Table Of ContentMethods in
Molecular Biology 1070
Miriam Dwek
Udo Schumacher
Susan A. Brooks Editors
Metastasis
Research
Protocols
Second Edition
M E T H O D S I N M O L E C U L A R B I O LO G Y ™
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfi e ld, Hertfordshire, AL10 9AB, UK
F or further volumes:
h ttp://www.springer.com/series/7651
M etastasis Research Protocols
Second Edition
E dited by
M iriam D wek
Molecular and Applied Biosciences, School of Life Sciences, University of Westminster, London, UK
U do S chumacher
Institute Anatomie und Experimentelle Morphologie, Universitätsklinikum Hamburg-Eppendorf,
Hamburg, Germany
S usan A. B rooks
Department of Biological and Medical Sciences, Oxford Brookes University, Headington, Oxford, UK
E ditors
M iriam D wek U do S chumacher
M olecular and Applied Biosciences I nstitute Anatomie und Experimentelle
School of Life Sciences Morphologie
U niversity of Westminster U niversitätsklinikum Hamburg-Eppendorf
L ondon, U K H amburg, G ermany
S usan A. B rooks
D epartment of Biological and Medical Sciences
O xford Brookes University
H eadington, Oxford, U K
I SSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4614-8243-7 ISBN 978-1-4614-8244-4 (eBook)
D OI 10.1007/978-1-4614-8244-4
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P refa ce
D iverse molecular, cellular, and environmental events must all come together to allow the suc-
cessful 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 the 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 rst 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 rst volume of M etastasis Research Protocols, Analysis of Cells and Tissues, takes the
reader through key cellular and molecular techniques relevant to exploration of cancer cells
and tissues. In this second volume, M odels of Metastasis, we move to the level of living cells and
tissues, and present methodologies applicable to behaviour in vitro, in animal models and in
mathematical constructs. It is concerned with the interaction between cancer cells and their
host/environment. The focus throughout is on the tools that have been shown to be help-
ful in unravelling the processes important in cancer metastasis.
I n comparison to the fi rst edition of M etastasis Research Protocols, here we have retained
important key methods in both volumes and introduced new and cutting-edge methods
which are making an impact in the arena of metastasis research. Consistent with the fi rst
edition, volume 1 includes standard techniques including immunochemistry (Chapters 1,
2, 3, and 4), PCR (Chapter 5), and SDS-PAGE (Chapter 6), the mainstay of many labora-
tories, and these have been revised and updated. Volume 1 has also been extended to incor-
porate newer techniques, for example, affi nity measurement of biomolecular interactions
(Chapter 11), methylation analysis of microRNA (Chapter 15), and RNAi technology
(Chapter 16). This volume, volume 2, similarly retains chapters from the fi rst edition,
where they represent methods commonly and usefully employed in metastasis research, and
these have been fully revised and updated. They include, for example, methods to assess
cancer cell adhesion to extracellular matrix components and endothelial cells (Chapters 1 ,
2 , 3 , 4 , and 5 ) , and syngeneic and xenograft animal models of metastasis (Chapters 9 , 1 0,
1 6 and 1 7) . In addition, new methods are presented, for example, for the production of in
vivo double knockout models (Chapter 1 4) and the application of fl uorescent imaging
techniques for monitoring the development of metastases in vivo (Chapters 1 1 and 1 2) .
Each chapter stands alone and we aim for there to be enough detail for them to be useful
to the newcomer and the experienced researcher alike.
O ne of the much-valued aspects of the M ethods 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
v
vi Preface
handy “hints and tips” format, which enables researchers who may be hesitant to adopt a
new procedure 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 nd this to be a continued useful aspect of the series.
F inally, we would like to thank all of our contributors who have worked tirelessly to
master their techniques, for sharing these with us and you, the reader. We hope that you
fi nd in these two volumes methods that will assist in helping you to make new observations
that will enhance our knowledge and understanding of the complexities of metastasis and
may, in turn, lead to developments in treatments aimed at combating cancer metastasis.
