搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM,a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells,GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However,the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature,which defines aggressiveness and invasion,and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1,which encode a key regulator of selective autophagy,p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs,DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation,thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast,autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally,DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism,in particular reduced ATP and lactate levels. Taken together,these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells. View Publication

The antitumor activity of LPS was first described by Dr. William Coley. However,its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 μg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs),regulatory T cells,myeloid-derived suppressor cells,and CD8(+) regulatory T cells. In contrast,high-dose LPS (10 μg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs,as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs,which were immunosuppressive after the low-dose LPS,and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration,whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion,our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice. View Publication

BACKGROUND AND OBJECTIVES: Leptin receptors can be expressed by acute myelogenous leukemia (AML) cells,but the functional effects of leptin on native AML blasts have not been characterized in detail. We investigated systemic leptin levels in AML patients and in vitro effects of leptin on cultured AML blasts. DESIGN AND METHODS: Serum leptin levels were compared for patients with untreated AML and healthy controls. Native AML blasts were derived from a large group of consecutive patients,and effects of leptin on proliferation (suspension cultures and colony formation),constitutive cytokine secretion,differentiation and apoptosis regulation were assayed in vitro. RESULTS: Systemic leptin levels were decreased in patients with untreated AML,and leptin levels in acute leukemia patients were not altered during severe chemotherapy-induced cytopenia and complicating febrile neutropenia. In vitro studies demonstrated that leptin increased AML blast release of interleukin (IL) 1beta,IL6,tumor necrosis factor (TNF) alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). This enhancing effect showed no correlation with CD34 expression and was not dependent on the presence of serum,induction of differentiation or alteration of caspase 3 activity with decreased in vitro apoptosis. Leptin also increased spontaneous AML blast proliferation,whereas divergent effects on blast proliferation were observed in the presence of exogenous cytokines. The in vitro effects were usually observed at concentrations exceeding the systemic levels. INTERPRETATION AND CONCLUSIONS: Our results suggest that systemic leptin levels alone do not have a major influence on native AML blasts,but the systemic levels in combination with local leptin release in the bone marrow may affect the functional characteristics of these cells. View Publication

BACKGROUND AND OBJECTIVES: Campath-1H is used in conditioning regimens and more recently as an anti-leukemic therapy in acute lymphoblastic leukemias (ALL). We therefore investigated CD52 expression and campath-1H-mediated lysis of ALL cells in vitro. DESIGN AND METHODS: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells and cell lines using human serum. Antibody-dependent cellular cytotoxicity (ADCC) was performed by calcein-AM release assays. RESULTS: CD52 was expressed in four out of eight ALL cell lines studied. Among 61 freshly isolated ALL samples CD52 was expressed at varying levels in 87% of cases. Whereas ADCC was equivalent in different CD52+ lines,complement-dependent cytotoxicity (CDC) was variable. The REH cell line bearing the t(12;21) translocation showed 47-60% lysis when treated with 10 microg/mL campath-1H compared to 0-6% for the other cell lines expressing equivalent amounts of CD52. Furthermore all nine ALL samples with t(12;21) showed very high CDC (mean 97%) compared to the other 24 CD52+cases (mean 24%)(ptextless0.0001). In t(12;21) samples,efficient CDC was obtained with as little as 1 microg/mL campath-1H. CDC correlated in part with CD52 levels,suggesting that CD52 expression and other yet undefined factors contribute to the particular sensitivity of t(12;21) cells. The resistance of non t(12;21) ALL cases could be overcome to a limited extent by increasing the concentration of campath-1H,blocking the CD55 and CD59 complement inhibitors,and more effectively by combining campath-1H with fludarabine. INTERPRETATION AND CONCLUSIONS: We conclude that most ALL samples express CD52 to a variable level and that campath-1H has cytotoxic activity against CD52+ALL,alone or in combination with cytotoxic drugs. View Publication

The concept of cancer stem cells (CSC) embodies two aspects: the stem cell as the initial target of the oncogenic process and the existence of two populations of cells in cancers: the CSC and derived cells. The second is discussed in this review. CSC are defined as cells having three properties: a selectively endowed tumorigenic capacity,an ability to recreate the full repertoire of cancer cells of the parent tumor and the expression of a distinctive repertoire of surface biomarkers. In operational terms,the CSC are among all cancer cells those able to initiate a xenotransplant. Other explicit or implicit assumptions exist,including the concept of CSC as a single unique infrequent population of cells. To avoid such assumptions,we propose to use the operational term tumor-propagating cells (TPC); indeed,the cells that initiate transplants did not initiate the cancer. The experimental evidence supporting the explicit definition is analyzed. Cancers indeed contain a fraction of cells mainly responsible for the tumor development. However,there is evidence that these cells do not represent one homogenous population. Moreover,there is no evidence that the derived cells result from an asymmetric,qualitative and irreversible process. A more general model is proposed of which the CSC model could be one extreme case. We propose that the TPC are multiple evolutionary selected cancer cells with the most competitive properties [maintained by (epi-)genetic mechanisms],at least partially reversible,quantitative rather than qualitative and resulting from a stochastic rather than deterministic process. View Publication

