Ulf Kahlert

Johns Hopkins University, Baltimore, Maryland, United States

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Publications (25)132.63 Total impact

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    ABSTRACT: Glioblastomas are highly aggressive tumors that contain treatment resistant stem-like cells. Therapies targeting developmental pathways such as Notch eliminate many neoplastic glioma cells, including those with stem cell features, but their efficacy can be limited by various mechanisms. One potential avenue for chemotherapeutic resistance is the induction of autophagy, but little is known how it might modulate the response to Notch inhibitors. We used the γ-secretase inhibitor (GSI) MRK003 to block Notch pathway activity in glioblastoma neurospheres and assessed effects on autophagy. A dramatic, several fold increase of LC3B-II/LC3B-I autophagy marker was noted on western blots, along with the emergence of punctate LC3B immunostaining in cultured cells. By combining the late stage autophagy inhibitor chloroquine (CQ) with MRK003, a significant induction in apoptosis and reduction in growth was noted as compared to Notch inhibition alone. A similar beneficial effect on inhibition of clonogenicity in soft agar was seen using the combination treatment. These results demonstrate that pharmacological Notch blockade can induce protective autophagy in glioma neurospheres, resulting in chemoresistance, which can be abrogated by combination treatment with autophagy inhibitors. This article is protected by copyright. All rights reserved.
    No preview · Article · Nov 2015 · Brain Pathology
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    ABSTRACT: Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.\ This article is protected by copyright. All rights reserved.
    No preview · Article · Sep 2015 · International Journal of Cancer
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    ABSTRACT: Wingless (Wnt) signaling is an important pathway in gliomagenesis and in the growth of stem-like glioma cells. Using immunohistochemistry to assess the translocation of β-catenin protein, we identified intranuclear staining suggesting Wnt pathway activation in 8 of 43 surgical samples (19%) from adult patients with glioblastoma and in 9 of 30 surgical samples (30%) from pediatric patients with glioblastoma. Wnt activity, evidenced by nuclear β-catenin in our cohort and high expression of its target AXIN2 (axis inhibitor protein 2) in published glioma datasets, was associated with shorter patient survival, although this was not statistically significant. We determined the effects of the porcupine inhibitor LGK974 on 3 glioblastoma cell lines with elevated AXIN2 and found that it reduced Wnt pathway activity by 50% or more, as assessed by T-cell factor luciferase reporters. Wnt inhibition led to suppression of growth, proliferation in cultures, and modest induction of cell death. LGK974 reduced NANOG messenger RNA levels and the fraction of cells expressing the stem cell marker CD133 in neurosphere cultures, induced glial differentiation, and suppressed clonogenicity. These data indicate that LGK974 is a promising new agent that can inhibit the canonical Wnt pathway in vitro, slow tumor growth, and deplete stem-like clonogenic cells, thereby providing further support for targeting Wnt in patients with glioblastoma.
    No preview · Article · Jul 2015 · Journal of Neuropathology and Experimental Neurology
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    ABSTRACT: Malignant gliomas, with an average survival time of 16-19 months after initial diagnosis, account for one of the most lethal tumours overall. Current standards in patient care provide only unsatisfying strategies in diagnostic and treatment for high-grade gliomas. Here we describe metabolic phenomena in the choline and glycine network associated with stem cell culture conditions in the classical glioma cell line 1187. Using high-resolution proton magnetic resonance spectroscopy of cell culture metabolic extracts we compare the metabolic composition of 1187 chronically propagated as adherent culture in medium supplemented with serum to serum-free neurosphere growth. We found that the switch to neurosphere growth, besides the increase of cells expressing the putative glioma stem cell marker CD 133, modulated a number of intracellular metabolites including choline, creatine, glycine, and myo-inositol that have been previously reported as potential diagnostic markers in various tumours. These findings highlight the critical influence of culture conditions on glioma cell metabolism, and therefore particular caution should be drawn to the use of in vitro system research in order to investigate cancer metabolism.
    No preview · Article · Jan 2015 · Folia Neuropathologica
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    ABSTRACT: Diffuse spread through brain parenchyma and the presence of hypoxic foci rimmed by neoplastic cells are two cardinal features of glioblastoma, and low oxygen is thought to drive movement of malignant gliomas in the core of the lesions. Transcription factors associated with epithelial-to-mesenchymal transition (EMT) have been linked to this invasion, and we found that hypoxia increased in vitro invasion up to 4-fold in glioblastoma neurosphere lines and induced the expression of ZEB1. Immunohistochemical assessment of 295 surgical specimens consisting of various types of pediatric and adult brain cancers showed that ZEB1 expression was significantly higher in infiltrative lesions than less invasive tumors such as pilocytic astrocytoma and ependymoma. ZEB1 protein was also present in human fetal periventricular stem and progenitor cells and ZEB1-inhibition impaired migration of in vitro propagated human neural stem cells. The induction of ZEB1 protein in hypoxic glioblastoma neurospheres could be partially blocked by the HIF1alpha inhibitor digoxin. Targeting ZEB1 blocked hypoxia-augmented invasion of glioblastoma cells in addition to slowing them in normoxia. These data support the role for ZEB1 in invasive and high grade brain tumors and suggest its key role in promoting invasion in the hypoxic tumor core as well as in the periphery. This article is protected by copyright. All rights reserved.
    Full-text · Article · Dec 2014 · Brain Pathology
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    ABSTRACT: The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.
    Full-text · Article · Nov 2014 · Oncotarget

