Nonobese Diabetic/severe Combined Immune-deficient Research Articles

EID3 Promotes Glioma Cell Proliferation and Survival by Inactivating AMPKα1.

EID3 (EP300-interacting inhibitor of differentiation) was identified as a novel member of EID family and plays a pivotal role in colorectal cancer development. However, its role in glioma remained elusive. In current study, we identified EID3 as a novel oncogenic molecule in human glioma and is critical for glioma cell survival, proliferation and invasion. A total of five patients with glioma were recruited in present study and fresh glioma samples were removed from patients. Four weeks old male non-obese diabetic severe combined immune deficiency (NOD/SCID) mice were used as transplant recipient models. The subcutaneous tumor size was calculated and recorded every week with vernier caliper. EID3 and AMP-activated protein kinase α1 (AMPKα1) expression levels were confirmed by real-time polymerase chain reaction and Western blot assays. Colony formation assays were performed to evaluate cell proliferation. Methyl thiazolyl tetrazolium (MTT) assays were performed for cell viability assessment. Trypan blue staining approach was applied for cell death assessment. Cell Apoptosis DNA ELISA Detection Kit was used for apoptosis assessment. EID3 was preferentially expressed in glioma tissues/cells, while undetectable in astrocytes, neuronal cells, or normal brain tissues. EID3 knocking down significantly hindered glioma cell proliferation and invasion, as well as induced reduction of cell viability, apoptosis and cell death. EID3 knocking down also greatly inhibited tumor growth in SCID mice. Knocking down of AMPKα1 could effectively rescue glioma cells from apoptosis and cell death caused by EID3 absence, indicating that AMPKα1 acted as a key downstream regulator of EID3 and mediated suppression effects caused by EID3 knocking down inhibition. These findings were confirmed in glioma cells generated patient-derived xenograft models. AMPKα1 protein levels were affected by MG132 treatment in glioma, which suggested EID3 might down regulate AMPKα1 through protein degradation. Collectively, our study demonstrated that EID3 promoted glioma cell proliferation and survival by inhibiting AMPKα1 expression. Targeting EID3 might represent a promising strategy for treating glioma.

Establishment and evaluation of retroperitoneal liposarcoma patient-derived xenograft models: an ideal model for preclinical study.

Retroperitoneal liposarcoma (RLPS) is one of the most common subtypes of retroperitoneal soft tissue sarcomas. It is characterized by poor sensitivity to radiotherapy and chemotherapy and a low success rate of complete surgical resection. However, there are few reliable preclinical RLPS models for target discovery and therapy research. In this study, we aimed to establish RLPS patient-derived xenograft (PDX) models that are useful for biological research and preclinical drug trials. A total of 56 freshly resected RLPS tissues were subcutaneously transplanted into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice, with subsequent xenotransplantation into second-generation mice. The tumor engraftment rate of first generation PDXs was 44.64%, and higher success rates were obtained from implantations of dedifferentiated, myxous, pleomorphic, high-grade liposarcomas and those with retroperitoneal organ infiltration. The first- and second- generation PDX models preserved the histopathological morphology, gene mutation profiles and MDM2 amplification of the primary tissues. PDX models can also provide the benefit of retaining original tumor biology and microenvironment characteristics, such as abnormal adipose differentiation, elevated Ki67 levels, high microvessel density, cancer-associated fibroblast presence, and tumor-associated macrophage infiltration. Overall survival (OS) and disease-free survival (DFS) of patients with successful first-generation PDX engraftment were significantly poorer than those with failed engraftment. Treatment with MDM2 inhibitor RG7112 significantly suppressed tumor growth of DDLPS PDX in mice. In conclusion, we successfully established RLPS PDX models that were histologically, genetically, and molecularly consistent with the original tissues. These models might provide opportunities for advancing RLPS tumor biology research, facilitating the development of novel drugs, particularly those targeting MDM2 amplification, adipose differentiation process, angiogenesis, cancer-associated fibroblasts, and so on.

Targeted Delivery of Colloidal Silver for MCF-7 Breast Cancer Treatment.

