Irradiated SCID Mice Research Articles

Isolation and (111)In-Oxine Labeling of Murine NK Cells for Assessment of Cell Trafficking in Orthotopic Lung Tumor Model.

A noninvasive in vivo imaging method for NK cell trafficking is essential to gain further understanding of the pathogenesis of NK cell mediated immune response to the novel cancer treatment strategies, and to discover the homing sites and physiological distribution of NK cells. Although human NK cells can be labeled for in vivo imaging, little is known about the murine NK cell labeling and its application in animal models. This study describes the isolation and ex vivo radiolabeling of murine NK cells for the evaluation of cell trafficking in an orthotopic model of human lung cancer in mice. Scid-Tg(FCGR3A)Blt transgenic SCID mice were used to isolate NK cells from mouse splenocytes using the CD49b (DX5) MicroBeads positive selection method. The purity and viability of the isolated NK cells were confirmed by FACS analysis. Different labeling buffers and incubation times were evaluated to optimize (111)In-oxine labeling conditions. Functionality of the radiolabeled NK cell was assessed by (51)Cr-release assay. We evaluated physiological distribution of (111)In-oxine labeled murine NK cells in normal SCID mice and biodistribution in irradiated and nonirradiated SCID mice with orthotopic A549 human lung tumor lesions. Imaging findings were confirmed by histology. Results showed that incubation with 0.011 MBq of (111)In-oxine per million murine NK cells in PBS (pH 7.4) for 20 min is the best condition that provides optimum labeling efficiency without affecting cell viability and functionality. Physiological distribution in normal SCID mice demonstrated NK cells homing mainly in the spleen, while (111)In released from NK cells was excreted via kidneys into urine. Biodistribution studies demonstrated a higher lung uptake in orthotopic lung tumor-bearing mice than control mice. In irradiated mice, lung tumor uptake of radiolabeled murine NK cells decreased between 24 h and 72 h postinjection (p.i.), which was accompanied by tumor regression, while in nonirradiated mice, radiolabeled NK cells were retained in the lung tumor lesions up to 72 h p.i. without tumor regression. In tumor-bearing mice that were only irradiated but did not receive radiolabeled murine NK cells, a high tumor burden was observed at 72 h p.i., which indicates that irradiation in combination with murine NK cell allocation, but not irradiation alone, induced a remarkable antitumor effect in the orthotopic A549 lung tumor bearing mouse model. In conclusion, we describe a method to evaluate murine NK cell trafficking and biodistribution, which can be used to determine potential effects of immune-mediated therapeutic agents on NK cell biodistribution.

Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

British Society for Gene and Cell Therapy (BSGCT) Conference AbstractsRoyal HollowayUniversity of London17–19 April, 2013

Genetic correction of β-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by over-expression of several transcription factors has the potential to cure many genetic and degenerative diseases currently recalcitrant to traditional clinical approaches. One such genetic disease is β-thalassemia major (Cooley's anemia). This disease is caused by either a point mutation or the deletion of several nucleotides in the β-globin gene, and it threatens the lives of millions of people in China. In the present study, we successfully generated iPSCs from fibroblasts collected from a 2-year-old patient who was diagnosed with a homozygous 41/42 deletion in his β-globin gene. More importantly, we successfully corrected this genetic mutation in the β-thalassemia iPSCs by homologous recombination. Furthermore, transplantation of the genetically corrected iPSCs-derived hematopoietic progenitors into sub-lethally irradiated immune deficient SCID mice showed improved hemoglobin production compared with the uncorrected iPSCs. Moreover, the generation of human β-globin could be detected in the mice transplanted with corrected iPSCs-derived hematopietic progenitors. Our study provides strong evidence that iPSCs generated from a patient with a genetic disease can be corrected by homologous recombination and that the corrected iPSCs have potential clinical uses.

Abstract 1484: Inhibition of the glycosylation enzyme Mgat1 blocks migration, invasion and metastasis of malignant cells

Abstract N-acetylglucosaminyltransferase I (Mgat1) is the enzyme that initiates the biosynthesis of hybrid and complex N-glycans in medial golgi. As such, it regulates N-glycosylation, a post-translational modification that alters the localization and function of cell surface proteins. Aberrant activity of the N-glycosylation pathway is seen frequently in malignant cells and contributes to their metastatic potential. Here, we have evaluated the effects of Mgat1 inhibition in malignant cells. We knocked down Mgat1 with two independent shRNA in Hela human cervical cancer cells. Target knockdown and inhibition was confirmed by demonstrating mRNA knockdown, decreased cell-surface lectin staining, and inhibition of enzymatic activity. Knockdown of Mgat1 did not induce cell death or inhibit proliferation. Complex N-glycans influence cell migration and invasion. Therefore, we investigated the effect of Mgat1 knockdown on these glycosylation-mediated processes. Hela cells with Mgat1 knockdown and control shRNA were seeded into invasion chambers. Twenty four hours after seeding, we measured cell migration through uncoated chambers and cell invasion through Matrigel-coated chambers. Compared to control shRNA, Mgat1 knockdown significantly decreased Hela cells migration and invasion. ιntegrins influence cell migration and invasion and are N-glycosylated. Therefore, to begin to understand the mechanism by which Mgat1 knockdown inhibits cell migration and invasion, we examined changes in the cell surface expression of β1 integrins. By confocal microscopy, knockdown of Mgat1 decreased cell surface expression of β1 integrins and increased their localization around the nucleus. To assess the effects of Mgat1 on metastasis in vivo, we knocked down Mgat1 with shRNA in RFP-labeled PC3N7 prostate cancer cells. Mgat1 knockdown or control cells were then injected orthotopically into the prostate gland of sublethally irradiated SCID mice. Four weeks after injection, mice were sacrificed and distant tumor formation was imaged with fluorescent microscopy. Mgat1 knockdown decreased the median number of tumors that had metastasized to the lung more than three-fold compared to control. Our preclinical studies, suggest a role for Mgat1 in cancer cell metastasis. Therefore, we examined the association between Mgat1 mRNA expression in primary tumors and the incidence of subsequent metastasis. By analyzing publically available gene expression array data sets from patients with breast cancer, we determined that patients with the lowest levels of Mgat1 mRNA in their primary tumors were least likely to develop metastatic disease after therapy. In summary, this work highlights the role of the N-glycosyltransferase Mgat1 in cancer cell metastasis. As such, Mgat1 may be a novel target for anti-cancer therapies and levels of Mgat1 in primary tumors may help predict the risk of developing metastatic disease. 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 1484. doi:10.1158/1538-7445.AM2011-1484