  • No preview · Article · Oct 2014 · Cancer Research
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    ABSTRACT: Introduction Although there are many experimental studies describing the methodology of the middle cerebral artery occlusion (MCAO) in the literature, only limited data on these distinct anatomical structures and the details of the surgical procedure in a step by step manner. The aim of the present study simply is to examine the surgical anatomy of MCAO model and its modifications in the rat. Materials and methods Forty Sprague-Dawley rats were used; 20 during the training phase and 20 for the main study. The monofilament sutures were prepared as described in the literature. All surgical steps of the study were performed under the operating microscope, including insertion of monofilament into middle cerebral artery through the internal carotid artery. Results After an extensive training period, we lost two rats in four weeks. The effects of MCAO were confirmed by the evidence of severe motor deficit during the recovery period, and histopathological findings of infarction were proved in all 18 surviving rats. Conclusion In this study, a microsurgical guideline of the MCAO model in the rat is provided with the detailed description of all steps of the intraluminal monofilament insertion method with related figures.
    Full-text · Article · Jun 2014 · Experimental and Translational Stroke Medicine
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    ABSTRACT: This is supplemental data of the my article published in Scientific Reports.
    Full-text · Dataset · Jan 2014
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    ABSTRACT: The impact of pressure waves on cells may provide several possible applications in biology and medicine including the direct killing of tumors, drug delivery or gene transfection. In this study we characterize the physical properties of mechanical pressure waves generated by a nanosecond laser pulse in a setup with well-defined cell culture conditions. To systematically characterize the system on the relevant length and time scales (micrometers and nanoseconds) we use photon Doppler velocimetry (PDV) and obtain velocity profiles of the cell culture vessel at the passage of the pressure wave. These profiles serve as input for numerical pressure wave simulations that help to further quantify the pressure conditions on the cellular length scale. On the biological level we demonstrate killing of glioblastoma cells and quantify experimentally the pressure threshold for cell destruction.
    Full-text · Article · Jan 2014 · Scientific Reports

