSPC of MM patients which could be restored by inhibition of TGF beta signaling. Proliferation and cell cycle analyses revealed a significantly decreased proliferation activity in CD34+ HSPC and, particularly, MEP. Again, this was reversible after inhibition of TGF beta signaling. In addition, the transcriptional analyses showed disturbance of pathways involved in the adhesion and migration of HSPC and the gene encoding for the principal hyaluronan receptor CD44 throughout the HSPC subsets. This was corroborated by immunofluorescence imaging of CD44 on HSPC subsets showing a marked downregulation in the patients' cells. In line, the adhesion of CD34+ HSPC subsets to hyaluronan and their migration towards SDF-1 was significantly inhibited. Subsequent xenotransplantation of CD34+ HSPC from MM patients and healthy donors into myeloma-free recipients revealed even increased long-term engraftment of CD34+ HSPC obtained from MM patients and normal differentiation capacities suggesting that the observed functional alterations in fact depend on the MM-related bone marrow microenvironment. Our data show that hematopoietic impairment in patients with multiple myeloma originates, at least in part, from functional alterations of hematopoietic stem and progenitor cells. These alterations seem to depend on the disease-related changes of the bone marrow microenvironment. Currently, experiments are underway to elucidate in more detail the role of the microenvironment and the responsible structures for the impairment of HSPC in MM patients. These data will be presented. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy.

Prostaglandin E2 Inhibits Apoptosis in Hematopoietic Stem and Progenitor Cells and Enhances Their Survival Following Sub-Lethal Radiation Injury,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3401-3401
Author(s):  
Rebecca L Porter ◽  
Mary A Georger ◽  
Laura M Calvi
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Radiation Injury ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Cox 2 ◽  
Apoptotic Genes

Abstract Abstract 3401 Hematopoietic stem and progenitor cells (HSPCs) are responsible for the continual production of all mature blood cells during homeostasis and times of stress. These cells are known to be regulated in part by the bone marrow microenvironment in which they reside. We have previously reported that the microenvironmentally-produced factor Prostaglandin E2 (PGE2) expands HSPCs when administered systemically in naïve mice (Porter, Frisch et. al., Blood, 2009). However, the mechanism mediating this expansion remains unclear. Here, we demonstrate that in vivo PGE2 treatment inhibits apoptosis of HSPCs in naïve mice, as measured by Annexin V staining (p=0.0083, n=6–7 mice/group) and detection of active-Caspase 3 (p=0.01, n=6–7 mice/group). These data suggest that inhibition of apoptosis is at least one mechanism by which PGE2 expands HSPCs. Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. Thus, these studies explored the role of PGE2 signaling in the bone marrow following myelosuppressive injury using a radiation injury model. Endogenous PGE2 levels in the bone marrow increased 2.9-fold in response to a sub-lethal dose of 6.5 Gy total body irradiation (TBI)(p=0.0004, n=3–11 mice/group). This increase in PGE2 correlated with up-regulation of microenvironmental Cyclooxygenase-2 (Cox-2) mRNA (p=0.0048) and protein levels at 24 and 72 hr post-TBI, respectively. Further augmentation of prostaglandin signaling following 6.5 Gy TBI by administration of exogenous 16,16-dimethyl-PGE2 (dmPGE2) enhanced the survival of functional HSPCs acutely after injury. At 24 hr post-TBI, the bone marrow of dmPGE2-treated animals contained significantly more LSK cells (p=0.0037, n=13 mice/group) and colony forming unit-spleen cells (p=0.037, n=5 mice/group). Competitive transplantation assays at 72 hr post-TBI demonstrated that bone marrow cells from irradiated dmPGE2-treated mice exhibited increased repopulating activity compared with cells from vehicle-treated mice. Taken together, these results indicate that dmPGE2 treatment post-TBI increases survival of functional HSPCs. Since PGE2 can inhibit apoptosis of HSPCs in naïve mice, the effect of dmPGE2 post-TBI on apoptosis was also investigated. HSPCs isolated from mice 24 hr post-TBI demonstrated statistically significant down-regulation of several pro-apoptotic genes and up-regulation of anti-apoptotic genes in dmPGE2-treated animals (3 separate experiments with n=4–8 mice/group in each), suggesting that dmPGE2 initiates an anti-apoptotic program in HSPCs following injury. Notably, there was no significant change in expression of the anti-apoptotic gene Survivin, which has previously been reported to increase in response to ex vivo dmPGE2 treatment of bone marrow cells (Hoggatt et. al., Blood, 2009), suggesting differential effects of dmPGE2 in vivo and/or in an injury setting. Additionally, to ensure that this inhibition of apoptosis was not merely increasing survival of damaged and non-functional HSPCs, the effect of early treatment with dmPGE2 post-TBI on hematopoietic recovery was assayed by monitoring peripheral blood counts. Interestingly, dmPGE2 treatment in the first 72 hr post-TBI significantly accelerated recovery of platelet levels and hematocrit compared with injured vehicle-treated mice (n=12 mice/group). Immunohistochemical analysis of the bone marrow of dmPGE2-treated mice also exhibited a dramatic activation of Cox-2 in the bone marrow microenvironment. This suggests that the beneficial effect of dmPGE2 treatment following injury may occur, both through direct stimulation of hematopoietic cells and also via activation of the HSC niche. In summary, these data indicate that PGE2 is a critical microenvironmental regulator of hematopoietic cells in response to injury. Exploitation of the dmPGE2-induced initiation of an anti-apoptotic program in HSPCs may represent a useful method to increase survival of these cells after sub-lethal radiation injury. Further, amplification of prostaglandin signaling by treatment with PGE2 agonists may also represent a novel approach to meaningfully accelerate recovery of peripheral blood counts in patients with hematopoietic system injury during a vulnerable time when few therapeutic options are currently available. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PTPN11D61Y/+; Vavcre+ Animals Commonly Succumb to Leukemia Relapse Despite Robust Engraftment of Transplanted Wild-Type Hematopoietic Stem and Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 496-496
Author(s):  
Stefan P. Tarnawsky ◽  
Mervin C. Yoder ◽  
Rebecca J. Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Stem And Progenitor Cells ◽  
Leukemia Relapse ◽  
Conditioning Regimens

