Nonobese Diabetic/severe Combined Immunodeficient Research Articles

Systemic Correction of Storage Disease in MPS I NOD/SCID Mice Using the Sleeping Beauty Transposon System

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.

Monoclonal Antibody-Mediated Targeting of CD123, IL-3 Receptor α Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor alpha chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34(+)CD38(-) cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

Expression of CD133, a possible marker for cancer stem cells (CSCs), in small cell lung cancer (SCLC) cell lines and non- small cell lung cancer (NSCLC) cell lines

e22100 Background: Cancer stem cell (CSCs) are believed to play important roles in tumor development, recurrence or metastasis. Identification of CSCs may have a therapeutic significance. CD133 expression has been shown on a minority of various human cancer cells with high capability of self-renewal and proliferation. Therefore, CD133 is thought to be one of possible markers for CSCs. Regarding human lung cancers, the existence, prevalence or roles of CD133 positive cells has not been fully understood. Methods: We examined CD133 mRNA by quantitative real-time PCR and sorted CD133-positive cells by fluorescence-activated cell sorting (FACS) using human small cell lung cancer(SCLC) and non-small cell lung cancer (NSCLC) cell lines. We evaluated differences of cell proliferation between CD133-positive and -negative cells by MTS assay in vitro and by subcutaneous injection for non- obese diabetic/severe combined immunodeficiency (NOD/SCID) mice in vivo. Results: CD133 expression was almost restricted in SCLC cell lines. CD133 mRNA expression or CD133-positive cell population was scarcely observed in NSCLC cell lines. In two SCLC cell lines examined (NCI-H82 and NCI-H69), CD133 positive cells had higher tumorgenicity both in vivo and in vitro than NSCLC cell lines. Conclusions: The expression status of CD133 is totally different between NSCLCs and SCLCs, probably reflecting the difference of these progenitor cells. Our results indicate that CD133-positive cells in SCLC cell are responsible for tumor growth. However, in view of their wide prevalence, CD133-positive cells do not seem to be a candidate for CSCs, at least in cell lines. To investigate the molecular and functional characteristics of CD133-positive cells may lead to a new therapeutic strategy for human lung cancers, especially for SCLCs. No significant financial relationships to disclose.

Diffusion-Weighted Imaging as Predictor of Therapy Response in an Animal Model of Ewing Sarcoma

To evaluate the potential of diffusion-weighted imaging (DWI) for monitoring dose-dependent tumor response in a mouse-xenograft model of Ewing sarcoma after administration of treosulfan in different dosages. Ewing sarcoma implanted orthotopically into the left thigh of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were evaluated with a 9.4 T MR unit by using a specific mouse whole body coil. Transverse T2-weighted fast spin-echo sequences, T1-weighted spin-echo sequences, and transverse echo-planar DWI examinations were performed at baseline, 24 hours and 72 hours after intraperitoneal injection of treosulfan in 2 different doses. The apparent diffusion coefficient (ADC) in the viable parts of the tumor was automatically calculated from DWI imaging findings. These findings were correlated with histopathologic results at each time point. Volumetric measurements were performed by summing up the regions of interest in consecutive slices. T1- and T2-weighted images before administration of treosulfan showed viable tumor tissue with small necrotic areas. At 24 hours after treosulfan injection, a significantly higher increase in ADC in the viable parts of the tumor could be observed in tumors of mice that had been injected with the higher dose of treosulfan compared with mice injected with the lower dose treosulfan, whereas no significant volumetric differences between the 2 different doses could be observed. Seventy-two hours after injection of treosulfan the ADC values in the viable parts of the tumor of mice treated with the high dose of treosulfan further increased and the tumor volume in the high-dose group was now significantly lower than in the low-dose group. Compared with volumetric measurements, DWI of the viable tumor parts could be used to discriminate between the effects of 2 different dosages at an earlier time point than volumetric measurements in an animal model in vivo. This method could be especially useful for monitoring drug effects in phase I/II clinical trials.

Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis

Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.

