Addition Of AMD3100 Research Articles

Upregulation of C-X-C motif chemokine 12 in the spinal cord alleviated the symptoms of experimental autoimmune encephalomyelitis in Lewis rats.

C-X-C motif chemokine 12 (CXCL12) is a chemokine that performs many functions. Studies have shown that CXCL12 can aggravate inflammatory symptoms in the central nervous system (CNS). Evidence also indicates that CXCL12 can promote the repair of myelin sheaths in the CNS in experimental autoimmune encephalomyelitis (EAE). Here, we investigated the function of CXCL12 in CNS inflammation by upregulating CXCL12 in the spinal cord and subsequently inducing EAE. CXCL12 upregulation in the spinal cords of Lewis rats was induced by the injection of adeno-associated virus 9 (AAV9)/eGFP-P2A-CXCL12 after intrathecal catheter implantation. Twenty-one days after AAV injection, EAE was induced and clinical score was collected; Immunofluorescence staining, WB and LFB-PAS staining were used to evaluate the effect of CXCL12 upregulation. In the in vitro study, oligodendrocyte precursor cells (OPCs) were harvested, cultured with CXCL12 and AMD3100, and subjected to immunofluorescence staining for functional assessment. CXCL12 was upregulated in the lumbar enlargement of the spinal cord by AAV injection. In each stage of EAE, upregulation of CXCL12 significantly alleviated clinical scores by inhibiting leukocyte infiltration and promoting remyelination. In contrast, the addition of AMD3100, which is a CXCR4 antagonist, inhibited the effect of CXCL12. In vitro, 10 ng/ml CXCL12 promoted the differentiation of OPCs into oligodendrocytes. AAV-mediated upregulation of CXCL12 in the CNS can alleviate the clinical signs and symptoms of EAE and significantly decrease the infiltration of leukocytes in the peak stage of EAE. CXCL12 can promote the maturation and differentiation of OPCs into oligodendrocytes in vitro. These data indicate that CXCL12 effectively promotes remyelination in the spinal cord and decreases the signs and symptoms of EAE.

Mesenchymal stromal cell-associated migrasomes: a new source of chemoattractant for cells of hematopoietic origin

BackgroundMultipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell–cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication.ResultsMigrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34+ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34+ progenitors, leukemic cells can take up migrasomes.ConclusionOverall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment.1ydn92XG6FRTxRi1UCUyVwVideo

Combination of indirect revascularization and endothelial progenitor cell transplantation improved cerebral perfusion and ameliorated tauopathy in a rat model of bilateral ICA ligation

ObjectiveEndothelial progenitor cells (EPCs) contribute to the recovery of neurological function after ischemic stroke. Indirect revascularization has exhibited promising effects in the treatment of cerebral ischemia related to moyamoya disease and intracranial atherosclerotic disease. The role of EPCs in augmenting the revascularization effect is not clear. In this study, we investigated the therapeutic effects of indirect revascularization combined with EPC transplantation in rats with chronic cerebral ischemia.MethodsChronic cerebral ischemia was induced by bilateral internal carotid artery ligation (BICAL) in rats, and indirect revascularization by encephalo-myo-synangiosis (EMS) was performed 1 week later. During the EMS procedure, intramuscular injection of EPCs and the addition of stromal cell-derived factor 1 (SDF-1), and AMD3100, an SDF-1 inhibitor, were undertaken, respectively, to investigate their effects on indirect revascularization. Two weeks later, the cortical microcirculation, neuronal damage, and functional outcome were evaluated according to the microvasculature density and partial pressure of brain tissue oxygen (PbtO2), regional blood flow, expression of phosphorylated Tau (pTau), TUNEL staining and the rotarod performance test, respectively.ResultsThe cortical microcirculation, according to PbtO2 and regional blood flow, was impaired 3 weeks after BICAL. These impairments were improved by the EMS procedure. The regional blood flow was further increased by the addition of SDF-1 and decreased by the addition of AMD3100. Intramuscular injection of EPCs further increased the regional blood flow as compared with the EMS group. The rotarod test results showed that the functional outcome was best in the EMS combined with EPC injection group. Western blot analysis showed that the EMS combined with EPC treatment group had significantly decreased expressions of phosphorylated Tau and phosphorylated glycogen synthase kinase 3 beta (Y216 of GSK-3β). pTau and TUNEL-positive cells were markedly increased at 3 weeks after BICAL induction. Furthermore, the groups treated with EMS combined with SDF-1 or EPCs exhibited marked decreases in the pTau expression and TUNEL-positive cells, whereas AMD3100 treatment increased TUNEL-positive cells.ConclusionThe results of this study suggested that indirect revascularization ameliorated the cerebral ischemic changes. EPCs played a key role in augmenting the effect of indirect revascularization in the treatment of chronic cerebral ischemia.