Abstract 5463: Development of a pharmacokinetic (PK)-pharmacodynamic (PD) model to guide combination-dosing strategies of aflibercept (VEGF Trap) and doxorubicin chemotherapy in a systemic preclinical model of human acute myeloid leukemia

Abstract Combination therapy utilizing an anti-angiogenic agent and cytotoxic chemotherapy represents a standard therapeutic approach in solid tumors; however the efficacy of this approach in hematological malignancies has not been established. In order to maximize anti-tumor effects, the in vivo mechanism of action as well as timing of administration of the anti-angiogenic agent with respect to the cytotoxic agent in such “liquid tumors” may be crucial. We performed detailed pharmacodynamic modeling to study the effects of the sequential combination of an anti-angiogenic (aflibercept) agent with doxorubicin (DOX) chemotherapy on tumor growth in a systemic model of human acute myeloid leukemia. Disease in sublethally irradiated SCID mice was established in marrow and other sites (e.g. central nervous system) by intraveneous injection of stable luciferase-transfected human AML (HEL) cells. Animals were then treated with aflibercept (25 mg/kg) or PBS (vehicle) i.p., on days 0, 4, and 7 (n=5 per group). On day 7, one dose of DOX 3mg/kg was also administered. In vivo leukemia disease burden was quantified on days 0, 5, 7, 14, 21 and 28 days by bioluminescent imaging. The total tumor load was calculated as the area under the curve of bioluminescence (photons/second/cm2/steradian) versus time using the trapezoidal rule (units: photons/second/cm2/steradian*days) for days 0-21 (AUC0-21) and days 0-28 (AUC0-21) in those animals surviving to day 28 (n=2 in PBS + DOX, n=3 in aflibercept + DOX). We found that the mean (AUC0-21) ± SE value was lower in animals treated with aflibercept+DOX (6.86e-08±3.2e-08) than in PBS + DOX (1.63e-09±7.9e-08) treated mice. Additionally, the fold change from baseline bioluminescence (ratio of day 21 to day 0) was higher in PBS + DOX treated animals (4.379 ± 0.5101) than aflibercept+DOX treated animals (3.077 ± 0.2037), although this difference was not statistically significant (p=0.05). In the animals that survived till day 28, the mean (AUC0-28) ± SE values were similar: PBS + DOX = 2.44e-09 ± 2.17e-09 and aflibercept + DOX = 7.21e-08 ± 3.39e-08. Conclusions: We conclude that there was a trend for improved inhibition of leukemia growth when aflibercept was combined with DOX over DOX treatment alone in SCID mice engrafted with systemic human AML disease. Although changes were noted over a 28 day period, this change was most remarkable at day 21. Integration of this PD data with drug PK in the same model system is ongoing. This PKPD modeling approach will enable us to predict the effects of different dosing frequency and schedules of anti-VEGF therapy in combination with cytotoxic agents in acute leukemia and will provide new insight into the development of rational dosing regimens of these agents in future clinical trials of patients with aggressive hematological cancer. 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 5463. doi:10.1158/1538-7445.AM2011-5463

The Anti-Malarial Mefloquine Demonstrates Preclinical Activity In Leukemia and Myeloma, and Is Dependent Upon Toll-Like Receptor Signaling for Its Cytotoxicity

