Metastasis Research Protocols PDF
Preview Metastasis Research Protocols
M E T H O D S I N M O L E C U L A R B I O L O G Y ™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfi e ld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 fdaioj Metastasis Research Protocols Second Edition Edited by Miriam Dwek Molecular and Applied Biosciences, School of Life Sciences, University of Westminster, London, UK Susan A. Brooks Department of Biological & Medical Sciences, Oxford Brookes University, Oxford, Headington, UK Udo Schumacher Institute Anatomie und Experimentelle Morphologie, Universitätsklinikum, Hamburg-Eppendorf, Germany Editors Susan A. Brooks Miriam Dwek Department of Biological & Medical Sciences Molecular and Applied Biosciences Oxford Brookes University School of Life Sciences Oxford, Headington, UK University of Westminster London, UK Udo Schumacher Institute Anatomie und Experimentelle Morphologie Universitätsklinikum Hamburg-Eppendorf Germany ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-61779-853-5 ISBN 978-1-61779-854-2 (eBook) DOI 10.1007/978-1-61779-854-2 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2012936663 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com) P reface D iverse molecular, cellular, and environmental events must all come together to allow the s uccessful formation of secondary cancers, metastases. The vast amount of knowledge that has been amassed in relation to the biology underlying cancer formation is being applied to enable a better understanding of the metastatic process. It is well accepted that elucidation of the key events in this process will lead to the next generation of laboratory tests for early-diagnosis of metastases and for the treatment of occult as well as more clinically advanced disease. T his second edition of M etastasis Research Protocols brings together updated versions of the seminal techniques that were presented in the fi r st edition and also includes new tech- niques that have recently been shown to be important in illuminating the processes under- lying this important area of biology. T he fi r st volume: A nalysis of Cells and Tissues takes the reader through key cellular and molecular techniques relevant to the exploration of cancer cells and tissues, the focus is on the tools that have been shown to be helpful in unravelling the molecular processes impor- tant in cancer metastasis. The second volume: M odels of Metastasis is concerned with the interaction between cancer cells and host/environment. This volume focuses on the diverse range of in vitro and in vivo models of metastasis. I n keeping with the fir st edition, volume one includes techniques such as immuno- chemistry (Chaps. 1 – 4 ) , PCR (Chap. 5 ) , and SDS-PAGE (Chap. 6 ) , the mainstay of many laboratories. As before, we make aim for each of the techniques to stand-alone and for these to be valuable to the newcomer as well as the experienced researcher. Volume one has been extended to include newer techniques, for example, affi n ity measurement of biomolecular interactions (Chap. 1 1) , methylation analysis of microRNA (Chap. 1 5) , and RNAi t echnology (Chap. 1 6) . Similarly, volume two retains chapters that describe in detail the methods often used to assess cancer cell adhesion to extracellular matrix components and endothelial cells (Chaps. 1 – 5 ) , syngeneic and xenograft models of metastasis (Chaps. 9 , 1 0, 1 6, and 1 7) . In addition, updated methods are presented, for example, for the production of in vivo double-knock out models (Chap. 1 4) and the application of flu orescent imaging techniques for monitoring the development of metastases in vivo (Chaps. 1 1 and 1 2 ) . W e have sought to keep the important key methods in both volumes and to introduce new methods which are making an impact in the area of metastasis research. One of the much-loved aspects of the Methods in Molecular Biology series is that it aims to impart knowledge of complex methodology to the end-user in an accessible manner. The “Notes” section found at the end of each chapter serves to demystify the techniques in a handy “hints and tips” format, this enables researchers who may be hesitant to adopt a new pro- cedure to try it out, thereby adding to their repertoire of laboratory techniques. We have tried to maintain this key element in the chapters presented in these two volumes and we hope that you fi n d this to be a continued useful aspect of this series. F inally, we would like to thank all our contributors who have worked tirelessly to master their techniques, for sharing these with us and you, the reader. We hope you fi n d that in these two volumes, methods that will assist in helping you to make new observations that may in turn lead to the development of new treatments aimed at combating cancer metastasis. v fdaioj Acknowledgements Miriam Dwek acknowledges the invaluable contribution of HRMD, thank you for all your input. vii fdaioj C ontents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Basic Immunocytochemistry for Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . 1 Susan A. Brooks 2 Lectin Histochemistry to Detect Altered Glycosylation in Cells and Tissues. . . . . . . 31 Susan A. Brooks and Debbie M.S. Hall 3 Histopathological Assessment of Metastasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Derek E. Roskell and Ian D. Buley 4 Immunocytochemical Detection and Characterisation of Individual Micrometastatic Tumour Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Stephan Braun and Klaus Pantel 5 The Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Hazel M. Welch 6 Sodium Dodecyl Sulphate–Polyacrylamide Denaturing Gel Electrophoresis and Western Blotting Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Christine Blancher and Rob Mc Cormick 7 2-DE-Based Proteomics for the Analysis of Metastasis-Associated Proteins . . . . . . . 111 Miriam Dwek and Diluka Peiris 8 Assessment of Gelatinases (MMP-2 and MMP-9) by Gelatin Zymography . . . . . . . 121 Marta Toth, Anjum Sohail, and Rafael Fridman 9 Determination of Cell-Specifi c Receptor Binding using a Combination of Immunohistochemistry and in vitro Autoradiography: Relevance to Therapeutic Receptor Targeting in Cancer . . . . . . . . . . . . . . . . . . . . . 137 Michael R. Dashwood and Marilena Loizidou 10 Fluorescence In Situ Hybridization for Cancer-Related Studies . . . . . . . . . . . . . . . . 149 Lyndal Kearney and Janet Shipley 11 Using a Quartz Crystal Microbalance Biosensor for the Study of Metastasis Markers on Intact Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Julien Saint-Guirons and Björn Ingemarsson 12 Cell Separations by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Derek Davies 13 Detection of Putative Cancer Stem Cells of the Side Population Phenotype in Human Tumor Cell Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Matthias Christgen, Matthias Ballmaier, Ulrich Lehmann, and Hans Kreipe 14 Genes Involved in the Metastatic Cascade of Medullary Thyroid Tumours . . . . . . . 217 Caroline Schreiber, Kirsten Vormbrock and Ulrike Ziebold 15 High-resolution Quantitative Methylation Analysis of microRNA Genes using Pyrosequencing™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Ulrich Lehmann, Cord Albat, and Hans Kreipe ix
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