Abstract PR-001: Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma

Abstract Introduction: Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The C-X-C chemokine receptor type 4 (CXCR4)/chemokine (C-X-C motif) ligand 12 (CXCL12) axis is a key immunosuppressive pathway where CXCL12 released by cancer-associated fibroblasts (CAF) binds to its receptor CXCR4 expressed by cytotoxic CD8+ T cells (CTL) which results in their decreased abundance within tumors. Encouraging preclinical and clinical data from mouse PDAC models and human PDAC tumor sections have shown T cell tumor accumulation after co-inhibition of CXCR4 and PD1/PDL1, however number of tumor regressions were limited. Results: To further explore the mechanism by which inhibition of the CXCR4/CXCL12 pathway modulates the immune TME in triggering an anti-tumor immune response in human PDAC, we generated an autologous co-culture system with patient-derived tumor organoids (PDTO) and peripheral blood mononuclear cells (PBMC). PDAC tumor tissue from surgical resection was used to generate lines of PDTO that recapitulate the histological and genetic characteristics of the original tumor. We demonstrate that tumor-specific reactive T cells can be obtained in PDAC by co-culture with autologous PDTO, with up to 5% of CD8+ T cells producing IFNγ after tumor organoid stimulation. No T cell reactivity was observed against normal pancreatic organoids, and blocking tumor MHC I/MHC II decreased the proportion of activated T cells. Ex vivo migration assays were established to better understand the role of CXCL12 in PDAC tumor T cell exclusion. Tumor organoids attracted autologous PBMC and addition of recombinant CXCL12 decreased PBMC migration, while treatment of PBMC with the CXCR4 inhibitor AMD3100, rescued the number of PBMCs that were able to migrate towards the tumor organoids. PBMC infiltrated PDTO-containing but not empty Matrigel domes. Addition of AMD3100 increased PBMC infiltration within the Matrigel dome containing PDTOs. Finally, we found that pre-treatment of PBMC with AMD3100 increased T cell reactivity against PDTO, and that T cell activation was further increased when stimulated with PDTO pre-treated with chemotherapy. Conclusions: We found that CXCL12 modulates the migration of T cells towards tumor organoids generated from pancreatic cancer tissue, and that inhibition of CXCR4 not only increases the migration potential, but also increases tumor-specific T cell activation. The use of ex vivo autologous co-culture and migration assays provide a unique strategy to identify modulators that may enhance T cell mediated neoplastic cell death to guide therapeutic intervention in PDAC. Citation Format: Emily Alouani, Ilenia Pellicciotta, Winston Wong, Alexander S. Thomas, Michael D. Kluger, Anna M. Chiarella, Anil K. Rustgi, Gulam A. Manji. Ex vivo co-culture system with patient-derived organoids to assess CXCR4 inhibitor as an immune modulating agent for human pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-001.

Icaritin promotes maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signal pathway

<b>Objective:</b> To investigate the effect of icaritin on maturation and mineralization of mouse osteoblast MC3T3-E1 cells and its mechanism. <b>Methods:</b> The cultured MC3T3-E1 cells were divided into blank control group, CXC chemokine receptor type 4 (CXCR4) inhibitor (AMD3100) group, icaritin group, and icaritin plus AMD3100 group. The expression of CXCR4, stromal cell-derived factor 1 (SDF-1) and osteogenesis-related genes and proteins were detected by real-time RT-PCR and Western blotting after drug treatment for 24 h. The alkaline phosphatase (ALP) activity was determined with ALP kit on d3 and d6; calcium nodules were detected by alizarin red staining after drug treatment for 14 d. <b>Results:</b> Real time RT-PCR showed that compared with the blank control group, relative expressions of <i>CXCR4, SDF-1</i> and osteogenesis-related genes in icaritin group were significantly increased (<i>P</i><0.05 or <i>P</i><0.01); After AMD3100 treatment, the relative expression of <i>CXCR4</i> gene was decreased (<i>P</i><0.05). Western blot showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related proteins in the icaritin group were significantly increased (all <i>P</i><0.01), but were decreased after AMD3100 was added (all <i>P</i><0.01). The ALP activity of icaritin group was significantly higher than that of blank control group (all <i>P</i><0.01) on d3 and d6 after drug treatment, while the activity of ALP was significantly decreased after AMD3100 treatment (all <i>P</i><0.01). At d14 after drug treatment, compared with the blank control group, the area of alizarin red staining was increased in the icaritin group, while it was significantly reduced after the addition of AMD3100. <b>Conclusion:</b> Icaritin may promote maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signaling pathway.