Amongst various cancer diseases, breast cancer is frequently diagnosed malignancy in women. Existing treatments are inadequate, painful and toxic. New ways of treatments need to be explored. The present work proposes preparation and targeted delivery of a formulation, F-1, for MCF-7 breast cancer treatment. The formulation, colloidal silver (0.76 ppm), was prepared by electrolytic deposition technique and multi surface coatings. Black tea extract (2.25%v/v) was used as a capping agent to tune the morphology of silver nanoparticle and potato extract (6.25%v/v) as a functionalizing agent for targeting MCF-7 breast cancer site. Characterization results show highly pure spherical silver nanoparticles with an average particle size of 15nm. The shift of peaks in the FTIR spectra of formulation confirms the interaction between nanoparticles and extracts. The UV-visible peak was obtained at 525nm, a typical characteristic of silver nanoparticles. In-vivo; anti-cancer study of formulation gave a moderate therapeutic effect in Non-Obese Diabetic Severe Combined Immune Deficiency (NOD-SCID) mice. It is observed that tumor volumes obtained in the case of Formulation-1 were moderately inhibited from days 5 to 9. However, one of the mice in the Formulation-1 group inhibited tumor volume to 1.52 cc similar to one of the mice of positive control group (Adriamycin 1.42cc).

Establishment and characterization of a DOT1L inhibitor-sensitive human acute monocytic leukemia cell line YBT-5 with a novel KMT2A-MLLT3 fusion.

Immortalized cell lines are useful for deciphering the pathogenesis of acute leukemia and developing novel therapeutic agents against this malignancy. In this study, a new human myeloid leukemia cell line YBT-5 was established. After more than 1-year cultivation from the bone marrow of a patient with acute monocytic leukemia, YBT cell line was established. Then a subclone, YBT-5, was isolated from YBT using single cell sorting. Morphological and cytogenetical characterizations of the YBT-5 cell line were determined by cytochemical staining, flow cytometry analysis, and karyotype analysis. Molecular features were identified by transcriptomic analysis and reverse transcription-polymerase chain reaction. To establish a tumor model, 5 × 106 YBT-5 cells were injected subcutaneously in nonobese diabetic/severe combined immune-deficiency (NOD/SCID) mice. DOT1L has been proposed as a potential therapeutic target for KMT2A-related leukemia; therefore, to explore the potential application of this new cell line, its sensitivity to a specific DOT1L inhibitor, EPZ004777 was measured ex vivo. The growth of YBT-5 does not depend on granulocyte-macrophage colony-stimulating factor. Cytochemical staining showed that α-naphthyl acetate esterase staining was positive and partially inhibited by sodium fluoride, while peroxidase staining was negative. Flow cytometry analysis of YBT-5 cells showed positive myeloid and monocytic markers. Karyotype analysis of YBT-5 showed 48,XY,+8,+8. The breakpoints between KMT2A exon 10 and exon 11 (KMT2A exon 10/11) and MLLT3 exon 5 and exon 6 (MLLT3 exon 5/6) were identified, which was different from all known breakpoint locations, and a novel fusion transcript KMT2A exon 10/MLLT3 exon 6 was formed. A tumor model was established successfully in NOD/SCID mice. EPZ004777 could inhibit the proliferation and induce the differentiation of YBT-5 cells. Therefore, a new acute monocytic leukemia cell line with clear biological and molecular features was established and may be used in the research and development of new agents targeting KMT2A-associated leukemia.