AbstractAbstract 290Known drugs with previously unrecognized anti-cancer activity can be rapidly repurposed for this new indication, given their prior safety and toxicity testing. To identify such compounds, we compiled and screened an in-house library of on-patent and off-patent drugs and screened them to identify agents cytotoxic to hematologic malignancies. From this screen, we identified mefloquine, a quinoline licensed for malaria treatment and prophylaxis. In secondary assays, leukemia and myeloma cell lines were treated with mefloquine for 72 hours and cell viability measured by MTS. Mefloquine decreased the viability of 10/10 human and murine leukemia (LD50 <8.0 μM) and 9/9 human myeloma (LD50 <5.0 μM) cell lines; cell death was confirmed by Annexin V staining. Mefloquine also reduced the viability of 6/6 primary AML samples with LD50 < 5 μ M. These concentrations of mefloquine appear pharmacologically achievable based on prior studies conducted in the context of malaria treatment. In contrast to the effects on malignant cells, mefloquine was significantly less cytotoxic to normal hematopoietic cells (LD50 31.83 ± 5.38 μM) and murine monocyte-derived dendritic cells (LD50 17.56 ± 2.69 μM), Given its in vitro activity, we evaluated the effects of oral mefloquine in mouse xenograft models of leukemia and myeloma. Sublethally irradiated SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells, MDAY-D2 murine leukemia cells, or LP1 human myeloma cells, and treated with 50 mg/kg mefloquine, or vehicle alone, by gavage. Oral mefloquine delayed tumor growth by up to 60% in all 4 mouse models without toxicity at doses that appear pharmacologically relevant to humans based on scaling for body surface area. Mefloquine's mechanism of action as an anti-malarial agent is unknown. Therefore, to determine the mechanism by which mefloquine induced cell death in malignant cells, we performed gene expression oligonucleotide array analysis of mefloquine-treated OCI-AML2 cells. At times preceding cell death, mefloquine altered the expression of genes associated with Toll-like receptor (TLR) signaling. For example, we detected 4.5-fold up-regulation of STAT1 and >10-fold up-regulation of its downstream targets, including OAS1, IFIT3 and TRIM22, by 24 hr after treatment. Upregulation of additional TLR targets IRF1, IRF7 and IL-8 was also noted by 8 hours after treatment. Mefloquine also induced early activation of NF-κB with a 2.5± 0.2-fold increase noted after 1 hr, using an ELISA-based DNA binding assay. In contrast to TLR activation in malignant cells, changes in TLR targets were not detected in mefloquine-resistant normal dendritic cells, suggesting that mefloquine's effects on TLR signaling were specific to malignant cells. We next investigated whether TLR activation was functionally important for mefloquine's cytotoxicity in malignant cells. STAT1 activity was required for mefloquine-mediated cell death, as U4A bladder sarcoma cells lacking JAK1 were resistant to mefloquine (LD50 14.6± 4.9 μM), compared to the mefloquine sensitive parental line (LD50 2.3± 0.4 μM). TLR signaling requires the immediate downstream adapter proteins MyD88 and TRIF1. To assess the functional importance of TLR activation for mefloquine induced cell death, we knocked down MyD88 and TRIF1 with siRNA. Double knockdown of MyD88 and TRIF1 completely abrogated mefloquine-induced cell death in K562 leukemia cells at concentrations where control cells exhibited up to 80% loss of viability. TLR signaling and up-regulation of STAT1 can increase reactive oxygen species (ROS) generation. Therefore, we measured ROS generation in leukemia cells after mefloquine treatment. Mefloquine increased ROS production in leukemia cells in a dose-dependent manner within 24 hr. Co-treatment with the ROS scavenger N-Acetyl-L-Cysteine abrogated mefloquine-induced ROS production and cell death. Mefloquine-induced ROS production was also abrogated in MyD88 and TRIF1 double knockdown cells. Our data suggest that the known anti-malarial mefloquine displays preclinical activity in leukemia and myeloma through a mechanism related to TLR activation. Thus, these results highlight TLR activation as a novel therapeutic strategy for the treatment of leukemia and myeloma. Moreover, given its prior toxicology and pharmacology testing, mefloquine could be rapidly advanced into clinical trial for patients with leukemia and myeloma. Disclosures:No relevant conflicts of interest to declare.

Abstract 2529: The anti-malarial agent Mefloquine preferentially demonstrates pre-clinical activity in leukemia and myeloma cells through STAT1-induced reactive oxygen species production

Abstract Off patent drugs with previously unrecognized anti-leukemia activity can be rapidly repurposed for this new indication, given their prior safety and toxicity testing. By screening a group of anti-malarial compounds for anti-cancer activity, we identified mefloquine, a quinoline licensed in oral formulation for the treatment of malaria. As an anti-cancer agent, we demonstrated that mefloquine decreased the viability of 9/9 leukemia cell lines with an LD50 &amp;lt;7.5 uM, and 9/9 myeloma cell lines with an LD50 &amp;lt;5.0 uM. Furthermore, mefloquine demonstrated induced cell death in primary AML samples (n = 3; LD50 &amp;lt;7.5 uM), but not normal peripheral blood stem cells. Given its in vitro activity, we evaluated the effects of oral mefloquine in mouse xenograft models of leukemia. Sublethally irradiated SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells or MDAY-D2 murine leukemia cells, and treated with 50 mg/kg mefloquine, or vehicle alone by oral gavage. Oral mefloquine decreased tumor weight and volume in all 3 mouse models without toxicity. Mechanistically, mefloquine induced reactive oxygen species (ROS) in leukemia cells at times preceding and concentrations associated with cell death. Blockade of ROS by N-acetyl-L-cysteine (NAC) abrogated mefloquine sensitivity, suggesting that mefloquine-mediated cell death in AML was ROS-dependent. To further understand the mechanism of mefloquine-mediated cytoxicity, whole genome gene expression oligonucleotide array analysis of AML cells treated with mefloquine was conducted. The gene expression pattern of cells treated with mefloquine strongly resembled gene signatures associated with activated Toll-like receptor and interferon response pathways. STAT1 and NF-κB, both downstream transcription factor components of TLR-IFN signaling, were activated, as were downstream targets IRF1, IRF7 and IL-8, at times that preceded mefloquine-induced cell death. Gene expression changes were validated by Q-RT-PCR, and are potential biomarkers of mefloquine activity in cells. This pathway appears functionally important for mefloquine-mediated cell death, as cell lines defective for STAT1 signaling components showed decreased cell death after mefloquine treatment. These lines also showed decreased ability to generate ROS in response to mefloquine treatment, suggesting that mefloquine-induced ROS was produced through a STAT1-dependent mechanism. Taken together, our data demonstrate that the anti-malarial mefloquine displays significant pre-clinical activity in leukemia and myeloma cells, likely through a STAT1-dependent induction of ROS that is triggered by activation of the TLR-IFN cytokine axis in cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2529.