Effect of Irradiation on Tumor Microenvironment and Bone Marrow Cell Migration in a Preclinical Tumor Model

To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts. Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2Gy/d for a total dose of 20Gy using a 5days on, 2days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40Gy in 4weeks using the same 2-Gy/d regimens. Over the 2weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31±0.02mm(3)/d and 0.55±0.04mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5%±3%), hypoxia (10%±3.5%), vascular endothelial growth factor (2.3%±1%), and stromal-derived factor-1α (2.5%±1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8%±2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6%±0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT. Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs.

224 Stromal Derived Factor 1 Alpha is a Mediator of Conditioning in Human and Rat Myocardium

BackgroundWe have recently demonstrated that stromal cell-derived factor 1α (SDF-1α) limits myocardial infarct size (IS) in a murine model of ischaemia-reperfusion injury (IRI) via its receptor CXCR4. This study aimed...

The role of SDF-1α–ECM crosstalk in determining neural stem cell fate

The consequences of central nervous system injury are far-reaching and debilitating and, while an endogenous repair response to neural injury has been observed in recent years, the mechanisms behind this response remain unclear. Neural progenitor/stem cell (NPSC) migration to the site of injury from the neural stem cell niches (e.g. subventricular zone and hippocampus) has been observed to be vasophilic in nature. While the chemotactic stimuli directing NPSC homing to injury is not well established, it is thought to be due in part to an increasing gradient of chemotactic cytokines, such as stromal cell-derived factor 1α (SDF-1α). Based on these recent findings, we hypothesize that critical crosstalk between SDF-1α and the extracellular matrix (ECM) drives injury-induced NPSC behavior. In this study, we investigated the effect of SDF-1α and ECM substrates (Matrigel, laminin, and vitronectin) on the migration, differentiation, and proliferation of NPSCs in vitro using standard assays. The results demonstrated that SDF-1α and laminin-based ECM (Matrigel and laminin) significantly and synergistically enhanced NPSC migration and acute neuronal differentiation. These effects were significantly attenuated with the addition of AMD3100 (an antagonist against the SDF-1α receptor, CXCR4). SDF-1α alone significantly increased NPSC proliferation regardless of ECM substrate, however no synergy was observed between SDF-1α and the ECM. These results serve to elucidate the relationship between adhesive and soluble signaling factors of interest and their effect on NPSC behavior following neural injury. Furthermore, these results better inform the next generation of biomaterials aimed at stimulating endogenous neural regeneration for neural injury and neurodegenerative diseases.

CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway

Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension.

Abstract 1511: CXCR4 inhibition sensitizes prostate cancer cells to radiotherapy in an in vitro co-culture model

Abstract Background: Tumor microenvironment is an important factor influencing radiosensitivity of cancer cells. Amongst multiple soluble factors released by tumor-associated stromal cells, chemokine CXCL12 was shown to confer pro-survival effects on cancer cells expressing chemokine receptor 4 (CXCR4). It has been recently shown that stromal microenvironment protects prostate cancer cells from chemotherapy-induced cell death via CXCR4 signaling, which was reversed by AMD3100, a specific CXCR4 inhibitor. In this study, AMD3100 was tested for its radiosensitizing capacity in prostate cancer. Methods: Both short-term (72 h) and long-term survival (17 days) of PC3 prostate cancer cells in the absence or presence of murine stromal cells (MS5) after increasing doses of gamma-irradiation (0-15 Gy) was assessed. To analyze the short-term effect of stromal cells on PC3 viability after radiation, fluorescently labeled PC3 cells were cultured with or without MS5 cells and exposed to gamma-irradiation (0-15 Gy) in the presence or absence of 25 µg/ml AMD3100. DAPI nuclear staining was used to assess the viability of PC3 after 72 hours, Long-term survival of PC3 cells was evaluated by clonogenic assay of PC3 cells seeded alone or on feeder layer of MS5-GFP stromal cells and irradiated with 0-15 Gy. Results: The short-term viability assay showed that PC3 in mono-culture show dose-dependent loss of viability after increasing doses of irradiation, showing only 18±6% (mean ± SD) viability when exposed to 15 Gy as compared to non-irradiated control cells. However, in the presence of stromal cells, viability of PC3 cells exposed to the same radiation dose remained high, reaching levels of 69±2% viability (P=0.004 vs 15 Gy in monoculture). This effect was potently reversed by AMD3100, which lowered viability of cells to 46±3% (P=0.001 vs 15 Gy in co-culture without AMD3100). Importantly, addition of AMD3100 to PC3 cultured in the absence of stromal cells did not change their viability after radiation. The long-term survival measured in clonogenic assay showed that in presence of MS5-GFP murine stromal cell line, 8% PC3-luc cells survived 6 Gy gamma-radiation dose, as compared to only 3.6% of PC3 cells that were cultured without the stromal cells (P=0.03). Conclusion: Murine stromal cells protect PC3 prostate cancer cells against irradiation-induced cell death in an in vitro co-culture system. Treatment with AMD3100 potently inhibits the protective effect of stromal cells on the prostate cancer cells exposed to gamma-irradiation. These data underscore the relevance of stromal microenvironment in tumor response to irradiation and warrant further research on the role of CXCR4 inhibitors as a therapeutic option to potentiate radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1511. doi:1538-7445.AM2012-1511