Direct comparison of the abilities of bone marrow mesenchymal versus hematopoietic stem cells to reverse hyperglycemia in diabetic NOD.SCID mice

ABSTRACTBoth bone marrow-derived hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) improve glycemic control in diabetic mice, but their kinetics and associated changes in pancreatic morphology have not been directly compared. Our goal was to examine the time course of improvements in glucose tolerance and associated changes in β-cell mass and proliferation following transplantation of equivalent numbers of HSC or MSC from the same bone marrow into diabetic non-obese diabetic severe combined immune deficiency (NOD.SCID) mice. We used transgenic mice with a targeted expression of yellow fluorescent protein (YFP) driven by the Vav1 gene promoter to genetically tag HSC and progeny. HSC were separated from bone marrow by fluorescence-activated cell sorting and MSC following cell culture. Equivalent numbers of isolated HSC or MSC were transplanted directly into the pancreas of NOD.SCID mice previously made diabetic with streptozotocin. Glucose tolerance, serum insulin, β-cell mass and β-cell proliferation were examined up to 28 days following transplant. Transplantation with MSC improved glucose tolerance within 7 days and serum insulin levels increased, but with no increase in β-cell mass. Mice transplanted with HSC showed improved glucose tolerance only after 3 weeks associated with increased β-cell proliferation and mass. We conclude that single injections of either MSC or HSC transiently improved glycemic control in diabetic NOD.SCID mice, but with different time courses. However, only HSC infiltrated the islets and were associated with an expanded β-cell mass. This suggests that MSC and HSC have differing mechanisms of action.

Embryonic stem-like cells from rabbit blastocysts cultured with melatonin could differentiate into three germ layers in vitro and in vivo.

The rabbit is considered an important model animal from which to obtain embryonic stem cells because of the utility of this animal in physiology and reproductive research. Here, we derived rabbit ES-like (rES-like) cells from blastocysts of superovulated Japanese white rabbits using culture medium containing 10-7 M melatonin, 10 ng/mL basic fibroblast growth factor, and 1,000 IU/mL human leukemia inhibitory factor. This concentration of melatonin had the most significant positive effects on the proliferation inner cell mass-derived cells (improving rates from 19.97% to 34.57%) and the longevity of passaging rES-like cells. Melatonin also enhanced the expression of pluripotent genes-including alkaline phosphatase, Pou5f1, Sox2, Klf4, c-Myc, Nanog, Line28a, and surface marker proteins-in fifth-passage rES-like cells. In vitro, these rES-like cells could spontaneously differentiate into some somatic cells, such as beating cardiomyocytes; formed embryoid bodies; expressed markers of the three germ layers after differentiation; and formed teratomas after injection into non-obese diabetic-severe combined immune deficient (NOD-SCID) mice. Thus, melatonin helped coax ES-like cells from rabbit blastocysts, which raises intriguing questions about the relationship between pluripotency and proliferation in rabbit embryonic stem cells. Mol. Reprod. Dev. 83: 1003-1014, 2016 © 2016 Wiley Periodicals, Inc.

Biological characteristics of side population cells in a self-established human ovarian cancer cell line.

The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×103 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self-renewal, differentiation, proliferation, tumorigenesis and stronger drug resistance capacities.

Clinical Utility of Patient-Derived Xenografts to Determine Biomarkers of Prognosis and Map Resistance Pathways in EGFR-Mutant Lung Adenocarcinoma.

Although epidermal growth factor receptor (EGFR) -mutated adenocarcinomas initially have high response rates to EGFR tyrosine kinase inhibitors (TKIs), most patients eventually develop resistance. Patient-derived xenografts (PDXs) are considered preferred preclinical models to study the biology of patient tumors. EGFR-mutant PDX models may be valuable tools to study the biology of these tumors and to elucidate mechanisms of resistance to EGFR-targeted therapies. Surgically resected early-stage non-small-cell lung carcinoma (NSCLC) tumors were implanted into nonobese diabetic severe combined immune deficient (NOD-SCID) mice. EGFR TKI treatment was initiated at tumor volumes of 150 μL. Gene expression analysis was performed using a microarray platform. Of 33 lung adenocarcinomas with EGFR activating mutations, only 6 (18%) engrafted and could be propagated beyond passage one. Engraftment was associated with upregulation of genes involved in mitotic checkpoint and cell proliferation. A differentially expressed gene set between engrafting and nonengrafting patients could identify patients harboring EGFR-mutant tumor with significantly different prognoses in The Cancer Genome Atlas Lung Adenocarcinoma datasets. The PDXs included models with variable sensitivity to first- and second-generation EGFR TKIs and the monoclonal antibody cetuximab. All EGFR-mutant NSCLC PDXs studied closely recapitulated their corresponding patient tumor phenotype and clinical course, including response pattern to EGFR TKIs. PDX models closely recapitulate primary tumor biology and clinical outcome. They may serve as important laboratory models to investigate mechanisms of resistance to targeted therapies, and for preclinical testing of novel treatment strategies.