The Antihelmintic Flubendazole Inhibits Microtubules through a Mechanism Distinct From Vinca Alkaloids and Displays Preclinical Activity in Leukemia and Myeloma.

Abstract 1017Poster Board I-39On patent and off-patent drugs with previously unrecognized anti-cancer activity, could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we compiled and screened a chemical library for potential anti-leukemia agents. From these screens, we identified the antihelmintic flubendazole that is currently used for the treatment of gastrointestinal and systemic parasites, but has not been evaluated for the treatment of malignancy. To explore its efficacy as an anti-cancer agent, leukemia and myeloma cell lines were treated with increasing concentrations of flubendazole. Seventy-two hours after incubation, cell viability was measured by the MTS assay. Flubendazole reduced cell viability with an LD50 ≤ 1 μM in 8/8 myeloma and 4/6 leukemia cell lines, a concentration that is pharmacologically achievable. Likewise, flubendazole reduced the clonogenic growth of primary AML samples at nanomolar concentrations. Cell death was confirmed by Trypan blue staining. Given the effects in leukemia and myeloma cells lines, we evaluated the effects of flubendazole in mouse models of leukemia and myeloma. Sublethally irradiated SCID mice were injected subcutaneously with OCI-AML2 leukemia or OPM2 myeloma cells. Mice were then treated intraperitoneally with flubendazole (20-50 mg/kg/day – doses more than 10-fold lower than the LD50) or buffer alone. Flubendazole decreased tumor weight and volume in both mouse models up to 5-fold compared to control without evidence of weight loss or gross organ toxicity. Mechanistically, flubendazole inhibited bovine-tubulin polymerization in cell-free assays and disrupted microtubule architecture in intact cells as visualized by confocal microscopy. We demonstrated that flubendazole bound tubulin at the colchicine binding site, a region distinct from where vinca-alkaloids bind. Flubendazole arrested cells in the G2 phase of the cell cycle and increased the number of multi-nucleated cells. We also demonstrated that cell death after flubendazole treatment was related to its ability to inhibit microtubule polymerization by using cell lines with tubulin mutations (gifts from Dr F. Loganzo, Wyeth, Pearl River, NY and Drs. S. Band Horwitz and C. Yang, Albert Einstein College of Medicine, Bronx, NY). Vinca-alkaloids are p-glycoprotein (Pgp) substrates and Pgp over-expression can limit the efficacy of these agents. Therefore, we tested the effects of Pgp over-expression on flubendazole's cytotoxicty. CEM-VBL cells over-expressing Pgp remained fully sensitive to flubendazole, but were over 1000-fold more resistant to vinblastine than wild type CEM cells. Therefore, flubendazole can overcome some forms of vinca-alkaloid resistance. Given that flubendazole binds tubulin at a site distinct from vinca-alkaloids, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. OCI-AML2 leukemia cells were treated with increasing concentrations of flubendazole and vinblastine. Flubendazole and vinblastine synergistically induced cell death with combination index (CI) values of 0.09, 0.017, 0.003 and 0.001 at the EC 50, 25, 10 and 5, respectively, where CI values <1 are considered synergistic. In contrast, cell death produced by the combination of flubendazole and colchicine was closer to additive with CI values of 0.54, 0.70, 0.897 and 1.07 at EC 50, 25, 10 and 5, respectively. As the combination of flubendazole and vinblastine were synergistic in cell culture, we evaluated the combination in vivo. Mice injected subcutaneously with OCI-AML2 cells were treated with flubendazole (15 mg/kg), vinblastine (0.3 mg/kg) or the combination of the two agents. The combination decreased tumor weight and volume greater than either agent alone without behavioural changes, weight loss, or gross organ toxicity. Similar effects were observed with the in vivo combination of flubendazole and vincristine. Since neurotoxicity is a dose limiting toxicity of vinca-alkaloids such as vincristine, we evaluated the neurotoxicity of flubendazole in mice using the Tail Flick assay. At doses up to 200mg/kg daily of flubendazole, no neurotoxicity was observed. Thus, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma. Given its prior safety record when evaluated for the treatment of parasitic disease, flubendazole could be rapidly repurposed for the treatment of hematologic malignancies. Disclosures:Off Label Use: Flubendazole is used to treat gastrointestinal and systemic parasite infections.