Abstract 3339: A role for CXCR4 signalling in the regulation of normal and malignant breast stem cell activity

Abstract CXCR4 is a chemokine receptor specific for stromal derived factor-1 (SDF-1) and is highly expressed in breast cancer cell lines and primary breast cancer samples. Elevated expression of CXCR4 in breast cancer is linked to poor prognosis. CXCR4 signalling has been reported to be important in breast cancer migration and metastasis formation but its role is yet to be fully elucidated. Our previously published data have shown that a stem cell-enriched population of cells can be obtained by harvesting anoikis resistant cells which survive in early mammosphere culture (Harrison et al., Cancer Res; 2010, 70 (2), 709-18). We collected anoikis resistant cells 12 hours after plating out into the mammosphere culture to obtain stem cell-enriched populations from 2 immortalised (MCF10A, 226L) and 3 malignant breast cell lines (MCF7, T47D, SKBR3). Anoikis resistant cells harvested at 12 hours had a significantly higher mammosphere colony-forming efficiency than their unsorted monolayer cells (2-4 fold increase, p &amp;lt; 0.05) suggesting stem cell enrichment. In limiting dilution transplantation assays into athymic Nude mice, MCF7 and T47D anoikis resistant cells had a 12 and 16-fold enrichment for tumor formation respectively, compared to the unsorted monolayer cells. Using an Agilent custom microarray, we found that CXCR4 expression was consistently upregulated in these stem cell-enriched populations compared with monolayer cells (2.0 and 6.8 fold; normal and malignant cell line averages respectively). We further investigated the function of CXCR4 in vitro using the mammosphere assay to assess stem cell activity. AMD3100 (Sigma), a CXCR4 antagonist and lentiviral CXCR4 shRNA were used to inhibit and recombinant soluble SDF-1 to stimulate the pathway. In the normal breast cell line MCF10a, SDF-1 addition significantly reduced mammosphere formation in a dose dependent manner (200ng/ml; 25% decrease, t = 4.320, p &amp;lt; 0.001) and this could be rescued by the addition of AMD3100. Inhibiting the CXCR4 pathway stimulated primary mammosphere formation in the breast cancer cell lines T47D and MCF7 using either 20uM AMD3100 (T47D; 34% increase, t = 3.017, p &amp;lt; 0.01) or lentiviral CXCR4 shRNA (T47D; 97% increase, t = 6.288 p &amp;lt; 0.005, MCF7; 48% increase, t = 3.026, p &amp;lt; 0.05). In vivo data has already shown the importance of the CXCR4/SDF-1 pathway in metastasis formation in breast cancer. Our data takes suggests that CXCR4 signalling, as well as its role in migration and homing, is important in maintaining stem cell activity. Thus CXCR4 targeted therapy, alongside current standards of care, may improve breast cancer outcomes. Funding: Biotechnology and Biological Sciences Research Council (BBSRC), Vertex Pharmaceuticals (Europe) Limited, Breast Cancer Campaign and Cancer Research UK (CRUK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3339. doi:10.1158/1538-7445.AM2011-3339

E0193 A critical role of ckit in CXCR4-mediated progenitor cell niche maintenance and mobilisation

Therapeutic mobilisation of bone marrow progenitor cells (BM PCs) is a novel strategy for cardiovascular repair. Both CXCR4 antagonism and c-kit blockade can rapidly and potently mobilise BM PCs; however,...

Inhibitory effect of CXC chemokine receptor 4 antagonist AMD3100 on bleomycin induced murine pulmonary fibrosis

CXC chemokine receptor 4 (CXCR4), which binds the stromal cell-derived factor-1 (SDF-1), has been shown to play a critical role in mobilizing the bone marrow (BM)-derived stem cells and inflammatory cells. We studied the effects of AMD3100, CXCR4 antagonist, on a murine bleomycin-induced pulmonary fibrosis model. Treatment of mice with AMD3100 in bleomycin-treated mice resulted in the decrease of SDF-1 in bronchoalveolar lavage (BAL) fluids at an early stage and was followed by the decrease of fibrocytes in the lung. AMD3100 treatment decreased the SDF-1 mRNA expression, fibrocyte numbers in the lung at an early stage (day 3) and CXCR4 expression at the later stage (day 7 and 21) after bleomycin injury. The collagen content and pulmonary fibrosis were significantly attenuated by AMD3100 treatment in later stage of bleomycin injury. AMD3100 treatment also decreased the murine mesenchymal and hematopoietic stem cell chemotaxis when either in the stimulation with bleomycin treated lung lysates or SDF-1 in vitro. In BM stem cell experiments, the phosphorylation of p38 MAPK which was induced by SDF-1 was significantly blocked by addition of AMD3100. Our data suggest that AMD3100 might be effective in preventing the pulmonary fibrosis by inhibiting the fibrocyte mobilization to the injured lung via blocking the SDF-1/CXCR4 axis.