The development of immunodeficient mouse xenograft models has greatly facilitated the investigation of some human hematopoietic malignancies,but application of this approach to the myelodysplastic syndromes (MDSs) has proven difficult. We now show that cells from most MDS patients (including all subtypes) repopulate nonobese diabetic-severe combined immunodeficient (scid)/scid-beta2 microglobulin null (NOD/SCID-beta2m(-/-)) mice at least transiently and produce abnormal differentiation patterns in this model. Normal marrow transplants initially produce predominantly erythroid cells and later predominantly B-lymphoid cells in these mice,whereas most MDS samples produced predominantly granulopoietic cells. In 4 of 4 MDS cases,the regenerated cells showed the same clonal markers (trisomy 8,n = 3; and 5q-,n = 1) as the original sample and,in one instance,regenerated trisomy 8(+) B-lymphoid as well as myeloid cells were identified. Interestingly,the enhanced growth of normal marrow obtained in NOD/SCID-beta2m(-/-) mice engineered to produce human interleukin-3,granulocyte-macrophage colony-stimulating factor,and Steel factor was seen only with 1 of 7 MDS samples. These findings support the concept that human MDS originates in a transplantable multilineage hematopoietic stem cell whose genetic alteration may affect patterns of differentiation and responsiveness to hematopoietic growth factors. They also demonstrate the potential of this new murine xenotransplant model for future investigations of MDS. View Publication

The steps to leukemia following an in utero fusion of MLL (HRX,ALL-1) to a partner gene in humans are not known. Introduction of the Mll-AF9 fusion gene into embryonic stem cells results in leukemia in mice with cell-type specificity similar to humans. In this study we used myeloid colony assays,immunophenotyping,and transplantation to evaluate myelopoiesis in Mll-AF9 mice. Colony assays demonstrated that both prenatal and postnatal Mll-AF9 tissues have significantly increased numbers of CD11b(+)/CD117(+)/Gr-1(+/-) myeloid cells,often in compact clusters. The self-renewal capacity of prenatal myeloid progenitors was found to decrease following serial replating of colony-forming cells. In contrast,early postnatal myeloid progenitors increased following replating; however,the enhanced self-renewal of early postnatal myeloid progenitor cells was limited and did not result in long-term cell lines or leukemia in vivo. Unlimited replating,long-term CD11b/Gr-1(+) myeloid cell lines,and the ability to produce early leukemia in vivo in transplantation experiments,were found only in mice with overt leukemia. Prenatal Mll-AF9 tissues had reduced total (mature and progenitor) CD11b/Gr-1(+) cells compared with wild-type tissues. Colony replating,immunophenotyping,and cytochemistry suggest that any perturbation of cellular differentiation from the prenatal stage onward is partial and largely reversible. We describe a novel informative in vitro and in vivo model system that permits study of the stages in the pathogenesis of Mll fusion gene leukemia,beginning in prenatal myeloid cells,progressing to a second stage in the postnatal period and,finally,resulting in overt leukemia in adult animals. View Publication

Bone marrow-derived endothelial precursor cells incorporate into neovasculature and have been successfully used as vehicles for gene delivery to brain tumors. To determine whether systemically administered Sca1+ bone marrow cells labeled with superparamagnetic iron oxide nanoparticles can be detected by in vivo magnetic resonance imaging in a mouse brain tumor model,mouse Sca1+ cells were labeled in vitro with ferumoxides-poly-L-lysine complexes. Labeled or control cells were administered intravenously to glioma-bearing severe combined immunodeficient (SCID) mice. Magnetic resonance imaging (MRI) was performed during tumor growth. Mice that received labeled cells demonstrated hypointense regions within the tumor that evolved over time and developed a continuous dark hypointense ring at a consistent time point. This effect was not cleared by administration of a gadolinium contrast agent. Histology showed iron-labeled cells around the tumor rim in labeled mice,which expressed CD31 and von Willebrand factor,indicating the transplanted cells detected in the tumor have differentiated into endothelial-like cells. These results demonstrate that MRI can detect the incorporation of magnetically labeled bone marrow-derived precursor cells into tumor vasculature as part of ongoing angiogenesis and neovascularization. This technique can be used to directly identify neovasculature in vivo and to facilitate gene therapy by noninvasively monitoring these cells as gene delivery vectors. View Publication

Cancer cells exhibit increased glycolysis for ATP production due,in part,to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration,how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (rho(-)) harboring mitochondrial DNA deletion exhibit dependency on glycolysis,increased NADH,and activation of Akt,leading to drug resistance and survival advantage in hypoxia. Similarly,chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism,leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents. View Publication

PURPOSE: To examine the role of cancer stem cells (CSC) in mediating metastasis in inflammatory breast cancer (IBC) and the association of these cells with patient outcome in this aggressive type of breast cancer. EXPERIMENTAL DESIGN: CSCs were isolated from SUM149 and MARY-X,an IBC cell line and primary xenograft,by virtue of increased aldehyde dehydrogenase (ALDH) activity as assessed by the ALDEFLUOR assay. Invasion and metastasis of CSC populations were assessed by in vitro and mouse xenograft assays. Expression of ALDH1 was determined on a retrospective series of 109 IBC patients and this was correlated with histoclinical data. All statistical tests were two sided. Log-rank tests using Kaplan-Meier analysis were used to determine the correlation of ALDH1 expression with development of metastasis and patient outcome. RESULTS: Both in vitro and xenograft assays showed that invasion and metastasis in IBC are mediated by a cellular component that displays ALDH activity. Furthermore,expression of ALDH1 in IBC was an independent predictive factor for early metastasis and decreased survival in this patient population. CONCLUSIONS: These results suggest that the metastatic,aggressive behavior of IBC may be mediated by a CSC component that displays ALDH enzymatic activity. ALDH1 expression represents the first independent prognostic marker to predict metastasis and poor patient outcome in IBC. The results illustrate how stem cell research can translate into clinical practice in the IBC field. View Publication