Inflammation and Cancer Methods and Protocols Methods in Molecular Biology 2691 2nd Edition Brendan J. Jenkins (Editor)

Inflammation and Cancer Methods and
Protocols Methods in Molecular

Biology 2691 2nd Edition Brendan J.
Jenkins (Editor)
Visit to download the full and correct content document:
https://ebookmeta.com/product/inflammation-and-cancer-methods-and-protocols-met
hods-in-molecular-biology-2691-2nd-edition-brendan-j-jenkins-editor/
More products digital (pdf, epub, mobi) instant
download maybe you interests ...
Cancer Cell Biology Methods and Protocols Methods in

Molecular Biology 2508 Sherri L. Christian (Editor)
https://ebookmeta.com/product/cancer-cell-biology-methods-and-
protocols-methods-in-molecular-biology-2508-sherri-l-christian-
editor/
Cancer Cell Culture Methods and Protocols Methods in

Molecular Biology 2645 Dania Movia (Editor)
https://ebookmeta.com/product/cancer-cell-culture-methods-and-
protocols-methods-in-molecular-biology-2645-dania-movia-editor/
Cancer Drug Resistance Methods and Protocols Methods in

Molecular Biology 2535 Marta Baiocchi (Editor)
https://ebookmeta.com/product/cancer-drug-resistance-methods-and-
protocols-methods-in-molecular-biology-2535-marta-baiocchi-
editor/
Ovarian Cancer Methods and Protocols Methods in

Molecular Biology 2424 Pamela K. Kreeger (Editor)
https://ebookmeta.com/product/ovarian-cancer-methods-and-
protocols-methods-in-molecular-biology-2424-pamela-k-kreeger-
editor/
Mouse Models of Cancer Methods and Protocols Methods in
Molecular Biology 2773 1st Edition Maja ■emažar
https://ebookmeta.com/product/mouse-models-of-cancer-methods-and-
protocols-methods-in-molecular-biology-2773-1st-edition-maja-
cemazar/
Whole-Body Regeneration: Methods and Protocols (Methods

in Molecular Biology, 2450) Blanchoud
https://ebookmeta.com/product/whole-body-regeneration-methods-
and-protocols-methods-in-molecular-biology-2450-blanchoud/
Circadian Regulation Methods and Protocols Methods in

Molecular Biology 2482 Guiomar Solanas
https://ebookmeta.com/product/circadian-regulation-methods-and-
protocols-methods-in-molecular-biology-2482-guiomar-solanas/
Rhodopsin Methods and Protocols Methods in Molecular

Biology 2501 Valentin Gordeliy (Editor)
https://ebookmeta.com/product/rhodopsin-methods-and-protocols-
methods-in-molecular-biology-2501-valentin-gordeliy-editor/
Ferroptosis Methods and Protocols Methods in Molecular

Biology 2712 Guido Kroemer (Editor)
https://ebookmeta.com/product/ferroptosis-methods-and-protocols-
methods-in-molecular-biology-2712-guido-kroemer-editor/
Methods in
Molecular Biology 2691
Brendan J. Jenkins Editor
Inflammation
and Cancer
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Inflammation and Cancer
Methods and Protocols
Second Edition
Edited by
Brendan J. Jenkins
Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research,
Clayton, VIC, Australia
Editor
Brendan J. Jenkins
Centre for Innate Immunity
and Infectious Diseases
Hudson Institute of Medical Research
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-3330-4 ISBN 978-1-0716-3331-1 (eBook)
https://doi.org/10.1007/978-1-0716-3331-1
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2018, 2023
This work is subject to copyright. All rights are solely and exclusively licensed 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,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
Dysregulated activation of both the innate and adaptive arms of the host immune system
contributes to the pathogenesis of a wide range of chronic diseases, in particular autoim-
mune and inflammatory disorders, infectious diseases, and cancer. With respect to cancer, at
least one third of all cancers are associated with chronic inflammatory responses, with prime
examples being cancers of the lung, stomach, pancreas, and colon, among a range of others.
Over the last two decades, laboratory-based investigations using in vivo disease models and
clinical samples, coupled to state-of-the-art molecular and cellular biological methodolo-
gies, along with next-generation sequencing, proteomics, and functional genomics technol-
ogies, have driven research efforts worldwide to understand the pathogenesis of these
disease states. Such knowledge is critical to our collective efforts to discover new immune-
based biomarkers and therapeutic strategies against disease, especially as we enter the phase
of precision medicine.
This book, entitled Inflammation and Cancer: Methods and Protocols, Second Edition, is
the latest instalment of the highly successful Methods in Molecular Biology laboratory
protocol-based book series, and is a follow-up to the initial 2018 edition of Inflammation
and Cancer: Methods and Protocols. Written by leading experts in the fields of inflammation
and cancer, this book comprises 25 individual chapters and provides a timely update on a
broad spectrum of research models, techniques, and protocols, which are employed by basic
and clinical research laboratories. Each chapter is divided into sections providing detailed
information on the background and context for the chosen topic of interest, a list of the
materials and reagents needed for each topic, the step-by-step methodology for the success-
ful and reproducible execution of each topic, as well as notes to provide tips, troubleshoot-
ing advice, and elaborate further on specific materials, reagents, or methods.
Considering the enormity of the rapidly evolving and large number of research models
(in vitro, ex vivo, and in vivo) and techniques which are collectively used by researchers, it is
impractical to provide their sufficient coverage in detail in a single book. Therefore, this
edition is divided into two parts: Part I, “Experimental Model Systems,” which provides an
up-to-date snapshot of the development and characterization of representative research
models for chronic immune-based (i.e. infectious, autoimmune, inflammatory) diseases
and inflammation-associated cancers, and Part II, “Experimental Techniques,” which covers
a range of biochemical, molecular, and cellular biological techniques that are commonly
utilized to dissect the molecular mechanisms and cellular processes (including cell type(s))
which drive the pathogenesis of certain disease states.
With a strong emphasis on practicality, Inflammation and Cancer: Methods and Proto-
cols, Second Edition will appeal to a readership with a very diverse range of laboratory-based
experience, ranging from undergraduate students with limited research exposure to estab-
lished basic and/or clinical researchers wishing to diversify their existing portfolio of
practical knowledge. In addition, this book aims to supplement researchers with the neces-
sary practical expertise and know-how to assist their efforts to publish their research find-
ings, dissemination of which in the literature will enhance our collective knowledge of the
processes which drive inflammation and cancer.
Clayton, VIC, Australia Brendan J. Jenkins
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I EXPERIMENTAL MODEL SYSTEMS
1 Identifying Adult Stomach Tissue Stem/Progenitor Cells