Pegylated Recombinant Murine GM-CSF Is a Potent Mobilizer of Murine Bone Marrow Progenitors, Synergizes with BIO5192 and Plerixafor (AMD3100), and Skews Mobilized Cells to a Tolerogenic Phenotype.

Abstract 2432Poster Board II-409Granulocyte-macrophage colony stimulating factor (GM-CSF) is a naturally occurring acidic glycoprotein growth factor that regulates proliferation and differentiation of hematopoietic stem and progenitor cells. Inflammation results in the release of GM-CSF from numerous cell types including T-cells, macrophages, and neutrophils. Murine GM-CSF (mGM-CSF), unlike human GM-CSF, has a very short half-life in vivo. Pegylation of mGM-CSF (peg-mGM-CSF) has been shown to prolong its half-life.There are numerous reports of GM-CSF use in humans to mobilize stem cells. It was observed by our group that patients who were transplanted with stem cell products mobilized by GM-CSF had a lower incidence of acute GVHD (Devine et al. BBMT 2005; 36:531). The reason for this is unclear. A recent report suggested that regulatory T-cells (T-regs) express the low affinity alpha subunit of the mGM-CSF receptor and can be expanded ex vivo in the presence of GM-CSF and independent of IL-2 (Kared et al. Blood 2008;112:2575). We hypothesized that peg-mGM-CSF may expand T-regs in vivo and act synergistically with the CXCR4 antagonist Plerixafor (AMD3100) and a small molecule inhibitor of VLA-4, BIO5192, to generate optimal peripheral blood stem cell products enriched in hematopoietic progenitors and T-regs.Peg-mGM-CSF was manufactured in a bioreactor as previously described (Sainathan et al. Prot Express and Purification 2005;44:94). C57/Bl6 mice were injected with peg-mGM-CSF 5ug IP, daily x 4 days then bled and analyzed for CFU progenitor assays (CFU-C) and FACS analysis. The same mice were then injected with AMD3100 (5mg/kg SC) with or without BIO5192 (1mg/kg IV) and serially bled at 30 minutes, 1 hour, 3 hours, and 6 hours. CFU-C were performed and blood and spleen were analyzed for T-cell and dendritic cell subsets by FACS. We observed that peg-mGM-CSF was a potent mobilizer of hematopoietic progenitors and resulted in leukocytosis (mean difference 17.98 K/uL +/- 2.15, 95%CI 13.4 - 22.53, p<0001, n=6) and splenomegally. Peg-mGM-CSF increased CFU-C by 26.5 fold compared to PBS treated mice (n=6, 95% CI = 10.25-42.73). Peg-mGM-CSF had synergistic effects on progenitor mobilization when combined with AMD3100 (89.8 fold, 95% CI 61.6-118, n=6) or BIO5192 (94 fold, 95% CI 46.5-141.6, n=6) compared to PBS treated mice. The combination of peg-mGM-CSF, AMD3100 and BIO5192 resulted in 119.7 fold (95% CI 63.7-175.6, n=6) increase in progenitor mobilization over PBS treated mice and peaked at 30 minutes. In addition, peg-mGM-CSF increased circulating CD4+ FoxP3+ T-regs from a mean of 10.66% +/- 2.65 to 23.86% +/- 4.7 at 5 days (n=15, p=0.001 by paired t-test). Splenic T-regs were also increased from 16.09% +/- 2.7 to 31.56% +/- 4.34 (n=9, p=0.001 by paired t-test). This effect was sustained out to 4 days after the last dose of peg-mGM-CSF. Furthermore, there was no significant change in thymic T-regs, and this effect was not significantly affected by the addition of AMD3100 or BIO5192. The function of these peg-mGM-CSF T-regs, as measured in a MLR was found to be equivalent to T- regs from PBS treated control mice (n=3, p=0.27, by paired t-test). Interestingly, analysis of the spleens from animals on day 5 after peg-mGM-CSF demonstrated an increase in absolute CD11c+ cells, as expected, and a 4 fold increase in the absolute number of plasmacytoid dendritic cells as determined by CD11c+ SiglecH+ staining. Additional studies are underway to assess the functional impact of murine peripheral blood stem cells mobilized with peg-mGM-CSF in combination with small molecule inhibitors of the CXCR4 and VLA-4 axes on engraftment and graft versus host disease in a murine acute GVHD parent to F1 model. Disclosures:DiPersio:Genzyme Corporation: Honoraria.