Effect of feeder cells transduced with Ad-hLIF on the expansion of CD34+ cells in indirect co-culture

Objective To establish Ad-hLIF transgenic feeder cells for the expansion of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of hematopoietic stem/progenitor cell (HSPC) transplantation. Methods The expression of objective gene in Ad-hLIF transgenic feeder cells was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting(MACS) was detected by the flow cytometry. After expanded with various combinant of cytokines and transgenic feeder layer cells for 28 d, the quantity of mono-nuclear cell (MNC) and CD34+ cells rate was detected in different time. MNC after expansion stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanize gene Alu was detected by RT-PCR and fluorescence microscope. Results The green fluorescence was observed in the transgenic cells infected with 50MOI( multiplicity of infection) Ad-hLIF, and the objective gene was confirmed by RT-PCR and ELISA. The purity of umbilical cord blood CD34+ HSPC separated by MACS could reach 95.60% ±2.58%, Ad-hLIF transgenic feeder cells and various cytokines system increased MNC by 356.95±0.87 fold, and maximal expansion of CD34+ cells was observed during 0-14 d of culture, then down-expansion gradually. Four weeks after transplanted in SCID mice, fluorescently-labeled humanize cells still can be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion Ad-hLIF transgenic feeder cells can effectively proliferate umbilical cord blood CD34+ HSPC in vitro and delay it differentiate, what's more, it has high transplant efficacy and haematogenesis activity. Key words: hLIF; Recombinant adenovirus vector; Hematopoietic stem/progenitor cell; SCID mice

Reprogrammed Murine Fibroblasts Differentiated into Hematopoietic Progenitors Are Able to Successfully Engraft and Repopulate the Bone Marrow

Cellular therapy using embryonic stem cells has always been an area of great interest due to the pluripotent characteristics of stem cells. In 2006, Takahashi and Yamanaka (Cell 126, 663–676) demonstrated that somatic cells can be reprogrammed into a stem cell-like state, termed induced pluripotent stem (iPS) cells, by ectopic expression of Oct4, Sox2, Klf4 and c Myc. A later report (Nakagawa et al. Nat. Biotechnol. 26:101–106, 2008) showed that iPS cells can be produced in the absence of the c Myc oncogene. We have used this latter strategy to successfully reprogram somatic cells derived from C57BL/6 mouse tail fibroblast to iPS cells. Retrovirus infected fibroblasts exhibited stem cell-like morphology by 14 days post infection. These iPS cells were then infected with a retrovirus that expressed HOXB4. Recombinant leukemia inhibitor factor (LIF) supplement was removed from media at this time and the cells allowed to differentiate into embryoid bodies. These cells were screened for specific differentiation stem cell markers, such as Oct4, Nanog, Sall4 and SSEA-1. iPS cells were converted into embryonic bodies and then infected with retroviruses expressing HOXB4. Embryoid bodies stably expressing HOXB4 were induced to hematopoietic differentiation by treatment of thrombopoietin (TPO), stem cell factor (SCF), vascular endothelial growth factor (VEGF), interferon gamma (IFNg) and fms-like tyrosine kinase (FLT3 ligand). Evaluation of iPS-derived hematopoietic cells on smears show strikingly similarity in morphology to the W4 mouse embryonic stem (ES) cells differentiated into hematopoietic cells as a control. Flow cytometry analysis of iPS-derived hematopoietic cells after 1 week exposure to cytokines revealed 7% B220+ cells (B cells), 11% Ter119+ cells (erythroid), and 13% Gr-1+ cells (granulocytes) similar to W4 ES cells. The iPS-derived hematopoietic cells were transplanted into irradiated immunodeficient mice via lateral tail vein injection. Transplantation of these iPS-derived hematopoietic progenitors tagged with GFP into irradiated SCID mice revealed that the hematopoietic progenitors were able to home to the bone marrow after 1 week of transplantation. Importantly, after 1 month, GFP+ engrafted cells remained in the bone marrow suggesting a long-term engraftment. This long term engraftment of the iPS-derived hematopoietic cells to the bone marrow constitutes an important step toward potential therapy of numerous patient-specific blood based diseases.

Pre-Clinical Data and Preliminary Patient Results of Intravenous MV-NIS To Treat Relapsed, Refractory Multiple Myeloma.

Background: MV-NIS is an Edmonston-lineage measles virus that expresses the human sodium-iodide symporter (hNIS). The virus is oncolytic, and its activity can be monitored by noninvasive imaging of radioiodine uptake by hNIS. The receptor is CD46, a membrane regulator of complement activation that is overexpressed on myeloma cells.Methods: Formal studies were done in collaboration with the RAID program to assess the efficacy, biodistribution and safety of MV-NIS in transgenic, measles susceptible mice and in non-human primates. The protocol therapy for the animal studies was modeled on the planned human clinical trial. Mouse efficacy studies were done in irradiated 6-week old female CB17 ICR SCID mice implanted subcutaneously with human KAS-6/1 cells. Mouse toxicity & biodistribution were done in 184 6–8 week IfnarKO x CD46 Ge mice treated with 105–107 TCID50 MV-NIS. Squirrel monkey toxicity studies were done in 12 measles-naïve adults with 108 TCID50. The clinical trial is a 3 patient/cohort dose escalation study which allows for 1 dose of MV-NIS/patient. Once MTD of MV-NIS is reached, subsequent cohorts will be treated with CTX 10 mg/kg 2 days prior to MV-NIS at MTD/100. Immune response to measles is tested pre & post therapy. PK & biodistribution are tested by measurements of MV-N in blood, urine, & gargle samples and by serial nuclear imaging, respectively. BM myeloma cells are tested for CD46 expression and MV infectivity.Results: Preclinical studies: The minimum effective dose in the mouse model was 4 x 106 TCID50 /kg. The MTD for MV-NIS with CTX was not reached in the mouse study. Pre-treatment with CTX changed the biodistribution of MV-NIS infected cells and the kinetics of virus elimination from infected tissues in mice and monkeys. In monkeys, MV-N mRNA was detected at day 1 in the buccal cavity with levels declining on day 2, then surging on day 8, suggesting viral replication. There were no unscheduled deaths or treatment related lesions on necropsy.Patients: Three patients were treated with a dose of 106 TCID50 (Table). No DLT was observed. Two patients had MV-N amplified from their gargle specimen. Accrual to dose level 2 has begun.Conclusions: IV administration of MV-NIS is feasible. The virus is capable of traveling to a distant site and replicating before being cleared by the immune system.Patient 2Patient 2Patient 3Anti-measles neutralizing Ab titer, pre-Rx / post Rx1:10 / 1:160< 1:10 / 1:20< 1:10 / 1:10AEs possibly relatedGr 2 pltsGr 1 plts, cough, fever, rigorsGr 2 Incr aPTTCD46 copies/cell CD138+ / CD138- BMPC79,771 / 10,322140,187 / 10,14528,258 / 11,228MV-N QRT-PCR: d3/ d8/ d15/ d22/ d29 (cp/ug RNA)*−/ −/ −/ nd / nd−/ −/ 16,440 / 1405 / −123-I uptake on SPECTNoUptake day 8No*copies per microgram RNA of cells derived from gargle sample at each time point: day +3, +8, +15, +22, and +29; no MV-N detected in urine or blood