L ondon, U K M iriam D wek
H amburg, G ermany U do S chumacher
O xford , U K S usan A . B rooks
C ontents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 In Vitro Invasion Assay Using Matrigel™:
A Reconstituted Basement Membrane Preparation . . . . . . . . . . . . . . . . . . . . . 1
Debbie M.S. Hall and Susan A. Brooks
2 Single Cell and Spheroid Collagen Type I Invasion Assay . . . . . . . . . . . . . . . . 13
Olivier De Wever, An Hendrix, Astrid De Boeck, Frank Eertmans,
Wendy Westbroek, Geert Braems, and Marc E. Bracke
3 Rocking Adhesion Assay System to Study Adhesion
and Transendothelial Migration of Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . 37
Deepashree Bapu, Munira Khadim, and Susan A. Brooks
4 Small-Cell Lung Cancer (SCLC) Cell Adhesion on E- and P-Selectin
Under Physiological Flow Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Ulrich Richter
5 Adhesion of Tumor Cells to Matrices and Endothelium. . . . . . . . . . . . . . . . . . 57
Clara M. Yates, Helen M. McGettrick, Gerard B. Nash, and G.Ed Rainger
6 Cell Aggregation Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Delphine Debruyne, Tom Boterberg, and Marc E. Bracke
7 Chick Heart Invasion Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Marc E. Bracke, Virinder S. Parmar, Anthony L. DePass,
Christian V. Stevens, Barbara W. Vanhoecke, and Marc M. Mareel
8 Computer Simulation of the Metastatic Progression . . . . . . . . . . . . . . . . . . . . 107
Gero Wedemann, Anja Bethge, Volker Haustein, and Udo Schumacher
9 Theoretical Considerations in Using Animal Models of Metastasis
and Brief Methodology for In Vivo Colorectal Cancer Models
in SCID and Nude Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Sue A. Watson and Rajendra Kumari
10 Syngeneic Murine Metastasis Models: B16 Melanoma . . . . . . . . . . . . . . . . . . . 131
Raffaella Giavazzi and Alessandra Decio
11 Imageable Clinically Relevant Mouse Models of Metastasis . . . . . . . . . . . . . . . 141
Robert M. Hoffman
12 Imaging Metastatic Cell Trafficking at the Cellular Level
In Vivo with Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Robert M. Hoffman
vii
viii Contents
13 Ultrasound Techniques for the Detection of Tumors and Metastases
in Small Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Michael Didié and Wolfram-Hubertus Zimmermann
14 The PFP/RAG2 Double-Knockout Mouse in Metastasis
Research: Small-Cell Lung Cancer and Prostate Cancer. . . . . . . . . . . . . . . . . . 191
Imke Müller and Sebastian Ullrich
15 Ultrasound-Guided Intracardial Injection and In Vivo Magnetic
Resonance Imaging of Single Cells in Mice as a Paradigm
for Hematogenous Metastases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Johannes Salamon and Kersten Peldschus
16 Magnetic Resonance Imaging of Metastases in Xenograft
Mouse Models of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Kersten Peldschus and Harald Ittrich
17 Spontaneous and Experimental Metastasis Models: Nude Mice . . . . . . . . . . . . 223
Janet E. Price
18 Identifying the Origin and Phenotype of Cells in Tumor Xenografts . . . . . . . . 235
Rosemary Jeffery, Pooja Seedhar, and Richard Poulsom
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
C ontributors
G ERHARD A DAM • Department of Diagnostic and Interventional Radiology, U niversity
Medical Center Hamburg-Eppendorf , H amburg, G ermany
D EEPASHREE B APU • Department of Biological and Medical Sciences, Oxford Brookes
University, H eadington, Oxford, U K
A NJA B ETHGE • University of Applied Sciences Stralsund, S tralsund, G ermany
A STRID D E B OECK • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research , G hent University Hospital, G hent, B elgium
T OM B OTERBERG • Laboratory of Experimental Cancer Research, Department of
Radiotherapy and Nuclear Medicine, G hent University Hospital, G hent, B elgium
M ARC E . BRACKE • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research , G hent University Hospital, G hent, B elgium
G EERT B RAEMS • Department of Gynaecology , G hent University Hospital, G hent, B elgium
S USAN A . B ROOKS • Department of Biological and Medical Sciences, Oxford Brookes University,
H eadington, Oxford, U K
D ELPHINE D EBRUYNE • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research , G hent University Hospital, G hent, Belgium
A LESSANDRA D ECIO • Department of Oncology, Laboratory of Biology and Therapy
of Metastasis, M ario Negri Institute for Pharmacological Research , B ergamo, I taly
A NTHONY L . D EPASS • Department of Biology, L ong Island University-Brooklyn,
B rooklyn , N Y, U SA
M ICHAEL D IDIÉ • Department of Pharmacology , G eorg-August-University ,
G oettingen, G ermany
F RANK E ERTMANS • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research , Ghent University Hospital, G hent, B elgium
R AFFAELLA G IAVAZZI • Department of Oncology, Laboratory of Biology and Therapy
of Metastasis, M ario Negri Institute for Pharmacological Research , B ergamo, I taly
D EBBIE M .S . H ALL • Department of Biological and Medical Sciences, Oxford Brookes
University, H eadington, Oxford, UK
V OLKER H AUSTEIN • System Engineering and Information Management, I nstitute for
Applied Computer Science, University of Applied Sciences Stralsund, S tralsund, G ermany
A N H ENDRIX • Department of Medical Oncology, Ghent University Hospital, G hent, B elgium
R OBERT M . H OFFMAN • AntiCancer, Inc. , San Diego, C A, U SA
H ARALD I TTRICH • Department of Diagnostic and Interventional Radiology, U niversity
Medical Center Hamburg-Eppendorf , Hamburg, G ermany
R OSEMARY J EFFERY • Molecular Pathology Facility , N ational Centre for Bowel Research
and Surgical Innovation, Centre for Digestive Diseases, Blizard Institute Barts
and The London School of Medicine and Dentistry, Queen Mary University of London,
L ondon, U K
M UNIRA K HADIM • Department of Biological and Medical Sciences, Oxford Brookes University,
H eadington, Oxford, UK
ix