Short-Term Culture of Umbilical Cord Blood–Derived CD34 Cells Enhances Engraftment Into NOD/SCID Mice Through Increased CXCR4 Expression

Human umbilical cord blood (CB) has been used successfully in stem cell transplantation. A subpopulation of CD34(+) cells expresses chemokine receptor CXCR4 which is critical for bone marrow engraftment in human hematopoietic stem cells. Here, we demonstrate the effect of short-term culture on CXCR4 expression on umbilical CB-derived CD34(+) cells and subsequent engraftment capability in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Surface CXCR4 expression on CD34(+) cells increased after incubating the cells in medium alone for 2 h; this effect was blocked by the addition of AMD3100. No difference in CXCR4 mRNA expression was noted after incubating CD34(+) cells in culture for 2 h, although these cells showed significantly increased transmigrational activity toward SDF-1 and homing activity in NOD/SCID mice. Furthermore, cultured human CD34(+) cells showed improved engraftment into the bone marrow of NOD/SCID mice compared to noncultured or AMD3100-treated CD34(+) cells. These observations suggest that increased cell surface expression of CXCR4 on CD34(+) cells improved the engraftment of human umbilical CB cells into bone marrow through enhanced homing activity.

Short-Term Culture of Umbilical Cord Blood-Derived Stem Cells Enhances the Engraftment into NOD/SCID Mice through the Increased Expression of CXCR4.

Human umbilical cord blood (CB) is rich source of hematopoietic stem cells (HSCs) and provides an attractive alternative to bone marrow or mobilized peripheral blood transplantation. However, major disadvantage of CB transplantation is the relatively low number of HSCs in each CB unit, which severely limits its usefulness in clinical transplantation. A part of CD34+ cells expresses cell surface CXCR4, which has been found to be critical for bone marrow engraftment by human hematopoietic stem cells. In the present study, we examined the effect of short-term culture on CXCR4 expression and engraftment in CB-derived CD34+ cells transplanted into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. CB-derived CD34+-enriched samples were divided into three aliquots and cultured. The first aliquot was incubated for 2 h in RPMI-1640 medium alone, whereas the second was incubated for 2h in RPMI-1640 medium containing the specific CXCR4 antagonist, AMD3100. As a control, the third sample was neither treated nor incubated. Surface CXCR4 expression on CD34+ cells significantly increased after incubating the cells in medium alone for 2 h (7.7 to 19.8%); this increase was blocked by the addition of AMD3100. No difference in CXCR4 mRNA expression was noted after incubating CD34+ cells for 2 h. The short-term incubation seems to influence the translocation of CXCR4 from the interior of the cell to the cell surface. CD34+ cells cultured for 2 h showed significantly increased transmigrational activity toward SDF-1 and homing activity in NOD/SCID mice. However, those were lower in non-cultured cells and cells treated with AMD3100. Next, we assessed the engraftment of cells cultured into NOD/SCID mice. Engraftment analysis was performed 8 weeks after transplantation. The presence of human cells was detected by the cell surface expression of CD45, CD34, CD33, and CD19. The CD34+ cells cultured demonstrated significantly increased engraftment of human cells in the bone marrow of NOD/SCID mice compared with that of CD34+ cells without culture or with culture in the presence of AMD3100 (p < 0.01). The recipients exhibited CD19+ B-lymphoid cells, CD33+ myeloid cells, and CD34+ immature cells, but neither CD3+ T cells nor CD45+ cells in bone marrow. Finally, we performed a secondary transplant of engrafted cells from previously transplanted mice to confirm long-term engraftment and self-renewal of cultured CB cells. The successful engraftment was observed in mice injected with primary cultured cells, demonstrating that these cultured cells are self-renewing and capable of long-term engraftment. These observations suggest that the increase of the cell surface expression of CXCR4 on CD34 cells resulted in the retention of human umbilical cord blood cells within the bone marrow through the engraftment of homing activity.

Interplay of CXCR4 and FLT3-ITD in the Regulation of Stem Cell Migration and Proliferation.