Long-term ability for hematopoiesis of CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria: experiment with mice

To obtain the evidence of long-term hematopoiesis by normal clone stem cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). 10 ml fresh bone marrow was collected from 2 PNH patients. Normal bone marrow was obtained from the ribs resected during operation of 2 patients without hematopathy. CD34(+)CD59(+) cells were isolated, purified, and expended. 12 total body irradiated SCID mice were divided into 2 equal group to undergo transplantation of the expanded CD34(+)CD59(+) cells from PNH patients (experiment group 1) or normal patients (control group 1). Four mice underwent transfusion of cell-free medium as blank control group 1. 30 and 60 days after the transplantation peripheral blood samples were collected and 90 days after the transplantation heart blood samples were collected. Spleens were taken out to prepare suspension of splenocytes and bacterium-free bone marrow cell suspension was prepared from the bilateral femurs. 90 days after the primary transplantation another 16 mice underwent total body irradiation and then divided into 2 equal groups to undergo transplantation of the bone marrow cells of the mice of the experiment group 1 and control group 1 (experiment group 2 and control group 2). Another 4 mice were transfused with cell-free medium fluid (blank control group 2). The levels of blood cells of all groups were calculated 30, 60, and 90 days after the primary and 30 days after the secondary transplantation. The cell percentages of peripheral blood, spleen, and bone marrow after the primary transplantation and 30 days after the secondary transplantation were detected by flow cytometry. PCR was used to detect whether the sex determining region Y existed in the female SCID mice after transplantation. Ninety days after primary transplantation, the peripheral blood cell levels of both experiment group 1 and control group 1 recovered to normal. Expression of CD45 was detected in the mice without significant difference between the 2 groups. Thirty days after the secondary transplantation, the peripheral blood cell levels of the experiment group 2 and control group 2. Expression of CD45 was detected in both the experiment group 2 and control group 2 without significant difference between these two groups. Human SRY gene could be detected by PCR in the female SCID mice after transplantation. CD34(+)CD59(+) cells isolated from PNH patients and expanded in vitro can be successfully engrafted with long-term ability in hematopoiesis not different from that of the normal CD34(+)CD59(+) cells.

Improved Homing of Antigen-Specific T Cells to Hodgkin's Disease (HD) Tumor Cells by Forced Expression of CCR4 Receptor.

