Methods in Molecular Biology 1501 Finian Martin Torsten Stein Jillian Howlin Editors Mammary Gland Development Methods and Protocols M ETHODS IN MOLECULAR BIOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB , UK For further volumes: http://www.springer.com/series/7651 Mammary Gland Development Methods and Protocols Edited by Finian Martin School of Biomolecular and Biomedical Science, University College Dublin, Belfield, Dublin, Ireland Torsten Stein Institute of Cancer Sciences, College of MVLS, University of Glasgow, Glasgow, UK Jillian Howlin Division of Oncology-Pathology, Lund University Cancer Center/Medicon Village, Lund, Sweden Editors Finian Martin Torsten Stein School of Biomolecular and Biomedical Science Institute of Cancer Sciences University College Dublin College of MVLS Belfield , Dublin , Ireland University of Glasgow Glasgow , UK Jillian Howlin Division of Oncology-Pathology Lund University Cancer Center/Medicon Village Lund , Sweden ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6473-4 ISBN 978-1-4939-6475-8 (eBook) DOI 10.1007/978-1-4939-6475-8 Library of Congress Control Number: 2016945163 © Springer Science+Business Media New York 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms 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 specifi c 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. Cover Illustration: Growing Mammary Ducts of a Pubertal Mouse - The image depicts the growing front of a pubertal mouse mammary ductal epithelium with its bulbous terminal end buds surrounded by the mammary fat pad. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Pref ace It is timely and exciting to compile some of the key molecular biological protocols and experimental strategies currently employed to study the various stages of mammary gland development. To this end we have invited leaders in the fi eld to contribute detailed descrip- tions of their methodologies. We begin with a comprehensive overview of mouse mammary gland development drawing particular attention to comparative human biology and then present our selection of core methodologies in four parts. Owing to the importance of transgenic and knock-out mouse models to the fi eld, we begin Part I with two reviews of genetically modifi ed mouse models that exhibit prenatal and pubertal mammary gland phenotypes. We discuss these phenotypes in the context of embryonic and postnatal gland development while emphasizing the study of the terminal end bud and the process of branching morphogenesis. We then present detailed descrip- tions of transplantation techniques, isolation and transcriptome analysis of the mammary terminal end bud during ductal morphogenesis, as well as transcriptome analysis of mam- mary fi broblasts isolated from sections taken at puberty. We fi nally describe how the post- lactational involuting gland can be used as a model to study epithelial cell death. In Part II we present a selection of 2D and 3D-model culture systems that have been employed to investigate a variety of mammary epithelial cell behaviors in vitro. We begin with a contrac- tile assay for the study of myoepithelial cells and then present methods for in vitro recapitu- lation of mammary epithelial cell organization in the now infamous 3D acinus formation assay. In particular, we focus on the intrinsic molecular requirements for acini formation such as the role of MAP kinases and transient EMT, but also that of the microenvironment, examining the role of other cell types and how mechanical forces affect aggregated epithe- lial cells. Part III deals with stem cells and the mammary gland and we present methodolo- gies for mammary stem cell isolation, reprogramming of progenitor populations, and a description of some strategies and methods for cell lineage tracing. Lastly, Part IV high- lights some translational applications that provide a bridge between experimental studies of mammary gland development and the study of human breast cancer, with tissue microar- rays for biomarker discovery and the increasingly popular practice of generating patient- derived xenographs for the study of cancer progression. Our hope is that this volume will have a wide readership: researchers whose primary inter- est is in mammary gland development; developmental biologists interested in related inter- nally branched epithelial organs, for instance the lung, kidney, and salivary gland; epithelial cell biologists and those with an interest in molecular mechanisms underlying breast cancer. Belfield, Dublin, Ireland Finian Martin Glasgow, UK Torsten Stein Lund, Sweden Jillian Howlin v Contents Preface. v Contributors . ix 1 Overview of Mammary Gland Development: A Comparison of Mouse and Human . 