Activating mutations of the FLT3-receptor tyrosine kinase such as the short internal tandem duplication (ITD) represent the single most common genetic aberration in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are present in about 25% of AML patients and are associated with high blast counts and a higher rate of relapse after conventional chemotherapy. CXCR4 and its ligand SDF-1 are of central importance for stem cell homing to the bone marrow. In FLT3-ITD positive AML, CXCR4 expression on stem cells is negatively correlated to overall survival. Using retroviral vector transduction we achieved overexpression of the human FLT-ITD transgene, the FLT3-wt or the GFP control gene in greater than 70% of cord blood hematopoietic progenitors (HP). In contrast to previously published data in murine cell lines, we found that FLT3-ITD positive human cells display strongly reduced migration towards the CXCR4 ligand SDF-1. Analysis of SOCS3 (suppressor of cytokine signaling 3) expression revealed strongly increased mRNA expression in FLT3-ITD overexpressing CD34+HP and FLT3-ITD positive AML blasts. Since SOCS3 is a known antagonist of CXCR4 signaling, increased SOCS3 levels might cause the observed inhibition of SDF-1 dependent migration. For individually tested CD34+HP from 6 cord blood donors, co-cultivation of FLT3-ITD overexpressing HP on the irradiated mouse stromal cell line M210-B4 or on primary mesenchymal stromal cells resulted in an increased formation of early cobble stones and a dramatic proliferation advantage for suspended cells compared to naïve, FLT3-wt or GFP-transduced HP. Addition of the CXCR4 chemokine inhibitor AMD3100 to the co-culture decreased the cobble stone formation and growth advantage of FLT3-ITD transduced cord blood HP. Similar results were obtained using FLT3-ITD positive AML cells from 3 patients. Using CFSE to track cell proliferation and PI/Annexin staining to analyze apoptosis, we demonstrate that the addition of AMD3100 had no effect on apoptosis but decreased the proliferation rate of FLT3-ITD positive blast cells. Using primary AML blasts and FLT3-ITD transgene overexpressing cord blood HP, we demonstrate that the robust FLT3-ITD mediated proliferation advantage can be partly inhibited by the CXCR4 antagonist AMD3100.

Efficacy of systemic administration of SDF-1 in a model of vascular insufficiency: Support for an endothelium-dependent mechanism

Studies have reported that administration of stromal cell-derived factor-1 (SDF-1), the ligand for the G-protein coupled receptor CXCR4, increased collateral blood flow in a mouse model of vascular insufficiency via recruitment of endothelial precursor cells (EPC). The present study investigated the contribution of mature endothelial cells in the actions of SDF-1. The regulation of SDF-1 and CXCR4 was examined in the rat cornea cauterization (CC) and aortic ring (AR) model. The functional significance of the SDF-1/CXCR4 pathway was explored in cultured endothelial cells, the AR model, and on collateral blood flow in a rat model of vascular insufficiency. In the present study, the CXCR4 transcript was dramatically upregulated in the rat CC and AR explants, systems containing and lacking bone marrow-derived EPCs, respectively. Addition of AMD3100, a selective CXCR4 antagonist, had no effect on vessel growth in the AR alone, but completely inhibited SDF-1 mediated increases in vascular sprouting. In cultured endothelial cells, SDF-1 alone or in combination with vascular endothelial growth factor (VEGF) significantly enhanced cell survival and migration. Finally, systemic administration of SDF-1 in a rat model of arterial insufficiency enhanced collateral blood flow above vehicle control and equal to that of VEGF after 2 weeks of treatment. These studies support activation of the SDF-1/CXCR4 axis as a means to promote blood vessel growth and enhance collateral blood flow, at least in part, via direct effects on vascular endothelial cells.

The CXCR4 Antagonist AMD3100 Mobilizes a More Primitive Subset of CD34+ Cells Than G-CSF.

The CXCR4-antagonist AMD3100 mobilizes peripheral blood progenitor cells (PBPC) in 8–10 hours while a standard mobilization regimen with granulocyte colony-stimulating factor (G-CSF) takes 4 days before significant numbers of PBPC can be detected. Recently it was shown that AMD3100-mobilized PBPC have a higher reconstitution potential in the NOD/SCID mouse model than G-CSF mobilized PBPC. Clinically they also mediate rapid and sustained engraftment after transplantation. Prospective comparative studies are yet lacking. It can be expected that the functional differences are also reflected on the phenotypic level. We used 6-color and 3-color flow cytometry to study the co-expression of primitive and lineage-associated markers on mobilized PBPC of patients with multiple myeloma and non-Hodgkin's lymphoma (n=5). Mobilization treatment consisted of 5 days G-CSF (10 μg/kg, s.c. AM) and a single dose of AMD3100 (240 μg/kg, s.c.) in the evening of day 4. CD34+ cells of day 4, before AMD3100-dosing (G-CSF group) and on day 5, 10-hours after AMD3100 (AMD3100+G-CSF group) were compared. Primitive CD34+/CD38- cells which solely mediate hematopoietic reconstitution in the NOD/SCID mouse model were increased after addition of AMD3100 in all patients by a median of 2,58 fold (range 1,19–6,66) while the myeloid lineage committed CD34+/CD117+ and the actively proliferating CD34+/CD71+ cell subsets were significantly downregulated (p<0,05 each). The myeloid CD34+/CD33+ cell population showed a similar trend (median 0,60-fold change, range 0,24–1,08). Adhesion molecules were significantly regulated (CD49b) or showed such a trend (CD49d). Quantification of aldehyde dehydrogenase (AlDH) positive cells in A+G vs. G-CSF-mobilized samples did not show significant differences between these groups. We conclude that AMD3100 mobilizes a more primitive subset of CD34+ progenitor cells than G-CSF alone. Whether this translates into lower CD34+ threshold values for transplantation to achieve a similarly rapid and sustained engraftment after transplantation or into a shorter reconstitution period if similar amounts of CD34+ cells are used as after standard G-CSF-mobilization needs further study.CD34 coexpression analysis: AMD3100+G-CSF mobilized CD34+ cells/G-CSF mobilized CD34+ cells (fold-change)CD34+/medianminmaxP valueCD38−2.581.192.090.073HLA-DR−1.140.142.090.568CD90+0.420.0526.470.417CD7+1.170.582.90.433CD10+0.870.116.660.513CD19+1.110.7212.690.368CD33+0.60.241.080.075CD45RA+5.841.547.460.155CD71+0.510.430.640.002CD117+0.360.021.110.046CD18+1.550.564.090.347CD49b+6.912.7411.750.08CD49d+0.630.250.890.061CD58+5.822.8321.880.261CD62L2.91.434.360.419CD184+0.990.251.790.955

