Advances in Experimental Medicine and Biology Volume 899 Editorial Board: IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA RODOLFO PAOLETTI, University of Milan, Milan, Italy More information about this series at http://www.springer.com/series/5584
Constantinos Koumenis Lisa M. Coussens Amato Giaccia Ester Hammond Editors Tumor Microenvironment Study Protocols
Editors Constantinos Koumenis Perelman School of Medicine University of Pennsylvania Philadelphia, PA, USA Amato Giaccia Department of Radiation Oncology Stanford University School of Medicine Stanford, CA, USA Lisa M. Coussens Cell & Developmental Biology Oregon Health & Science University Portland, OR, USA Ester Hammond Department of Oncology University of Oxford Oxford, Oxfordshire, UK ISSN 0065-2598 ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-3-319-26664-0 ISBN 978-3-319-26666-4 (ebook) DOI 10.1007/978-3-319-26666-4 Library of Congress Control Number: 2015959711 Springer International Publishing Switzerland 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Springer imprint is published by Springer Nature The registered company is Springer International Publishing AG Switzerland
Preface It is universally accepted that the tumor microenvironment is extremely relevant to both the study of cancer biology and the search for improved therapies. However, it is also accepted that the study of biological mechanism in conditions which accurately mimic this environment is both technically challenging and highly specialized. At a recent tumor microenvironment meeting held in Mykonos, Greece, we decided that it would aid the field in general to publish detailed protocols, far exceeding the level of detail usually reported in papers. We hope that these prove useful and that we as a community can continue to share our collective expertise. Philadelphia, PA, USA Portland, OR, USA Stanford, CA, USA Oxford, UK Constantinos Koumenis Lisa M. Coussens Amato Giaccia Ester Hammond v
Contents 1 Staining Against Phospho-H2AX (γ-h2ax) as a Marker for DNA Damage and Genomic Instability in Cancer Tissues and Cells... 1 Anika Nagelkerke and Paul N. Span 2 Measuring DNA Replication in Hypoxic Conditions... 11 Iosifina P. Foskolou, Deborah Biasoli, Monica M. Olcina, and Ester M. Hammond 3 Isolation of Proteins on Nascent DNA in Hypoxia and Reoxygenation Conditions... 27 Monica M. Olcina, Amato J. Giaccia, and Ester M. Hammond 4 In Vivo Interrogation of the Hypoxic Transcriptome of Solid Tumors: Optimizing Hypoxic Probe Labeling with Laser Capture Microdissection for Isolation of High-Quality RNA for Deep Sequencing Analysis... 41 Lauren K. Brady, Vladimir Popov, and Constantinos Koumenis 5 Gas Chromatography Coupled to Mass Spectrometry (GC MS) to Study Metabolism in Cultured Cells... 59 Fiona Grimm, Louise Fets, and Dimitrios Anastasiou 6 Probing Cancer Cell Metabolism Using NMR Spectroscopy... 89 Kate E.R. Hollinshead, Debbie S. Williams, Daniel A. Tennant, and Christian Ludwig 7 Measuring the Impact of Microenvironmental Conditions on Mitochondrial Dehydrogenase Activity in Cultured Cells... 113 Ramon C. Sun, Albert Koong, Amato Giaccia, and Nicholas C. Denko 8 Measuring Autophagy in the Context of Cancer... 121 Michael J. Morgan and Andrew Thorburn vii
viii Contents 9 Methods for Studying Autophagy Within the Tumor Microenvironment... 145 Sengottuvelan Murugan and Ravi K. Amaravadi 10 Methods: Using Three-Dimensional Culture (Spheroids) as an In Vitro Model of Tumour Hypoxia... 167 Russell Leek, David Robert Grimes, Adrian L. Harris, and Alan McIntyre 11 Isolation of Glioma-Initiating Cells for Biological Study... 197 Jing Hu, Geoffrey Markowitz, and Xiao-Fan Wang 12 Isolation of Mouse and Human Tumor-Associated Macrophages... 211 Luca Cassetta, Roy Noy, Agnieszka Swierczak, Gaël Sugano, Harriet Smith, Lisa Wiechmann, and Jeffrey W. Pollard 13 Mouse Models of Brain Metastasis for Unravelling Tumour Progression... 231 Manuel Sarmiento Soto and Nicola R. Sibson 14 Quantification of Lung Metastases from In Vivo Mouse Models... 245 Joan Chang and Janine T. Erler 15 A Multimodal Data Analysis Approach for Targeted Drug Discovery Involving Topological Data Analysis (TDA)... 253 Muthuraman Alagappan, Dadi Jiang, Nicholas Denko, and Albert C. Koong 16 Efficient Protocol for the Identification of Hypoxic Cell Radiosensitisers... 269 Cho Rong Hong, Jingli Wang, Kevin O. Hicks, and Michael P. Hay Index... 291