Stem cell factor is essential for preserving NOD/SCID reconstitution capacity of ex vivo expanded cord blood CD34+ cells

Stem cell factor (SCF) is essential in the haematopoietic stem cells (HSCs) niche, and is therefore used extensively in haematopoietic stem and progenitor cells (HSPCs) exvivo expansion. However, in the literature, dose and schedule of SCF feeding varies widely. We previously proposed a novel SCF feeding regimen with proven effectiveness for HSPCs expansion; however, physiological function of expanded cells with this SCF feeding regimen required further research. CD34(+) cells were cultured with or without SCF supplementation in serum-free medium for 10days. Expanded cells were transplanted into sublethally irradiated non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice. Engraftment and multilineage reconstitution of transplanted cells were determined. Also, clonogenic potential of engrafted cells was analysed. Cells, both cultured with and without SCF supplementation, successfully engrafted and reconstituted blood cell lineages in NOD/SCID mice. However, level of engraftment and multilineage reconstitution reduced when cells were expanded without SCF supplementation. Meanwhile, frequencies of colony-forming cells (CFCs) amongst bone marrow cells were higher in mice transplanted with CD34(+) cells expanded with SCF supplementation. Reconstitution capacity reduced when CD34(+) cells were expanded without SCF supplementation, though this feeding regimen did not have any effect on cell expansion. This finding suggested that SCF was essential for preserving NOD/SCID reconstitution capacity of exvivo expanded CD34(+) cells.

Abstract 1184: Modeling mechanisms of resistance of epidermal growth factor receptor (EGFR) mutations to targeted drugs through patient-derived xenografts (PDX) of non-small cell lung cancer (NSCLC)

Abstract Background: Resistance to small molecule EGFR tyrosine kinase inhibitors (TKIs) is seen in some NSCLC patients with activating EGFR mutations. We evaluated in vivo PDX models for their utility in studying EGFR-targeted drug resistance mechanisms. Methods: Surgically resected early stage NSCLC tumors were implanted into non-obese diabetic severe combined immune deficient (NOD-SCID) mice. Tumors were passaged and expanded in new mouse hosts once the humane endpoint of 1.5 cm maximum diameter was reached. EGFR TKI treatment was initiated at an average tumor volume of 150mm3. Treatments included daily oral gavage with first and second generation EGFR TKIs, and with weekly intraperitoneally administered cetuximab. Results: Of the 55 NSCLC tumors with EGFR activating mutations, only 6 engrafted (11%) and could be propagated beyond the first passage, and 4 have been studied for their responsiveness to EGFR-targeted agents. Model 148, developed from a patient who received pre-operative erlotinib, showed intrinsic pan-resistance to all EGFR-targeted therapies despite having an L858R mutation. The corresponding patient did not respond to erlotinib, relapsed after surgery and did not receive additional TKI therapy. Model 137, with an exon19 E746-A750 deletion, recapitulated the patient's response to gefitinib at relapse; this model was sensitive to first and second generation EGFR TKIs. Model 192 also has the exon19 E746-A750 deletion, however it did not recapitulate the patient's observed sensitivity to erlotinib. Selection for a MET amplified population during engraftment may be the cause for the disparate drug sensitivities. Model 164 has a double exon19 L747-T751 deletion and T790M EGFR mutation. Neither patient nor xenograft responded to erlotinib; the xenograft responded to cetuximab. Resistance developed over time to a second generation EGFR TKI; this resistant phenotype was not stable as each subsequent passage of the ‘resistant’ tumor exhibited the same initial response pattern. Two potential mechanisms for this transient sensitivity are currently being investigated: epigenetic mechanisms and intratumoural mutational heterogeneity. Conclusions: PDX models may provide important insight into biomarkers and mechanisms of resistance to targeted therapies, and provide a means to test novel treatment strategies to improve future treatment efficacies. Citation Format: Erin L. Stewart, Celine Mascaux, Shingo Shakashita, Devang Panchal, Dennis Wang, Ming Li, Nhu-An Pham, Natasha Leighl, Geoffrey Liu, Frances A. Shepherd, Ming-Sound Tsao. Modeling mechanisms of resistance of epidermal growth factor receptor (EGFR) mutations to targeted drugs through patient-derived xenografts (PDX) of non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1184. doi:10.1158/1538-7445.AM2014-1184