CXCR4 mediates the homing of B cell progenitor acute lymphoblastic leukaemia cells to the bone marrow via activation of p38MAPK

The mechanisms regulating the migration of leukaemic cells between the blood and bone marrow compartments remain obscure, but are of fundamental importance for the dissemination of the disease. This study investigated the in vivo homing of human B cell progenitor acute lymphoblastic leukaemia (ALL) cells to the femoral bone marrow of non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. It was demonstrated that patient ALL cells use the chemokine axis, chemokine (CXC motif) receptor 4 (CXCR4)/ chemokine (CXC motif) ligand 12 (CXCL12), to home to the femoral marrow. CXCL12-mediated signalling through p38 mitogen-activated protein kinase (MAPK) was required for optimal homing. In contrast, the homing of normal peripheral blood CD34(+) cells and the cytokine-dependent CD34(+) cell line Mo7e was independent of p38MAPK, consistent with the dependence of these cells, as well as normal CD34(+) CD19(+) B cell progenitors, on PI-3K/AKT signalling. Altogether, our data provide clarification of the direct role of CXCL12 in the bone marrow homing of ALL cells and demonstrate unique signalling molecule usage that may have therapeutic implications for this disease.

Follicular Loss of the Cryopreserved Canine Ovary after Xenotransplantation

Abstract: The effect of cryopreservation and subsequent xenotransplantation on the follicular reserve of the canine ovary by using non obese diabetic-severe combined immunodeficient (NOD-SCID) mice was examined. Vitrified-warmed canine ovarian tissues were placed into the ovarian bursa of mice, and then were removed and subjected to histological examination at 4 weeks after the transplantation. Over 30% of primordial follicles and 65% of early primary follicles survived after cryopreservation. However, regardless of breed or age, percentages of survived primordial follicles and early primary follicles after the transplantation ranged from 0–7% and from 0–15%, respectively. These results indicate that the majority of primordial follicles and early primary follicles in vitrified-warmed canine ovarian tissues disappear after xenotransplantation. Further studies will be required to be able to enhance the survival of transplanted cryopreserved ovarian follicles in canine.

Isolation and Identification of Cancer Stem-Like Cells in Esophageal Carcinoma Cell Lines

Side population (SP) cells may play an important role in tumorigenesis and cancer therapy. We isolate and identify the cancer stem-like cells in human esophageal carcinoma (EC) cell lines, EC9706 and EC109 cells labeled with Hoechst 33342. Both the cell lines contained SP cells, and the cells that had the strongest dye efflux activity ("Tip"-SP cell) in EC9706 had higher clone formation efficiency than non-SP cells. When transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, "Tip"-SP cells showed at least 50 times higher tumorigenicity than non-SP cells. Microarray analysis discriminated a differential gene expression profile between "Tip"-SP and non-SP cells, which is further tested using quantitative real-time RT-PCR. It is ascertained that several important stem cell-related genes (including OCT-4, SOX-2, BMI-1, and ZFX), two ATP-binding cassette (ABC) transporter genes (ABCG2 and ABCA5), and three Wnt and two Notch signal pathway-related genes (such as FZD10, PTGS2, KLF5, TTK, and RBM15) were upregulated in "Tip"-SP cells. Western blot showed a higher expression of beta-catenin protein in "Tip"-SP cells. All these indicated that the minority population described as "Tip"-SP cells possessed cancer stem cell character. Further understanding of tumor stem cell-specific traits will offer insights on the early stages of tumorigenesis for prevention and enhanced selectivity of antitumor therapeutics.

High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle

AbstractEvidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (G0 residency) of murine and human hematopoietic cells. In human cord blood, quiescent fractions (CD34+CD38−HoechstloPyronin Ylo) express more GATA-2 than cycling counterparts. Enforcing GATA-2 expression increased quiescence of cord blood cells, reducing proliferation and performance in long-term culture-initiating cell and colony-forming cell (CFC) assays. Gene expression analysis places GATA-2 upstream of the quiescence regulator MEF, but enforcing MEF expression does not prevent GATA-2–conferred quiescence, suggesting additional regulators are involved. Although known quiescence regulators p21CIP1 and p27KIP1 do not appear to be responsible, enforcing GATA-2 reduced expression of regulators of cell cycle such as CCND3, CDK4, and CDK6. Enforcing GATA-2 inhibited human hematopoiesis in vivo: cells with highest exogenous expression (GATA-2hi) failed to contribute to hematopoiesis in nonobese diabetic–severe combined immunodeficient (NOD-SCID) mice, whereas GATA-2lo cells contributed with delayed kinetics and low efficiency, with reduced expression of Ki-67. Thus, GATA-2 activity inhibits cell cycle in vitro and in vivo, highlighting GATA-2 as a molecular entry point into the transcriptional program regulating quiescence in human hematopoietic stem and progenitor cells.

Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex

Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rgamma(null)) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rgamma(null) mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 x 10(6) PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-alpha signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.

Maitake Beta-Glucan Enhances Umbilical Cord Blood Stem Cell Transplantation in the NOD/SCID Mouse

Beta glucans are cell wall constituents of yeast, fungi and bacteria, as well as mushrooms and barley. Glucans are not expressed on mammalian cells and are recognized as pathogen-associated molecular patterns (PAMPS) by pattern recognition receptors (PRR). Beta glucans have potential activity as biological response modifiers for hematopoiesis and enhancement of bone marrow recovery after injury. We have reported that Maitake beta glucan (MBG) enhanced mouse bone marrow (BMC) and human umbilical cord blood (CB) cell granulocyte-monocyte colony forming unit (GM-CFU) activity in vitro and protected GM-CFU forming stem cells from doxorubicin (DOX) toxicity. The objective of this study was to determine the effects of MBG on expansion of phenotypically distinct subpopulations of progenitor and stem cells in CB from full-term infants cultured ex vivo and on homing and engraftment in vivo in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse. MBG promoted a greater expansion of CD34+CD33+CD38- human committed hematopoietic progenitor (HPC) cells compared to the conventional stem cell culture medium (P = 0.002 by ANOVA). CD34+CXCR4+CD38- early, uncommitted human hematopoietic stem cell (HSC) numbers showed a trend towards increase in response to MBG. The fate of CD34+ enriched CB cells after injection into the sublethally irradiated NOS/SCID mouse was evaluated after retrieval of xenografted human CB from marrow and spleen by flow cytometric analysis. Oral administration of MBG to recipient NOS/SCID mice led to enhanced homing at 3 days and engraftment at 6 days in mouse bone marrow (P = 0.002 and P = 0.0005, respectively) compared to control mice. More CD34+ human CB cells were also retrieved from mouse spleen in MBG treated mice at 6 days after transplantation. The studies suggest that MBG promotes hematopoiesis through effects on CD34+ progenitor cell expansion ex vivo and when given to the transplant recipient could enhance CD34+ precursor cell homing and support engraftment.

AKR‐501 (YM477) a novel orally‐active thrombopoietin receptor agonist

Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. We have searched for small molecule compounds that mimic the action of TPO by using human TPO receptor-expressed in Ba/F3 cells, resulting in the discovery of AKR-501 (YM477). AKR-501 specifically targeted the TPO receptor and stimulated megakaryocytopoiesis throughout the development and maturation of megakaryocytes just as rhTPO did. AKR-501, however, was shown to be effective only in humans and chimpanzees with high species specificity. Therefore, we examined the in vivo platelet-increasing effect of AKR-501 in human platelet producing non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver CD34(+) cells. Daily oral administration of AKR-501 dose-dependently increased the number of human platelets in these mice, with significance achieved at doses of 1 mg/kg and above. The peak unbound plasma concentrations of AKR-501 after administration at 1 mg/kg in NOD/SCID mice were similar to those observed following administration of an active oral dose in human subjects. These results suggest that AKR-501 is an orally-active TPO receptor agonist that may be useful in the treatment of patients with thrombocytopenia.

Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2–mediated resistance

AbstractDefects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. Here, we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells, whereas they do not affect viability of normal peripheral blood lymphocytes, suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential, and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note, XIAP inhibitors even overcome Bcl-2–mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo, they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL.