  • No preview · Article · Aug 2013 · Cancer Research
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    ABSTRACT: The cellular reprogramming factor LIN28A promotes tumorigenicity in cancers arising outside the central nervous system, but its role in brain tumors is unknown. We detected LIN28A protein in a subset of human gliomas observed higher expression in glioblastoma (GBM) than in lower grade tumors. Knockdown of LIN28A using lentiviral shRNA in GBM cell lines inhibited their invasion, growth and clonogenicity. Expression of LIN28A in GBM cell lines increased the number and size of orthotopic xenograft tumors. LIN28A expression also enhanced the invasiveness of GBM cells in vitro and in vivo. Increasing LIN28A was associated with down-regulation of tumor suppressing microRNAs let-7b and let-7g and up-regulation of the chromatin modifying protein HMGA2. The increase in tumor cell aggressiveness in vivo and in vitro was accompanied by an upregulation of pro-invasive gene expression, including SNAI1. To further investigate the oncogenic potential of LIN28A, we infected hNSC with lentiviruses encoding LIN28A together with dominant negative R248W-TP53, constitutively active KRAS and hTERT. Resulting subclones proliferated at an increased rate and formed invasive GBM-like tumors in orthotopic xenografts in immunodeficient mice. Similar to LIN28A-transduced GBM neurosphere lines, hNSC-derived tumor cells showed increased expression of HMGA2. Taken together, these data suggest a role for LIN28A in high grade gliomas and illustrate an HMGA2-associated, pro-invasive program that can be activated in GBM by LIN28A-mediated suppression of let-7 microRNAs.
    Full-text · Article · Jul 2013 · Oncotarget
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    ABSTRACT: Purpose: The concept of transplantation of neuronal cells to treat Huntington's and Parkinson's diseases is based on the proven principle that dopaminergic and GABA-ergic progenitor neurons (from the human developing ventral mesencephalon and whole ganglionic eminence) can survive, differentiate and functionally integrate into an allogenic host brain. However, several donor and host-specific variables play a major role in the safety and outcome of this procedure. In this paper, we seek to summarize an updated neural transplantation protocol, based on our institutional experience and many years of collaboration with other neurotransplantation centers. Methods: We present a detailed clinical neurotransplantation protocol for Parkinson's (PD) and Huntington's (HD) diseases with special emphasis in understanding the anatomical relationships of the human fetal tissue that are relevant for selection of the desired cell populations. Results: Two detailed step-wise neurotransplantation protocols are presented, outlining strategies facilitating the avoidance of possible procedure-related complications. Conclusions: In this paper we delineated some crucial technical factors enabling the execution of a safe and effective neural transplantation. The protocols presented here might contribute to further development of the experimental clinical neurotransplantation towards a routine therapeutic procedure.
    Full-text · Article · Jun 2013