SDF-1/CXCL12 Enhances Survival of Murine Embryonic Stem Cells and Their Production of Myeloid Progenitors, but Not Hemangioblasts.

Stromal cell derived factor (SDF)-1/CXCL12 has been shown to promote the survival of embryonic retinal ganglion cells, peritoneal B1a (PerBla) lymphocytes and chronic lymphocytic leukemia B cells. Our previous studies implicated SDF-1 as an important factor in enhancing survival of murine bone marrow (BM) hematopoietic stem cells and, human cord blood and adult human BM myeloid progenitors. Since pluripotent embryonic stem (ES) cells can give rise to differentiated cell types derived from all three primary germ layers (endoderm, mesoderm, and ectoderm) and adult stem cells are generated during embryoid body (EB) formation, we investigated whether SDF-1 has effects on survival of ES cells and EB generation of hematopoietic progenitor cells. In order to establish SDF-1 expression patterns during EB formation, we screened supernatants during day 1–5 EB formation for SDF-1 production by three murine ES cell lines (E14, R1 and CCE). We observed low but detectable SDF-1 secreted in cultures of ES cells and day 1 stage EBs. SDF-1 was increased in the media from day 2 stage EBs and continued to increase through days 4–5. CXCR4, SDF-1 receptor, expression was also analyzed. CXCR4 mRNA expression was low in ES cells and day 1–3 EBs, and increased significantly from Day 4 EBs, reaching maximum levels at day 5, and decreasing after day 6. Surface CXCR4 expression was consistent with mRNA data. To determine if SDF-1 had an effect on ES cell survival, we cultured ES cells without serum, and added serum at either 0, 24, 48 or 96 hrs to each of the following groups: A) Control, B) SDF-1 (100ng/ml) or C) AMD3100 (1 μM), an SDF-1 receptor (CXCR4) antagonist. Colonies were scored 7 days after the addition of serum. SDF-1 enhanced survival of ES cells, while AMD3100 decreased survival. We also checked the apoptosis of ES cells after withdrawing serum for 24, 48, 72 and 96 hours in four groups: A) control, B) SDF-1 (100ng/ml), C) AMD3100 (1 μM) or D) AMD3100 (1 μM) and SDF-1 (100ng/ml). SDF-1 decreased apoptosis and AMD3100 blocked the SDF-1 effect. AMD3100 alone increased apoptosis compared to control. This suggests that AMD3100 blocked endogenous SDF-1 effects. To determine if SDF-1 had an effect on differentiation of hematopoietic progenitor cells, we added SDF-1 (100 ng/ml), AMD3100, or AMD3100 plus SDF-1 (100 ng/ml) at the beginning of EB formation, immediately after removal of LIF, and quantitated primitive erythroid (p-BFU-E), definitive erythroid (d-BFU-E), granulocyte-macrophage (CFU-GM) and multipotential Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) colony formation. In comparison to control cells (cultured without SDF-1 and AMD3100), SDF-1 increased numbers of p-BFU-E, d-BFU-E, CFU-GM, and CFU-GEMM colonies. Addition of AMD3100 with SDF-1 blocked the enhancing effect of SDF-1. In addition, significantly decreased numbers of colonies were also observed in the presence of AMD3100 alone. This suggests that AMD3100 blocks endogenous SDF-1 actions, consistent with our data on SDF-1 production during EB formation. In order to determine when SDF-1 starts affecting hematopoiesis, hemangioblast colony assays were used. Neither SDF-1 nor AMD3100 influenced hemangioblast colony formation or expression of Flk-1 mRNA, a marker of hemangioblasts. The results suggest a role for SDF-1 in ES cell growth and differentiation.