Transplantation of allogeneic T cells alters iron homeostasis in NOD/SCID mice

AbstractIron overload is common in patients undergoing allogeneic hematopoietic cell transplantation (HCT), but the mechanisms leading to overload are unknown. Here, we determined iron levels and the expression of iron regulatory proteins in the liver and gut of nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice that underwent transplantation with syngeneic (histocompatible) or allogeneic (histoincompatible) T lymphocytes. Infusion of histoincompatible T cells resulted in a significant rise in serum iron levels and liver iron content. Iron deposition was accompanied by hepatocyte injury and intestinal villous damage. Feeding of low- or high-iron diet was associated with appropriate ferroportin 1 and hepcidin responses in mice given histocompatible T cells, whereas mice given histoincompatible T cells showed inappropriate up-regulation of duodenal ferroportin 1 and a loss of expression of hepatic hepcidin. These findings suggest that alloreactive T cell–dependent signals induced dysregulation of intestinal iron absorption, which contributed to liver iron overload after HCT.

Blockade of maternal anti‐HPA‐1a–mediated platelet clearance by an HPA‐1a epitope–specific F(ab′)2 in an in vivo mouse model of alloimmune thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT) is most commonly caused by transplacental passage of maternal human platelet-specific alloantigen (HPA)-1a antibodies that bind to fetal platelets (PLTs) and mediate their clearance. SZ21, a monoclonal antibody (MoAb) directed against PLT glycoprotein IIIa, competitively inhibits the binding of anti-HPA-1a alloantibodies to PLTs in vitro. The purpose of this investigation was to determine whether SZ21 F(ab')(2) fragments might be therapeutically effective in inhibiting or displacing maternal HPA-1a antibodies from the fetal PLT surface and preventing their clearance from circulation. Resting human PLTs from HPA-1ab heterozygous donors were injected into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purified F(ab')(2) fragments of SZ21 or control immunoglobulin G (IgG) were injected intraperitoneally 30 minutes before introduction of HPA-1a antibodies. Blood samples were taken periodically and analyzed by flow cytometry to determine the percentage of circulating human PLTs. Anti-HPA-1a IgG from NAIT cases were able to efficiently clear HPA-1a-positive PLTs from murine circulation. Administration of SZ21 F(ab')(2) fragments not only inhibited binding of HPA-1a antibodies to circulating human PLTs, preventing their clearance, but also displaced bound HPA-1a antibodies from the PLT surface. F(ab')(2) fragments of HPA-1a-selective MoAb SZ21 effectively inhibit anti-HPA-1a-mediated clearance of human PLT circulating in an in vivo NOD/SCID mouse model. These results suggest that agents that inhibit binding of anti-HPA-1a to PLTs may have therapeutic potential in the treatment of NAIT.

Detection of Fetal Cells in the Maternal Kidney during Gestation in the Mouse

It has been reported that fetal cells migrate into maternal blood and organs. Since these fetal chimeric cells could be involved in maternal allogeneic tolerance to the fetus, the fetal chimeric cells might be implicated in maternal-fetal immunology and development of maternal autoimmune diseases. However, the mechanism and role of fetal microchimerism remains unclear. We aimed to describe the mechanism by which fetal cells become associated with maternal organs during pregnancy, using a mouse fetal microchimerism model. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) female mice, which are useful for tracking the behavior of fetal cells in the maternal body, were mated with transgenic males expressing enhanced green fluorescent protein (GFP), and the presence of GFP-positive cells were examined in peripheral blood and organs of pregnant mothers. By flow cytometry, we showed that 0.95 +/- 0.48% of mononuclear cells detected in the maternal peripheral blood were GFP-positive, and thus of fetal origin, during the first gestational week. This value decreased to 0.10 +/- 0.13% during the third gestational week (p < 0.05). GFP-positive cells were detected in the extraglomerular mesangial region and among the epithelial cells of the proximal renal tubule of the maternal kidney. These GFP-positive cells also expressed angiotensin II receptor subtype 2 (AT2), which is known to participate in regulating organogenesis and vasoreactivity. Fetal cells expressing AT2 may therefore be involved in the regulation of vascular tone in the maternal kidney. These observations suggest that fetal cells could influence maternal renal function through activation of the AT2 signaling.

Efficient tumour formation by single human melanoma cells.