Sorting and identification of side population cells in the human cervical cancer cell line HeLa

BackgroundSeveral reports have revealed that cancer stem cells (CSCs) exist in many types of solid tumors. Some studies have demonstrated that side population (SP) cells isolated from diverse cancer lines harbor cancer stem-like properties, but there are few reports examining the characteristic of SP cells in human cervical cancer. The aim of this study is 1) to find out a feasible way to detect the tumor stem-like cells in cervical cancer, and 2) to analyze the properties of the SP cells being sorted.MethodsIsolated SP and non-SP cells from human cervical cancer cell line Hela by Hoechst 33342 dying method and flow cytometry analysis. Observing morphology of SP and non-SP cells. The expression of various biomarkers putatively related to cancer stem cells were investigated by immucytochemistry of SP and non-SP cells. We also analyzed cell cycle and cell apoptosis for sorted cells. The oncogenicity of the SP and non-SP cells were analyzed by tumor formation in nonobesediabeti- c/severe combined immune- deficient (NOD/SCID) mice. The drug-resistant and radiation-resistant index between SP, non-SP and Hela cells was estimated by MTS assay.ResultsThe fraction of SP cells in Hela was approximately 1.07 ± 0.32%. SP cells were smaller and rounder in shape than non-SP cells, and mostly showed colony-like growth. Immunocytochemistry showed that stem cell makers (Oct3/4, CD133, BCRP) were highly expressed in SP cells. Moreover, the number of apoptotic cells among non-SP cells (17.6 ± 3.7%) was significantly higher compared with that among SP cells (4.4 ± 1.2%). The HE staining of in vivo grown tumors result from SP cells showed more poor differentiation, though no significant differences were shown between SP and non-SP cells in NOD/SCID mice tumorigenicity. Furthermore, SP cells demonstrated a higher degree of drug resistance against trichostatin A (TSA) compared with that of non-SP and Hela cells. SP cells were also found to be more resistant against radiotherapy.ConclusionsSP cells possess some characteristics of CSCs, namely high proliferation ability, chemoresistance and radioresistance, which may be helpful to elucidate novel targets for effective clinical treatments of cervical cancer in the future.

Priming of mononuclear cells with a combination of growth factors enhances wound healing via high angiogenic and engraftment capabilities

Recently, we demonstrated that a specific combination of growth factors enhances the survival, adhesion and angiogenic potential of mononuclear cells (MNCs). In this study, we sought to investigate the changes of the angiogenic potential of MNCs after short-time priming with a specific combination of growth factors. MNCs were isolated using density gradient centrifugation and incubated with a priming cocktail containing epidermal growth factor (EGF), insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, FMS-like tyrosine kinase (Flt)-3L, Angiopoietin (Ang)-1, granulocyte chemotactic protein (GCP)-2 and thrombopoietin (TPO) (all 400 ng/ml) for 15, 30 and 60 min. Wounds in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice were created by skin excision followed by cell transplantation. We performed a qRT-PCR analysis on the growth factor–primed cells. The angiogenic factors vascular endothelial growth factor (VEGF)-A, FGF-2, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and interleukin (IL)-8 and the anti-apoptotic factors IGF-1 and transforming growth factor-β1 were significantly elevated in the MNCs primed for 30 min. (T30) compared with the non-primed MNCs (T0). The scratch wound assay revealed that T30- conditioned media (CM) significantly increased the rate of fibroblast-mediated wound closure compared with the rates from T0-CM and human umbilical vein endothelial cells (HUVEC)-CM at 20 hrs. In vivo wound healing results revealed that the T30-treated wounds demonstrated accelerated wound healing at days 7 and 14 compared with those treated with T0. The histological analyses demonstrated that the number of engrafted cells and transdifferentiated keratinocytes in the wounds were significantly higher in the T30-transplanted group than in the T0-transplanted group. In conclusion, this study suggests that short-term priming of MNCs with growth factors might be alternative therapeutic option for cell-based therapies.