1 Sara McNally and Torsten Stein PART I IN VIVO MODELS 2 Prenatal Mammary Gland Development in the Mouse: Research Models and Techniques for Its Study from Past to Present. 21 Jacqueline M. Veltmaat 3 Pubertal Mammary Gland Development: Elucidation of In Vivo Morphogenesis Using Murine Models . 77 Jean McBryan and Jillian Howlin 4 Analysis of Mammary Gland Phenotypes by Transplantation of the Genetically Marked Mammary Epithelium. 115 Duje Buric and Cathrin Brisken 5 Pubertal Ductal Morphogenesis: Isolation and Transcriptome Analysis of the Terminal End Bud . 131 Joanna S. Morris and Torsten Stein 6 RNA Profiling of Non-cultured Fibroblasts Isolated from Pubertal Mouse Mammary Gland Sections . 149 Ayman M. Ibrahim , Claire Cairney , Joanna S. Morris , and Torsten Stein 7 Analysis of the Involuting Mouse Mammary Gland: An In Vivo Model for Cell Death. 165 Bethan Lloyd-Lewis , Timothy J. Sargeant , Peter A. Kreuzaler , Henrike K. Resemann , Sara Pensa , and Christine J. Watson PART II IN VITRO 2D & 3D-MODELS 8 Contractility Assay for Established Myoepithelial Cell Lines. 189 Stéphanie Cagnet , Marina A. Glukhova , and Karine Raymond 9 Using 3D Culture of Primary Mammary Epithelial Cells to Define Molecular Entities Required for Acinus Formation: Analyzing MAP Kinase Phosphatases. 199 Malgorzata Gajewska and Sara McNally 10 A 3D Fibroblast-Epithelium Co-culture Model for Understanding Microenvironmental Role in Branching Morphogenesis of the Mammary Gland. 217 Zuzana Koledova and Pengfei Lu vii viii Contents 11 Next Generation RNA Sequencing Analysis Reveals Expression of a Transient EMT Profile During Early Organization of MCF10A Cells in 3D . 233 Benedikt Minke , Drieke Vandamme , Thomas Schwarzl , Walter Kolch , and Finian Martin 12 A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells . 245 Alexandra S. Piotrowski-Daspit , Allison K. Simi , Mei-Fong Pang , Joe Tien , and Celeste M. Nelson PART III MAMMARY STEM CELLS 13 Purification of Distinct Subsets of Epithelial Cells from Normal Human Breast Tissue . 261 Mona Shehata and John Stingl 14 Techniques for the Reprogramming of Exogenous Stem/Progenitor Cell Populations Towards a Mammary Epithelial Cell Fate . 277 Gilbert H. Smith and Corinne A. Boulanger 15 Lineage Tracing of Mammary Stem and Progenitor Cells . 291 Anoeska A. A. van de Moosdijk , Nai Yang Fu , Anne C. Rios , Jane E. Visvader , and Renée van Amerongen PART IV TRANSLATIONAL APPLICATIONS 16 Assessment of Significance of Novel Proteins in Breast Cancer Using Tissue Microarray Technology . 311 Laoighse Mulrane , William M. Gallagher , and Darran P. O’Connor 17 Patient-Derived Xenografts of Breast Cancer . 327 Damir Varešlija , Sinead Cocchiglia , Christopher Byrne , and Leonie Young Conclusion . 337 Index . 347 Contributors RENÉE VAN AMERONGEN • Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences , University of Amsterdam , Amsterdam , The Netherlands CORINNE A. BOULANGER • Mammary Stem Cell Section, Basic Research Laboratory, National Cancer Institute , National Institutes of Health , Bethesda , MD , USA CATHRIN BRISKEN • The Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology (EPFL) , Lausanne , Switzerland DUJE BURIC • The Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology (EPFL) , Lausanne , Switzerland CHRISTOPHER BYRNE • Endocrine Oncology Research Group, Department of Surgery , Royal College of Surgeons in Ireland , Dublin , Ireland STÉPHANIE CAGNET • Institut Curie, Centre de Recherche, Paris, France; CNRS, UMR144, Paris,