  • No preview · Article · Apr 2013 · Cancer Research
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    U.D. Kahlert · G Nikkhah · J Maciaczyk
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    ABSTRACT: Tumor dissemination and metastatic behavior account for the vast majority of cancer associated mortality. Epithelial tumors achieve this progressive state via epithelial-to-mesenchymal transition (EMT); however, the importance of this process in the neuroepithelial context is currently very controversially discussed. The review describes the current research status concerning EMT-like changes in malignant gliomas including the role of TWIST1, ZEB1/ZEB2 and SNAIl1/SNAIl2 as inducers for cell-invasiveness in GBMs. Furthermore, WNT/β-catenin signaling with its key-component FRIZZLED4 activating an EMT-like program in malignant gliomas and its relationship to the stem-like phenotype as well as discoveries on micro-RNA-level regulating the EMT-like process are discussed.
    Full-text · Article · Dec 2012 · Cancer letters
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    ABSTRACT: In addition to intrinsic regulatory mechanisms, brain tumor stemlike cells (BTSCs), a small subpopulation of malignant glial tumor-derived cells, are influenced by environmental factors. Previous reports showed that lowering oxygen tension induced an increase of BTSCs expressing CD133 and other stem cell-related genes and more pronounced clonogenic capacity in vitro. We investigated the mechanisms responsible for hypoxia-dependent induction of CD133-positive BTSCs in glioblastomas. We confirmed that cultures exposed to lowered oxygen levels showed a severalfold increase of CD133-positive BTSCs. Both the increase of CD133-positive cells and deceleration of the growth kinetics were reversible after transfer to normoxic conditions. Exposure to hypoxia induced BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3)-dependent apoptosis preferentially in CD133-negative cells. In contrast, CD133-positive cells proved to be more resistant to hypoxia-induced programmed cell death. Application of the demethylating agent 5'-azacitidine resulted in an increase of BNIP3 expression levels in CD133-positive cells. Thus, epigenetic modifications led to their better survival in lowered oxygen tension. Moreover, the, hypoxia-induced increase of CD133-positive cells was inhibited after 5'-azacitidine treatment. These results suggest the possible efficacy of a novel therapy for glioblastoma focused on eradication of BTSCs by modifications of epigenetic regulation of gene expression.
    Full-text · Article · Nov 2012 · Journal of Neuropathology and Experimental Neurology
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    ABSTRACT: Susceptibility differences among tissues were recently used for highlighting complementary contrast in MRI different from the conventional T(1) , T(2) , or spin density contrasts. This method, based on the signal phase, previously showed improved image contrast of human or rodent neuroarchitecture in vivo, although direct MR phase imaging of cellular architecture was not available until recently. In this study, we present for the first time the ability of microcoil-based phase MRI to resolve the structure of human glioma neurospheres at significantly improved resolutions (10 × 10 μm(2) ) with direct optical image correlation. The manganese chloride property to function as a T(1) contrast agent enabled a closer examination of cell physiology with MRI. Specifically the temporal changes of manganese chloride uptake, retention and release time within and from individual clusters were assessed. The optimal manganese chloride concentration for improved MR signal enhancement was determined while keeping the cellular viability unaffected. The presented results demonstrate the possibilities to reveal structural and functional observation of living glioblastoma human-derived cells. This was achieved through the combination of highly sensitive microcoils, high magnetic field, and methods designed to maximize contrast to noise ratio. The presented approach may provide a powerful multimodal tool that merges structural and functional information of submilimeter biological samples. Magn Reson Med, 2012. © 2011 Wiley Periodicals, Inc.
    Full-text · Article · Jul 2012 · Magnetic Resonance in Medicine
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    ABSTRACT: Susceptibility differences among tissues were recently used for highlighting complementary contrast in MRI different from the conventional T(1), T(2), or spin density contrasts. This method, based on the signal phase, previously showed improved image contrast of human or rodent neuroarchitecture in vivo, although direct MR phase imaging of cellular architecture was not available until recently. In this study, we present for the first time the ability of microcoil-based phase MRI to resolve the structure of human glioma neurospheres at significantly improved resolutions (10 × 10 μm(2)) with direct optical image correlation. The manganese chloride property to function as a T(1) contrast agent enabled a closer examination of cell physiology with MRI. Specifically the temporal changes of manganese chloride uptake, retention and release time within and from individual clusters were assessed. The optimal manganese chloride concentration for improved MR signal enhancement was determined while keeping the cellular viability unaffected. The presented results demonstrate the possibilities to reveal structural and functional observation of living glioblastoma human-derived cells. This was achieved through the combination of highly sensitive microcoils, high magnetic field, and methods designed to maximize contrast to noise ratio. The presented approach may provide a powerful multimodal tool that merges structural and functional information of submilimeter biological samples.
    No preview · Article · Jul 2012 · Magnetic Resonance in Medicine
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    ABSTRACT: Here we show that activation of the canonical WNT/β-catenin pathway increases the expression of stem cell genes and promotes the migratory and invasive capacity of glioblastoma. Modulation of WNT signaling alters the expression of epithelial-to-mesenchymal transition activators, suggesting a role of this process in the regulation of glioma motility. Using immunohistochemistry in patient-derived glioblastoma samples we showed higher numbers of cells with intranuclear signal for β-catenin in the infiltrating edge of tumor compared to central tumor parenchyma. These findings suggest that canonical WNT/β-catenin pathway is a critical regulator of GBM invasion and may represent a potential therapeutic target.
    Full-text · Article · May 2012 · Cancer letters
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    ABSTRACT: Glioblastoma multiforme (GBM), as many other solid tumours, contains a subpopulation of cells termed cancer stem-like cells responsible for the initiation and propagation of tumour growth. However, a unique immunophenotype/surface antigen composition for the clear identification of brain tumour stem cells (BTSC) has not yet been found. Here we report a novel code of cell surface markers for the identification of different cell subpopulations in neurospheres derived from a GBM with a primitive neuroectodermal tumour (PNET)-like component (GBM-PNET). These subgroups differ in their CD133/CD15 expression pattern and resemble cells with different stem-like genotype and developmental pathway activation levels. Strikingly, clonogenic analysis of cultures differentially expressing the investigated markers enabled the identification of distinct subpopulations of cells endowed with stem cell characteristics. High clonogenicity could be found in CD133-/CD15- and CD133+/CD15+ but not in CD133-/CD15+ cells. Moreover, cell subpopulations with pronounced clonogenic growth were characterized by high expression of stem cell-related genes. Interestingly, these observations were unique for GBM-PNET and differed from ordinary GBM cultures derived from tumours lacking a PNET component. This work elucidates the complex molecular heterogeneity of in vitro propagated glioblastoma-derived cells and potentially contributes to the development of novel diagnostic modalities aiming at the identification of the brain tumour stem-like cell population in a subgroup of GBMs.
    Full-text · Article · Jan 2012

Publication Stats

175 Citations
132.63 Total Impact Points

Institutions

  • 2013-2015
    • Johns Hopkins University
      Baltimore, Maryland, United States
  • 2014
    • Johns Hopkins Medicine
      • Department of Pathology
      Baltimore, Maryland, United States
  • 2011-2013
    • University of Freiburg
      Freiburg, Baden-Württemberg, Germany
  • 2012
    • Universitätsklinikum Freiburg
      Freiburg an der Elbe, Lower Saxony, Germany