A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells (0.1% to 0.0001%) have tumorigenic potential when transplanted into NOD/SCID mice. However, the extent to which NOD/SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/SCID IL2Rγnull mice, can increase the detection of tumorigenic melanoma cells by several orders-of-magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumors under these more permissive conditions. In single cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumors. Xenotransplantation assay modifications can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.

Correction of the Abnormal Trafficking of Primary Myelofibrosis CD34+ Cells by Treatment with Chromatin Modifying Agents

Primary myelofibrosis (PMF) is a myeloproliferative disorder characterized by abnormal trafficking of CD34+ cells, resulting in their constitutive mobilization. This abnormal trafficking of PMF CD34+ cells has been related to the reduced expression of the chemokine receptor CXCR4 (Shi et al, Cancer Research 67: 6417, 2007). We further tested this hypothesis by studying the homing of PMF CD34+ cells to the marrow and the spleens of sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and determining if this defect could be corrected by treatment with chromatin modifying agents. Following the infusion of PMF (n=10) or G-CSF mobilized peripheral blood (mPB) (n=4) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and assayable human CFU-GM were detected in the marrow of these mice (139±47 PMF CD34+ cells/106 BMCs vs. 1493±946 mPB CD34+ cells/106 BMCs, 2.0±0.8% of input PMF CFU-GM vs. 12.6±6.6% of input mPB CFU-GM, P<0.05). The CFU-GM were shown to be of human origin by plucking individual colonies and analyzing by RT-PCR for the human 17-alpha satellite gene and the murine GAPDH gene. Of the total CD34+ cells homing to the spleen and marrow of the NOD/SCID mice, 25% of PMF CD34+ cells and 2.4% of mPB CD34+ cells homed to the spleen. The abnormal trafficking of PMF CD34+ cells was associated with reduced expression of CXCR4 (15.3% vs.4.8%, P<0.05). PMF CD34+ cells from patients with (n=6) or without (n=4) the JAK2V617F mutation exhibited similar degrees of defective bone marrow(BM) homing, suggesting that the impairement of PMF CD34+ cell trafficking was not related to the JAK2 mutation. We have previously shown that the sequential treatment of PMF CD34+ cell with the DNA methyltransferase inhibitor, decitabine [5-aza-2′-deoxycytidine (5azaD)], followed by the histone deacetylase inhibitor, trichostatin A (TSA), resulted in the upregulation of CXCR4 (Shi et al, Cancer Research 67: 6417, 2007). We next determined if this upregulation of CXCR4 was associated with correction of the impaired trafficking of PMF CD34+ cell to the marrow of NOD/SCID mice. The percentage of CD34+ cells expressing CXCR4 was significantly enhanced by exposure of PMF CD34+ cells to 5azaD/TSA (primary CD34+ cells: 2.55±0.87%, cytokines alone: 6.51±1.80%, cytokines plus 5azaD/TSA: 12.47±3.65%). Treatment of PMF CD34+ cells in vitro with cytokines plus 5azaD/TSA for 9 days resulted in a 3-fold increase in the absolute numbers of CD34+CXCR4+ cells when compared to CD34+ cells exposed to cytokines alone and 61-fold increase of CD34+CXCR4+ cells when compared to primary PMF CD34+ cells. Treatment of PMF CD34+ cells with cytokines alone resulted in 3- to 9-fold greater homing to NOD/SCID marrow than that of primary PMF CD34+ cells (P=0.12), while equal number of PMF CD34+ cells treated with chromatin modifying agents had a 7- to 20-fold greater ability to home to mouse marrow than that of primary PMF CD34+ cells (P<0.05) and a 2- to 3-fold greater marrow homing ability than PMF CD34+ cells treated with cytokines alone (P<0.05). Treatment with 5azaD/TSA reduced by 36% the homing of PMF CD34+ cells to the spleen as compared to the treatment with cytokines alone. We conclude that PMF CD34+ cells preferentially home to the spleen rather than the marrow of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin modifying agents. Chromatin modifying agents represent a novel means of correcting the abnormal cellular trafficking characteristic of PMF, a therapeutic strategy which is about to be evaluated in the clinic.

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.