One crucial requirement for the success of any adoptive T cell transfer is that the effector T cells should migrate efficiently to the tumor site. Such an effect has been documented following adoptive transfer of Epstein-Barr virus specific cytotoxic T cells (EBV-CTLs). Antigenic stimulus from (normal and malignant) cells persistently infected with EBV led to expansion and sustained survival, and was also associated with activity against the EBV+ HD tumors. Since most HD patients have tumors that are EBV- but CD30+, we attempted to extend this approach by incorporating a CD30 chimeric receptor (CD30CAR) into the EBV-CTLs. Pre-clinical animal studies showed that CD30CAR+ EBV-CTLs readily migrated to EBV+/CD30+ tumors, but had limited capacity to localize to EBV−/CD30+ tumor cells. The likeliest explanation for this observation is that EBV- HD tumors produce the chemokine TARC, which attracts Th2 and regulatory T cells, but has little effect on EBV-CTLs, since these express low levels of the TARC receptor, CCR4. We hypothesized that forced expression of CCR4 by redirected EBV-CTLs would improve their homing to the EBV−/CD30+ HD cells. The full length of CCR4 receptor was cloned into the SFG retroviral vector and used to transduce both activated T cells and EBV-CTLs obtained from 6 and 4 healthy donors, respectively. Expression of CCR4 was 12±8% on activated T cells (mainly on CD4+ cells, 12±6%) and 4±5% on EBV-CTLs. After transduction with a CCR4, but not a control vector, expression of CCR4 increased to 53±24% (CD4+ 27±15% and CD8+ 17±9%) on activated T cells and 30±19% on EBV-CTLs. We then evaluated the capacity of control and transgenic T cells and EBV-CTLs to migrate in response to TARC, using a trans-well migration assay. Migration was tested against different CD30+ tumor lines producing TARC at low (Karpas wild type, <32 pg/ml) or high levels (Karpas engineered to produce TARC, HDLM-2 and L428, all TARC >2000pg/mL), measured by ELISA. The percent of cells migrating in the trans-well assay was significantly increased for CCR4 transgenic CD8+ selected T cells (54±11% with Karpas/TARC vs. 8±2% with media vs. 8±4% with Karpas-wt). Migration of control OKT3/28 blasts was less than 15% in all the conditions. Migration was significantly inhibited by the addition of antibody blocking TARC (<10%). An improved migration of CCR4+ CD8+ cells was observed also towards HDLM-2 (48±7%) and L428 (45±16%) as compared to control T cells (21±7% and 23±7%, respectively). Similarly, CCR4+ EBV-CTL showed improved migration towards Karpas/TARC (26±14%) as compared to Karpas/wt (12±8%) or media (10±7%). This genetic modification did not modify either the phenotype or the antigen specificity of EBV-CTLs, which retained the capacity to kill autologous EBV+ lymphoblastoid cells. We then determined if forced expression of CCR4 receptor was functional in vivo. To track homing of transgenic T cells, we transduced them with the FireFlyluciferase (FFLuc) vector and followed signal with a bioluminescent system (IVIS, Xenogen). Sublethally irradiated SCID mice were implanted s.c. with Karpas/wt on the left side and Karpas/TARC on the right side. When tumors were measurable, FFLuc+CCR4+ T cells were injected iv. By 24 hours post injection, signal was detectable only at the side of TARC secreting tumor cells, and signal persisted at 10 to 16 fold higher levels on the TARC+ side for >7 days. These data suggest that the migration of CARCD30 EBV-CTL to EBV−/CD30+ HD can be augmented by co-expressing the CCR4 receptor.

B Cell Development in the Absence of PU.1.

Mice deficient of the ETS-family transcription factor PU.1 lack B cells as well as macrophages. While most macrophage specific genes are known to be regulated by high levels of PU.1, the reason for the defect in B cell formation is not known. Here we analyzed a mouse strain in which a floxed version of the PU.1 gene, surrounding exon 4 and 5, which encode the DNA, binding and PEST domains (developed by C. Somoza and D. Tenen), was excised by Cre mediated recombination. As expected, this strain lacks both B cells and macrophages and die at birth. Surprisingly, however, we were able to establish lymphoid cell lines from fetal livers of these mice (day 14 to day 18), which proliferated on S17 stromal cells supplemented with IL-7 and stem cell factor. These cells expressed the B lineage cell surface markers CD19, CD43, BP-1 and CD24, but not B220. They also expressed B cell transcription factors, EBF, E47, Pax5, and their target genes, Rag1, IL7R, λ5 and v-preB, as detected by RT-PCR, exhibited DJ and VDJ immunoglobulin heavy chain rearrangements, and expressed IgM after IL-7 withdrawal. We then tested the effect of PU.1 deletion in B cells in adult animals by crossing the floxed PU.1 strain with a CD19 Cre mouse line. The spleen and peripheral blood (but not bone marrow) of these mice contained B cells that were CD19+ IgMlow, IgDhigh but B220 negative and instead expressed CD43. Thus PU.1 is not essential for immunoglobulin production and late B cell development. Although PU.1−/− fetal liver cells can give rise to cells, resembling Pre-B in vitro, the process of B cell formation was delayed by almost 12 days, compared with wt fetal liver, and the efficiency was reduced approximately 25-fold. In addition, PU.1 deficient B cells demonstrated an impaired ability to engraft into the bone marrow, when injected into irradiated SCID mice. We have found that PU.1 deficient B progenitors showed reduced or undetectable levels of the SDF1 receptor CXCR4, a receptor that has been implicated in B cell homing. Taken together, our observations suggest that PU.1 plays two different roles during B cell development: for early B cell formation and for proper migration and engraftment, which might be mediated through regulation of CXCR4 expression.

Flk1+ cells derived from mouse embryonic stem cells reconstitute hematopoiesis in vivo in SCID mice

Embryonic stem (ES) cells are pluripotent and can differentiate into any cell type, including the hematopoietic lineage. We examined whether hematopoietic progenitor cells derived from ES cells reconstitute hematopoiesis in irradiated SCID mice. ES cells (E14.1, H2K(b)) were cultured for 4 days in semisolid medium containing methylcellulose. Irradiated SCID mice were used as recipients of hematopoietic progenitor cells. Cell surface antigen expression was analyzed by flow cytometry. The spleens of the recipient mice were studied by hematoxylin and eosin staining and immunohistochemical staining. After cell culture of ES cells in methylcellulose for 4 days, the cells expressing Flk1 (VEGF receptor 2), a tentative marker of hemangioblasts, were increased, whereas cells expressing CD31 (PECAM-1) and E-cadherin (nonmesodermal adhesion molecule) were dramatically reduced. Flk1+ cells expressed c-kit predominantly. Circulating leukocytes and thrombocytes were increased in irradiated SCID (H2K(d)) mice transplanted with ES cell-derived Flk1+ cells compared with vehicle-injected control mice. H2K(b+) and VE-cadherin(+) vascular endothelial cells were prominent in spleens of the recipient mice. Flow cytometric analysis demonstrated that H2K(b+) cells were increased in the bone marrow of recipient mice. In addition, Flk1+ cells accompanying enhanced c-kit expression preferentially repopulated in the bone marrow, and leukopoiesis and thrombopoiesis of the recipient mice were evident. The Flk1+ hematopoietic cells derived from ES cells reconstitute hematopoiesis in vivo and may become an alternative donor source for bone marrow transplantation.