Juvenile Myelomonocytic Leukemia (JMML) is a rare childhood myelodysplastic / myeloproliferative overlap disorder. JMML exhibits myeloid populations with mutations in Ras-Erk signaling genes, most commonly PTPN11, which confer growth hypersensitivity to GM-CSF. While allogeneic hematopoietic stem cell transplant (HSCT) is the treatment of choice for children with JMML, 50% of children succumb to leukemia relapse; however, the mechanism leading to this high relapse rate is unknown. We hypothesized that the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, leading to poor engraftment of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and thus promoting leukemia relapse. Using Vav1 promoter-directed Cre, we generated a mouse model of JMML that conditionally expresses gain-of-function PTPN11D61Yin utero during development. While PTPN11D61Y/+; VavCre+embryos did not demonstrate in utero lethality, we observed a modest reduction of PTPN11D61Y/+; VavCre+ mice at the time of weaning compared to predicted Mendelian frequencies. Further, surviving PTPN11D61Y/+; VavCre+ mice developed elevated peripheral blood leukocytosis and monocytosis as early as 4 weeks of age compared to PTPN11+/+; VavCre+ controls. To address the hypothesis that an aberrant bone marrow microenvironment in the PTPN11D61Y/+ mice leads to poor engraftment of wild-type donor cells following transplant, we examined engraftment of wild-type hematopoietic stem and progenitor cells (HSPCs) in the PTPN11D61Y/+; VavCre+ mice and monitored animals for disease relapse. 16-24 week-old diseased PTPN11D61Y/+; VavCre+ and control PTPN11+/+; VavCre+ mice were lethally irradiated (11 Gy split dose) and transplanted with 5 x 105 CD45.1+ wild-type bone marrow low density mononuclear cells (LDMNCs), which simulates a limiting stem cell dose commonly available in a human HSCT setting. 6 weeks post-HSCT, PTPN11D61Y/+; VavCre+recipients demonstrated an unexpected elevated CD45.1+ donor cell contribution in peripheral blood compared to the control PTPN11+/+; VavCre+ recipients. However, despite superior engraftment in the PTPN11D61Y/+; VavCre+ recipients, these mice had a significantly shorter median survival post-HSCT due to a resurgence of recipient CD45.2-derived leukemic cells. We repeated the experiment using a high dose of CD45.1+ LDMNCs (10 x 106 cells) to determine if providing a saturating dose wild-type cells could prevent the relapse of recipient-derived leukemogenesis and normalize the survival of the PTPN11D61Y/+; VavCre+recipients. While this saturating dose of wild-type cells resulted in high peripheral blood chimerism in both the PTPN11D61Y/+; VavCre+ and PTPN11+/+; VavCre+ recipients, the PTPN11D61Y/+; VavCre+ animals nevertheless demonstrated significantly reduced overall survival. When we examined the cause of mortality in the HSCT-treated PTPN11D61Y/+; VavCre+mice, we found enlarged spleens, hypercellular bone marrow, and enlarged thymuses. Flow cytometry revealed that the majority of cells in the peripheral blood, bone marrow, and spleen were recipient-derived CD45.2+ CD4+ CD8+ T cells. To verify that the disease was neoplastic in origin, secondary transplants into CD45.1/.2 recipients were performed from two independent primary PTPN11D61Y/+; VavCre+and two independent primary PTPN11+/+; VavCre+ controls. Secondary recipients of bone marrow from PTPN11D61Y/+; VavCre+ animals rapidly succumbed to a CD45.2-derived T-cell acute lymphoid leukemia (T-ALL). Previous studies demonstrated that wild-type PTPN11 is needed to protect the integrity of the genome by regulating Polo-like kinase 1 (Plk1) during the mitosis of the cell cycle (Liu et al., PNAS, 2016). We now demonstrate that even when PTPN11 mutant animals are provided with saturating doses of wild-type HSCs, dysregulated residual recipient cells are able to produce relapsed disease. Collectively, these studies highlight the propensity of residual mutant PTPN11 cells to transform after being subjected to mutagenic agents that are commonly used for conditioning regimens prior to allogeneic HSCT. These findings suggest that modified pre-HSCT conditioning regimens bearing reduced mutagenicity while maintaining adequate cytoreductive efficacy may yield lower post-HSCT leukemia relapse in children with PTPN11mutations. Disclosures No relevant conflicts of interest to declare.

Suprasynergistic Peripheral Blood Stem Cell Mobilization in Normal and Fanconi Anemia Knockout Mice by the Combination of G-CSF Plus the CXCR4 Antagonist AMD3100 and the CXCR2 Agonist GRO β

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3185-3185
Author(s):  
Louis M. Pelus ◽  
D. Wade Clapp ◽  
Gary Bridger
Keyword(s):  
Gene Therapy ◽  
Fanconi Anemia ◽  
Peripheral Blood ◽  
Wild Type ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Large Numbers ◽  
Stem And Progenitor Cells ◽  
Cell Mobilization ◽  
Combination Regimens

Abstract In mice, the CXCR4 antagonist AMD3100 and the CXCR2 agonist GROβ rapidly mobilizes short and long term repopulating hematopoietic stem and progenitor cells (HSPC). Synergy in mobilization is observed using GRO plus G-CSF or AMD plus G-CSF, and we have recently shown synergy in rapid mobilization using AMD plus GROβ. In general, a common feature of mobilization is that only a relatively small percentage of HSPC egress from marrow. We therefore evaluated whether added benefit in HSPC mobilization could be attained by using all three mobilizers in combination. Although this alters the paradigm of rapid mobilization, it addresses shortcomings of poor mobilization response, requirements for multiple aphereses and the need for large numbers of HSPC in transplant and gene therapy applications. We mobilized BALB/c mice with AMD (5 mg/kg SC, 60 min), GROβ (2.5 mg/kg SC, 15 min), G-CSF (100 ug/kg/day, bid, SC x 4 days) or the G-CSF regimen followed by GROβ, AMD or GRO+AMD administered on day 5 and harvest of peripheral blood 15 (GRO; GRO+AMD) or 60 (AMD) min later. Significant CFU-GM/mL blood were mobilized by G-CSF (4362±996), GRO (2562±396) and AMD3100 (991±121) used alone as expected. Single administration of GRO or AMD to mice mobilized by G-CSF and harvest of blood 15 (GRO) and 60 (AMD) min later, resulted in synergistic mobilization of (12,246±2751) and (12,379±953) CFU-GM, respectively. Rapid mobilization by simultaneous injection of GRO+AMD was similar in magnitude (10,709±1041) at 15 min post administration to mobilization by GRO or AMD in combination with a multiday G-CSF regimen. Administration of the combination of GRO+AMD to mice mobilized by G-CSF resulted in suprasynergistic mobilization of 32,510±3569 CFU-GM/mL after 15 min, representing ~5% of total marrow CFU-GM, with no adverse effects. Fanconi Anemia patients mobilize poorly to G-CSF. FancC −/− mice present a phenotype similar to FancC patients and mobilize poorly to G-CSF, which can be improved by the addition of AMD. We evaluated mobilization by GRO, AMD and G-CSF alone and in combination in +/+ C57Bl and FancC −/− mice using the regimens described above. Mobilization by G-CSF was 45% lower in FancC −/− mice (858±21) compared to +/+ controls (1451±80) and AMD+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice (5078±597) than controls (2981±267). Similarly, CFU-GM mobilization by GRO was lower in FancC −/− mice and GRO+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice. The combination of GRO+AMD mobilized CFU-GM within 15 min that was similar in magnitude to mobilization by AMD+G-CSF in wild type (2077±541 vs 2511±176) as well as FancC −/− mice (4924±577 vs 5078±1597). Mobilization by addition of the rapid acting combination of GRO+AMD to mice mobilized by G-CSF was suprasynergistic reaching 44,669±2974 and 41,068±5630 CFU-GM/mL blood in wild type and −/− mice, respectively. In preliminary studies, transduction of mobilized blood cells with FancC and transplant in FancC −/−mice demonstrated durable engraftment. These studies identify highly effective, rapid GRO+AMD mobilization regimens for standalone application in normal donors and combination regimens for potential application in patients who respond poorly to G-CSF or when large quantities of HSPC are required, for example in gene therapy applications.

Patient Characteristics Associated with Successful Mobilization of Peripheral Blood Stem and Progenitor Cells Following Cytotoxic Chemotherapy and Peg-G-CSF Stimulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4920-4920
Author(s):  
Ingmar Bruns ◽  
Johannes C. Fischer ◽  
Akos G. Czibere ◽  
Cangül Kilic ◽  
Roland Fenk ◽  
...  
Keyword(s):  
Cell Count ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Serum Levels ◽  
Cytotoxic Chemotherapy ◽  
Cell Yield ◽  
Hematopoietic Stem ◽  
Peripheral Blood Cd34 ◽  
Stem And Progenitor Cells ◽  
Cell Mobilization

Abstract Mobilized peripheral blood stem and progenitor cells are nowadays widely used for transplantation of hematopoietic stem and progenitor cells (PBSCT). These cells can be mobilized into the peripheral blood with cytotoxic chemotherapy, cytokines or both. Currently, G-CSF is most frequently used due to its high efficacy and lack of serious toxicity. However, a serious patient-to-patient variation in the yield of peripheral blood stem and progenitor cells is a feature common of all mobilizations schemes. Therefore, factors determining the collection efficacy have been identified for G-CSF mobilization. Recently a polyethylenglycole-conjugated G-CSF (Peg-G-CSF) has been introduced which has a 12-fold longer half-life than the original compound and therefore leads to long-lasting G-CSF serum-levels after a single injection. Studies on Peg-G-CSF included only small cohorts and no attempts have been made to identify factors influencing the mobilization of blood stem and progenitor cells. Therefore, we retrospectively analyzed 101 unselected patients (66 with multiple myeloma, 26 with non-Hodgkin-lymphoma, 7 with Hodgkin’s disease, 1 with Ewing sarcoma, 1 with malignant germ cell tumor). 27% of patients had active disease, while all others where at least in partial remission after conventional chemotherapy. Patients were treated with a broad range of chemotherapy regimens. The number of cytotoxic chemotherapy cycles administered prior to the mobilization therapy ranged from 1 to 11 (median 4). Mobilization chemotherapy was followed by 6 mg or 12 mg Peg-G-CSF (median 6 mg). Median peripheral blood CD34+ cell maximum in all patients was 65.3/μl (range 0.2–1084 per μl). 12 mg Peg-G-CSF led to a significantly earlier CD34+ cell maximum in the peripheral blood compared to 6 mg Peg-G-CSF (median 13 days vs 15 days, respectively; p=0.01). Overall, a median yield of 8.5 x 10^6 CD34+ cells/kg bodyweight (range 0.2–72.4 x 10^6) was reached with a single apheresis (median, range 1–4). To search for predictors of hematopoietic stem and progenitor cell mobilization, multiple regression analysis was used and revealed CD34+ cell count/μl peripheral blood at the day of apheresis and the processed blood volume during apheresis as predictors for the CD34+ cell yield per kilogram bodyweight. Age, sex, disease type and status were not significantly related to the CD34+ cell count/μl peripheral blood nor the CD34+ cell yield. Interestingly, the number of previous chemotherapy cycles was correlated with the CD34+ cell maximum (p=0.027) with fewer chemotherapy cycles leading to a higher peripheral blood CD34+ cell count and vice versa. In contrast, radiation therapy prior to CD34+ cell mobilization led to a significantly later occurrence of the CD34+ cell maximum in the peripheral blood. Our results confirm the feasibility and efficacy of PBPC mobilization with single dose Peg-G-CSF after cytotoxic chemotherapy shown in previous clinical trials analyzing the largest patient cohort to date and predictors for successful stem cell mobilization with Peg-G-CSF could be identified.

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