Reovirus as a successful ex vivo purging modality for multiple myeloma

Autologous stem cell rescue (ASCT) following high-dose myeloablative chemotherapy is considered to be a therapeutic option for many multiple myeloma (MM) patients; however relapse post ASCT presents a major challenge. The oncolytic potential of reovirus has been previously demonstrated and is currently undergoing phase I monotherapy clinical trials for MM and phase II/III clinical trials for solid tumors. Here we tested the hypothesis that reovirus can successfully purge MM in a murine model that partially recapitulates human MM. RPMI 8226, MM1S, H929 and U266 human myeloma cell lines were exposed to reovirus and oncolysis was assessed. Apheresis product admixed with MM cells was purged with live reovirus (LV) or dead virus (DV) and purging efficacy was monitored via flow cytometry, reverse transcribed-PCR (RT-PCR) and disease relapse in non obese diabetic/severe combined immune deficient (NOD/SCID) mice. Significant LV purging was seen with MM1S, H929 and U266 and the complete ex vivo purging achieved with RPMI 8226 was confirmed by flow cytometry, RT-PCR and absence of disease relapse in vivo. Mice that received LV-purged autografts exhibited 100% survival in comparison to mice that received DV-purged controls. Reovirus's unique ability to kill MM while sparing hematopoietic stem cells places it as an attractive purging agent for MM during ASCT.

Renoprotective Effect of Human Umbilical Cord–Derived Mesenchymal Stem Cells in Immunodeficient Mice Suffering from Acute Kidney Injury

It is unknown whether human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) can improve the renal function of patients suffering from acute kidney injury. Moreover, before beginning clinical trials, it is necessary to investigate this renoprotective effect of hUC-MSCs in a xenogeneic model of acute kidney injury. However, no previous studies have examined the application of hUC-MSCs to immunodeficient mice suffering from acute kidney injury. The objectives of this study were to examine whether hUC-MSCs could improve renal function in nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice suffering from acute kidney injury, and to investigate the mechanism(s) for hUC-MSCs to improve renal function in this xenogeneic model. Early (3 hr) and late (12 hr) administrations of hUC-MSCs (106 cells) were performed via the external jugular vein into NOD-SCID mice suffering from either folic acid (FA) (250 mg/kg body weight) or vehicle. The results showed that early administration of hUC-MSCs improved the renal function of NOD-SCID mice suffering from FA-induced acute kidney injury, as evidenced by decreased serum urea nitrogen and serum creatinine levels, as well as a reduced tubular injury score. The beneficial effects of hUC-MSCs were through reducing apoptosis and promoting proliferation of renal tubular cells. These benefits were independent of inflammatory cytokine effects and transdifferentiation. Furthermore, this study is the first one to show that the reduced apoptosis of renal tubular cells by hUC-MSCs in this xenogeneic model is mediated through the mitochondrial pathway, and through the increase of Akt phosphorylation.