In vivo ligation of CD3 on neonatal scid thymocytes blocks γ-irradiation-induced TCRβ rearrangements and thymic lymphomagenesis

Several studies have shown that the developmental arrest of severe combined immune deficiency (scid) thymocytes during the CD4 −CD8 − double negative (DN) to CD4 +CD8 + double positive (DP) transition can be overcome by a sub-lethal dose of ionizing radiation (IR). Concurrent with this developmental progression, IR also induces variable (diversity) joining (V(D)J) recombination at T cell receptor (TCR), δ, β, and γ, but not α loci. In addition, all irradiated scid mice succumb to thymic lymphoma. In this study, we demonstrate that scid neonates treated with anti-CD3ε antibody become more resistant to the development of thymoma upon exposure to IR. It is known that the anti-CD3ε antibody treatment induces T cell progression to the DP stage bypassing TCRβ rearrangement. We show here that the resistance to tumor development is correlated with a reduction of TCRβ rearrangements that are induced by IR. However, TCRγ rearrangements were not altered by the antibody treatment. The particular effect of anti-CD3ε antibody on TCRβ rearrangements is likely attributed to a decline of the double negative thymocyte subset (DN3), in which TCRβ rearrangements predominantly occur. These results suggest that the developmental stage of scid thymocytes can influence the effect of IR on TCR rearrangements as well as lymphomagenesis.

Oncogene-dependent engraftment of human myeloid leukemia cells in immunosuppressed mice

We have developed an in vivo model of differentiated human acute myeloid leukemia (AML) by retroviral infection of the cytokine-dependent AML cell line TF-1 with the v-Src oncogene. When injected either intravenously or intraperitoneally into 300 cGy irradiated SCID mice, animals formed multiple granulocytic sarcomas involving the adrenals, kidneys, lymph nodes and other organs. The mean survival time was 34+/-10 days (n = 40) after intravenous injection and 24+/-3 days (n = 5) after intraperitoneal injection of 20 million cells. The cells recovered from leukemic animals continued to express interleukin-3 receptors and remained sensitive to the diphtheria fusion protein DT388IL3. Further, these granulocytic sarcoma-derived cells grew again in irradiated SCID mice (n = 10). The cytogenetic abnormalities observed prior to inoculation in mice were stably present after in vivo passage. Similar to the results with v-Src transfected TF-1 cells, in vivo leukemic growth was observed with TF-1 cells transfected with the human granulocyte-macrophage colony-stimulating factor gene (n = 5) and with TF-1 cells recovered from subcutaneous tumors in nude mice (n = 5). In contrast, TF-1 cells expressing v-Ha-Ras (n = 5), BCR-ABL (n = 5), or activated Raf-1 (n = 44) did not grow in irradiated SCID mice. This is a unique, reproducible model for in vivo growth of a differentiated human acute myeloid leukemia and may be useful in the assessment of anti-leukemic therapeutics which have human-specific molecular targets such as the interleukin-3 receptor.

Efficient gene transfer to hematopoietic progenitor cells using SV40-derived vectors.

We used recombinant SV40 (rSV40)-derived vectors to deliver transgenes to human and simian hematopoietic progenitor cells in culture, and in vivo after transduction ex vivo. rSV40 are highly efficient vectors that are made in very high titers. They infect almost all cells, whether resting or dividing. Two rSV40s were used: SV(HBS), carrying hepatitis B surface antigen as a marker; and SV(Aw) carrying IN#33, a single chain Fv antibody against HIV-1 integrase. CD34+ cells derived from human fetal bone marrow (HFBM) and rhesus macaque bone marrow were transduced once with SV(HBS) without selection. On average 60% of colonies derived from transduced CD34+ cells carried and expressed HBsAg, as assessed by PCR and immunochemistry. Transgene carriage persisted following differentiation of transduced rhesus CD34+ cells into T lymphocytes. In an effort to increase the percentage of gene-marked cells, three sequential treatments of CD34+ cells were done using SV(Aw), without selection. Two weeks later, >95% of colonies expressed IN#33. Unselected SV(Aw)-transduced CD34+ cells from HFBM were transplanted into sublethally irradiated SCID mice. Bone marrow harvested 3 months later showed that >50% of bone marrow cells expressed IN#33. This is comparable with the percentage of human cells in these animals' bone marrow as judged by immunostaining for human CD45. The stability and longevity of transduction in this setting suggests that rSV40 vectors integrate into the cellular genome. This possibility was supported by finding that PCR of genomic DNA using primer pairs with one cellular and one viral primer yielded PCR products only in transduced, but not control, cells. These PCR products hybridized with an SV40 DNA fragment. Thus, rSV40 vectors transduce normal human and primate bone marrow progenitor cells effectively without selection, and maintain transgene expression in vivo following reimplantation. Such high efficiency transduction may be useful in treating diseases of CD34+ cells and their derivatives.

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

IMPLICATIONS OF RECENT INSIGHTS INTO THE PATHOPHYSIOLOGY OF PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA