German Cancer Research Center (DKFZ) View Institution's Website 14 articles published in JoVE Immunology and Infection Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions Megan L. Stanifer1,2, Steeve Boulant2,3,4 1Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, 2Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, 3Department of Infectious Diseases, Virology, Heidelberg University Hospital, 4Research Group “Cellular Polarity and Viral Infection”, German Cancer Research Center (DKFZ) This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology. Cancer Research Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq Ashwin Narayanan1, Enrique Blanco-Carmona2, Engin Demirdizen1, Xueyuan Sun1,3, Christel Herold-Mende4, Matthias Schlesner2, Sevin Turcan1 1Neurology Clinic and National Center for Tumor Diseases, University Hospital Heidelberg, 2Bioinformatics and Omics Data Analytics, German Cancer Research Center (DKFZ), 3Clinical Cooperation Unit Neurooncology, German Cancer Research Center (DKFZ), 4Division of Experimental Neurosurgery, Department of Neurosurgery, University of Heidelberg, INF 400, Heidelberg, Germany Intra-tumoral heterogeneity is an inherent feature of tumors, including gliomas. We developed a simple and efficient protocol that utilizes a combination of buffers and gradient centrifugation to isolate single nuclei from fresh frozen glioma tissues for single nucleus RNA and ATAC sequencing studies. Cancer Research Machine Learning Algorithms for Early Detection of Bone Metastases in an Experimental Rat Model Stephan Ellmann1, Lisa Seyler1, Clarissa Gillmann2, Vanessa Popp1, Christoph Treutlein1, Aline Bozec3, Michael Uder1, Tobias Bäuerle1 1Department of Radiology, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg, 2Department of Medical Physics in Radiation Oncology, German Cancer Research Center, 3Department of Internal Medicine 3, University Hospital Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg This protocol was designed to train a machine learning algorithm to use a combination of imaging parameters derived from magnetic resonance imaging (MRI) and positron emission tomography/computed tomography (PET/CT) in a rat model of breast cancer bone metastases to detect early metastatic disease and predict subsequent progression to macrometastases. Cancer Research Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry Ramon Lopez Perez1,2, Franziska Münz1,2, Jonas Kroschke1,2, Jannek Brauer1,2, Nils H. Nicolay1,2,3, Peter E. Huber1,2 1CCU Molecular and Radiation Oncology, German Cancer Research Center, 2Department of Radiation Oncology, Heidelberg University Hospital, 3Department of Radiation Oncology, Freiburg University Medical Center The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure. Developmental Biology CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer Weijun Feng*1, Lena Herbst*2, Peter Lichter2, Stefan M. Pfister3,4,5, Hai-Kun Liu1, Daisuke Kawauchi3,4 1Division of Molecular Neurogenetics, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, 2Division of Molecular Genetics, German Cancer Research Center (DKFZ), and German Cancer Consortium (DKTK), 3 Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum. Cancer Research Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer Susan Kochall1, May-Linn Thepkaysone1, Sebastián A. García1, Alexander M. Betzler1, Jürgen Weitz1,2,3, Christoph Reissfelder1, Sebastian Schölch1,2,3 1Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 2German Cancer Consortium (DKTK), 3German Cancer Research Center (DKFZ) We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model. Cancer Research A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer Alexander M. Betzler1, Susan Kochall1, Linda Blickensdörfer2, Sebastian A. Garcia1, May-Linn Thepkaysone1, Lahiri K. Nanduri1, Michael H. Muders3, Jürgen Weitz1,4,5, Christoph Reissfelder1, Sebastian Schölch1,4,5 1Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 2Department of General, Gastrointestinal and Transplant Surgery, University of Heidelberg, 3Department of Pathology, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 4German Cancer Consortium (DKTK), 5German Cancer Research Center (DKFZ) A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented. Medicine Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery Christoph Emontzpohl1,2, David Simons3, Sandra Kraemer4, Andreas Goetzenich4, Gernot Marx1, Jürgen Bernhagen5,6, Christian Stoppe1 1Department of Intensive Care Medicine, University Hospital Aachen, 2Institute of Biochemistry and Molecular Biology, University Hospital Aachen, 3Department of Radiology, German Cancer Research Center, 4Department of Thoracic and Cardiovascular Surgery, University Hospital Aachen, 5Department of Vascular Biology, Institute for Stroke and Dementia Research (ISD), Klinikum der Universität München, 6Deutsches Zentrum für Herz-/Kreislaufkrankheiten (DZHK), Munich Heart Alliance Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients. Developmental Biology 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression Sheena Pinto*1, Hans-Jürgen Stark*2, Iris Martin2, Petra Boukamp2, Bruno Kyewski1 1Division of Developmental Immunology, German Cancer Research Center (DKFZ), 2Genetics of Skin Carcinogenesis, German Cancer Research Center (DKFZ) Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression. Bioengineering Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers Joseph D. Unsay1,2,3, Katia Cosentino1,2, Ana J. García-Sáez1,2 1Interfaculty Institute for Biochemistry, 2Max Planck Institute for Intelligent Systems, 3German Cancer Research Center We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy. Medicine Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors Hanibal Bohnenberger1, Philipp Ströbel1, Sebastian Mohr2, Jasmin Corso3, Tobias Berg2, Henning Urlaub3,4, Christof Lenz3,4, Hubert Serve2,5,6, Thomas Oellerich2,5,6 1Institute of Pathology, University Medical Center, Göttingen, 2Department of Hematology/Oncology, Goethe University of Frankfurt, 3Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, 4Bioanalytics, Institute of Clinical Chemistry, University Medical Center, Göttingen, 5German Cancer Consortium, 6German Cancer Research Center In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry. Neuroscience In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation Wilson Pak-Kin Lou1, Avni Baser1, Stefan Klußmann1, Ana Martin-Villalba1 1Department of Molecular Neurobiology, German Cancer Research Center (DKFZ) This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions. Medicine Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound Tobias Bäuerle1, Dorde Komljenovic1, Martin R. Berger2, Wolfhard Semmler1 1Department of Medical Physics in Radiology, German Cancer Research Center, Heidelberg, Germany, 2Unit of Chemotherapy and Toxicology, German Cancer Research Center, Heidelberg, Germany In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound. Immunology and Infection Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems Nazim El-Andaloussi*1,2, Barbara Leuchs*1, Serena Bonifati1,2, Jean Rommelaere1,2, Antonio Marchini1,2 1Tumour Virology Division F010, German Cancer Research Center (DKFZ), 2Inserm Unit 701, German Cancer Research Center (DKFZ) Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).