Reovirus as a Viable Therapeutic Option for the Treatment of Multiple Myeloma

Despite the recent advances made in the treatment of multiple myeloma, the disease still remains incurable. The oncolytic potential of reovirus has previously been shown and is currently in phase III clinical trials for solid tumors. We tested the hypothesis that reovirus can successfully target human multiple myeloma in vitro, ex vivo, and in vivo without affecting human hematopoietic stem cell (HHSC) re-population/differentiation in a murine model that partially recapitulates human multiple myeloma. Human myeloma cell lines and ex vivo tumor specimens were exposed to reovirus and oncolysis and mechanisms of cell death were assessed. RPMI 8226(GFP+) cells were injected intravenously to non-obese diabetic/severe combined immune deficient (NOD/SCID) mice and treated with live reovirus (LV) or dead virus (DV). Multiple myeloma disease progression was evaluated via whole-body fluorescence and bone marrow infiltration. HHSCs exposed to LV/DV were injected to NOD/SCID mice and re-population/differentiation was monitored. A total of six of seven myeloma cell lines and five of seven patient tumor specimens exposed to reovirus showed significant in vitro sensitivity. Tumor response of multiple myeloma by LV, but not DV, was confirmed by comparison of total tumor weights (P = 0.05), and bone marrow infiltration (1/6, LV; 5/6, DV). Mice injected with LV- or DV-exposed HHSCs maintained in vivo re-population/lineage differentiation showing a lack of viral effect on the stem cell compartment. Reovirus oncolysis was mediated primarily by activation of the apoptotic pathways. The unique ability of reovirus to selectively kill multiple myeloma while sparing HHSCs places it as a promising systemic multiple myeloma therapeutic for clinical testing.

Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells

To isolate and characterize human leiomyoma stem cells by the side population (SP) method. Prospective experimental human and animal study. University research laboratory-affiliated infertility clinic. Women undergoing laparoscopic myomectomy. Female non-obese diabetic severe combined immune deficiency (NOD-SCID) mutation mice. Obtainment of human leiomyoma SP cells as candidate tumor-initiating cells and establishment of two leiomyoma SP lines. Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cDNA microarrays hybridization, cell culture, karyotype, molecular analysis, immunocytochemistry, invitro differentiation, xenotransplantation assays, immunohistochemistry. SP cells from human leiomyomas were isolated, identified, and characterized. Two leiomyoma's SP cell lines with a normal karyotype were thus established. Undifferentiated status was confirmed by the expression of OCT-4, NANOG, DNMT3B, GDF3. Presence of typical mesenchymal markers (CD90, CD105, CD73) and absence of hematopoietic stem cell markers (CD34, CD45) supported their mesodermal origin. Mesenchymal lineage commitment was also demonstrated by their ability to differentiate invitro into adipogenic and osteogenic lineages. Finally, their functional capability was established in an animal model by leiomyoma tissue reconstruction. SP cells from human leiomyoma exhibit key features of tumor-initiating cells, opening up new possibilities of understanding the origin and developing new nonsurgical approaches for leiomyomas.

Preclinical Rationale for the Use of the Combined Treatment with the AKT Inhibitor Perifosine and the Multikinase Inhibitor Sorafenib in Hodgkin Lymphoma

Abstract 1653 Introduction:A significant proportion of Hodgkin lymphoma (HL) patients refractory to first-line chemotherapy or relapsing after autologous transplantation are not cured with currently available treatments and require new treatments. The PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL. These pathways can be targeted using the AKT inhibitor perifosine (Æterna Zentaris GmBH, Germany, EU), and the RAF/MEK/ERK inhibitor sorafenib (Nexavar®, Bayer, Germany, EU). We hypothesized that perifosine in combination with sorafenib might have a therapeutic activity in HL by overcoming the cytoprotective and anti-apoptotic effects of PI3K/Akt and RAF/MEK/ERK pathways. Since preclinical evidence supporting the anti-lymphoma effects of the perifosine/sorafenib combination are still lacking, the present study aimed at investigating in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS:Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. RESULTS:While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of apoptosis. In responsive cell lines, WB analysis showed that anti-proliferative events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. CONCLUSIONS:Perifosine/sorafenib combination resulted in potent anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation in HL patients. Disclosures:No relevant conflicts of interest to declare.