Preclinical Rationale for the Use of the Multikinase Inhibitor Sorafenib in the Treatment of Human Lymphomas

Introduction: The multikinase inhibitor Sorafenib (Nexavar, Bayer) exerts a remarkable activity against a variety of nonhematological tumors by blocking tumor cell proliferation and angiogenesis through the inhibition of the RAF/MEK/ERK pathway, as well as the receptor tyrosine kinases vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptor (PDGFR), c-KIT, Flt3, and RET. Several lines of evidence suggest that sorafenib might have a relevant clinical impact in the therapy of malignant lymphomas by overcoming the cytoprotective effects of ERK, Mcl-1, and Bcl-XL. However, preclinical data establishing a rationale for the clinical use of sorafenib in lymphomas are still lacking. The present studies aimed to investigate the activity and the mechanism(s) of action of sorafenib in human lymphomas.Methods: The effects of sorafenib were evaluated in vitro using a panel of six human cell lines of different phenotypes, including JVM-2 (B-Chronic Lymphocytic Leukemia), Granta-519 (Mantle Cell Lymphoma), DOHH2 (Follicular Lymphoma), SU-DHL-4V (Diffuse Large B-Cell Lymphoma), HD-MY-Z (Hodgkin Lymphoma), and KMS-11 (Multiple Myeloma) cell lines. Additionally, the antitumor efficacy and mechanism of action of sorafenib were investigated in vivo by means of five lymphoma xenograft models in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice.Results: In vitro, the response of cell lines to sorafenib (1–10 μM for 24–48 hours) was evaluated by detecting apoptotic cell death with the annexin-V/propidium iodide double staining assay, and viable cell countings with the Trypan blue dye exclusion test. All 6 cell lines responded to sorafenib with values of 50% inhibitory concentrations ranging from 1 to 7.5 μM. In contrast, normal CD34+ cells remain insensitive to the drug up to 15 μM. Despite significant rates of sorafenib-induced apoptosis were seen in all cell lines, activation of caspase-3 analyzed by fluorescent-activated cell sorter was only detected in DOHH-2 and JVM-2 cell lines. The phosphorylation status of mitogen-activated protein kinase (MAPK) was investigated using the human phospho-MAPK Array kit (R&D systems), analyzed with the open source imaging software ImageJ (http://rsb.info.nih.gov/ij/), and then validated by Western blotting. Sorafenib induced a significant reduction of pAkt1, pAkt2, and pAkt3 in SU-DHL-4V, Granta-519, and JVM-2 cell lines, whereas p38 phosphorylation levels were significantly reduced in all but one cell line (KMS-11). Reduced levels of pMEK, pERK1 and pERK2 were detected in SU-DHL-4V, KMS-11, Granta-519, and HD-MY-Z cell lines. Down-regulation of MCL-1 was seen in HD-MY-Z, JVM-2, and DOHH-2 cell lines. In vivo, the activity of sorafenib was evaluated in NOD/SCID mice bearing subcutaneous tumor nodules. Animals with tumors averaging from 140 to 160 mg were randomly grouped to receive sorafenib (90 mg/kg body weight, IP, once daily for 15 days) or control vehicle. Sorafenib significantly (P ≤0.001) reduced the growth of subcutaneous HD-MY-Z, KMS-11, Granta-519, SU-DHL-4V, and JVM-2 nodules, with values of tumor growth inhibition of 70%, 52%, 40%, 37%, and 24%, respectively. In control mice, TUNEL staining of tumor sections showed large areas of viable cells without significant necrosis, whereas a 2- to 5-fold increase of necrotic areas was detected in sorafenib-treated mice bearing the different lymphoma xenografts. Analysis of tumor vasculature by means of in vivo biotinylation of endothelial cells with sulfo-NHS-LC-biotin showed a 30% to 60% reduction of vessel density in sorafenib-treated mice bearing the different lymphoma xenografts.Conclusions: Sorafenib efficiently targets a variety of human lymphomas representative of different phenotypes by inhibiting tumor angiogenesis and directly affecting tumor cell survival. Our preclinical data establish a rationale for exploring the clinical activity of sorafenib in human lymphomas.