Comparison of childhood leukemia initiating cell populations in NOD/SCID and NSG mice

Leukemia initiating cells (LICs) are defined by their ability to generate leukemias in animal models and to self-renew by repopulating serial recipients. Recent findings suggest that multiple sub-populations of acute leukemia cells are capable of engrafting immune deficient mice raise important questions about the biology of these diseases.1, 2, 3 If several cell populations have LIC properties, what are the relationships of these stem cell populations to each other and which populations are most important to target with therapy? To address these questions, it is crucial to utilize assays that are capable of identifying any leukemia cells that possess stem cell properties. The recently developed NOD/LtSz-scid IL-2Rγc null (NSG) mouse is now considered a more permissive host for the growth and development of normal and acute myeloid leukemia cells than the nonobese diabetic severe combined immune deficient (NOD/SCID) strain.4, 5 In acute lymphoblastic leukemia (ALL) engraftment of high-risk cases has been reported to be slower in NOD/SCID than NSG but otherwise leukemias developed in both strains.6 However, another study reported that engraftment of T-ALL cells was improved using NSG mice.7 Observations that different populations of cells can engraft different mouse strains raise questions as to how the reports that used only NOD/SCID mice should be interpreted. NSG and NOD/SCID strains have not previously been directly and quantitatively compared for engraftment potential of pediatric LIC sub-populations using the same source of cells. Such studies should provide clear information as to the permissiveness of each strain and which sub-populations have LIC potential.

Inflammation Confers Dual Effects on Nociceptive Processing in Chronic Neuropathic Pain Model

Although inflammation induces pain, immune cells also produce mediators that can effectively counteract it. To further elucidate the role of the immune response, we analyzed the relationship of pain behavior, several inflammatory signals, and opioid peptides using partial sciatic nerve ligation in mice at different levels of immunocompromise. Sciatic nerves of C57BL/6C, nonobese diabetic (NOD), or nonobese diabetic-severe combined immune deficiency (NOD-SCID) mice were partially ligated. Responses to mechanical and radiant heat stimuli were observed. Inflammation was detected by immunohistochemistry and flow cytometry. Inflammatory cytokines and opioid peptides were analyzed using real-time polymerase chain reaction and enzyme-linked immunosorbent assay or immunostaining. Inflammation in immunocompromised mice was subordinate when compared with that seen in C57BL/6C mice. In addition, immunocompromised mice had less pain hypersensitivity at early stages. Whereas proinflammatory tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and interferon-γ (IFN-γ), as well as antiinflammatory interleukin 1 receptor antagonist (IL-1Ra), interleukin 4 (IL-4), interleukin 10 (IL-10), and interleukin 13 (IL-13) cytokine expression and protein were increased in C57BL/6C mice, they were lower in immunocompromised mice. Although enkephalin, dynorphin, and β-endorphin messenger RNA expression also increased in C57BL/6C mice, peaking on day 14, this result was not observed in immunocompromised mice. The contribution of inflammation to nerve injury is complex with biphasic modulation. During the early phase, a wide range of proinflammatory cytokines are released, leading to enhanced pain. In contrast, the analgesic effect of opioid peptides and antiinflammatory cytokines was more predominate in the later phases of injury, leading to attenuated pain responses.

The tetraspanin CD9 regulates migration, adhesion, and homing of human cord blood CD34+ hematopoietic stem and progenitor cells

AbstractThe stromal cell–derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing and engraftment of hematopoietic stem/progenitor cells (HSCs) during bone marrow transplantation. To investigate the transcriptional regulation provided by this axis, we performed the first differential transcriptome profiling of human cord blood CD34+ cells in response to short-term exposure to SDF-1 and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family of proteins, was expressed in CD34+CD38−/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously down-regulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expression was modulated via CXCR4, G-protein, protein kinase C, phospholipase C, extracellular signal-regulated kinase, and Janus kinase 2 signals. Pretreatment of CD34+ cells with the anti-CD9 monoclonal antibody ALB6 significantly inhibited SDF-1–mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells was enhanced. Pretreatment of CD34+ cells with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice. Sorted CD34+CD9− cells displayed lower bone marrow homing capacity compared with that of total CD34+ cells. CD9 expression on homed CD34+ cells was significantly up-regulated in vivo. Our results indicate that CD9 might possess specific functions in HSC homing.