Using the Iqgap3-2A-CreERT2 Mouse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Junichi Matsuo, Linda Shyue Huey Chuang, Jasmine Jie Lin Tong,
Daisuke Douchi, and Yoshiaki Ito
2 In Vitro and In Vivo Models for Metastatic Intestinal Tumors
Using Genotype-Defined Organoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Atsuya Morita, Mizuho Nakayama, Hiroko Oshima,
and Masanobu Oshima
3 On Target: An Intrapulmonary Transplantation Method for Modelling
Lung Tumor Development in its Native Microenvironment . . . . . . . . . . . . . . . . . . 31
Jackson A. McDonald, Leanne Scott, Jessica Van Zuylekom,
Steven Holloway, Benjamin J. Blyth, and Kate D. Sutherland
4 Endoscopic Ultrasound-Guided Fine-Needle Biopsies to Generate Preclinical
Disease Models to Study Inflammation in Pancreatic
Ductal Adenocarcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Joanne Lundy and Daniel Croagh
5 Modeling Intestinal Carcinogenesis Using In Vitro Organoid Cultures . . . . . . . . 55
Wing Hei Chan, Diana Micati, Rebekah M. Engel,
Genevieve Kerr, Reyhan Akhtar, Thierry Jardé,
and Helen E. Abud
6 An In Vitro Model for Assessing Acute Lung Injury During Pancreatitis
Development Using Primary Mouse Cell Co-cultures . . . . . . . . . . . . . . . . . . . . . . . 71
Mohamed I. Saad and Brendan J. Jenkins
7 Tracking the Host Response to Infection in Peritoneal Models
of Acute Resolving Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
David Millrine, Christopher M. Rice, Javier U. Fernandez,
and Simon A. Jones
8 Assessing Lung Inflammation and Pathology in Preclinical Models
of Chronic Obstructive Pulmonary Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Ross Vlahos, Hao Wang, and Steven Bozinovski
9 Preclinical Mouse Model of Silicosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Maggie Lam, Ashley Mansell, and Michelle D. Tate
vii
viii Contents
PART II EXPERIMENTAL TECHNIQUES
10 Tracking Cardiovascular Comorbidity in Models of Chronic

Inflammatory Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Aisling S. Morrin, Simon Eastham, Anwen S. Williams,
and Gareth W. Jones
11 Live Imaging of Pyroptosis in Primary Murine Macrophages . . . . . . . . . . . . . . . . . 139
Caroline L. Holley and Kate Schroder
12 A Streamlined Method for Detecting Inflammasome-Induced ASC
Oligomerization Using Chemical Crosslinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Sebastian A. Hughes and James E. Vince
13 DNA Methylation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Naoko Hattori, Yu-Yu Liu, and Toshikazu Ushijima
14 Flow Cytometry Identification of Cell Compartments
in the Murine Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Joel J. D. Moffet, Zachery Moore, Shannon J. Oliver,
Tahnee Towers, Misty R. Jenkins, Saskia Freytag,
James R. Whittle, and Sarah A. Best
15 Expression and Purification of Inflammasome Sensor NOD-Like Receptor
Protein-1 Using the Baculovirus-Insect Cell Expression System . . . . . . . . . . . . . . 199
Hariharan Sivaraman and Wilson Wong
16 Dissecting Interleukin-6 Classic and Trans-signaling in Inflammation
and Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Christoph Garbers and Stefan Rose-John
17 Selecting Therapeutic Antisense Oligonucleotides with Gene Targeting
and TLR8 Potentiating Bifunctionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Sunil Sapkota and Michael P. Gantier
18 Exploring Allosteric Inhibitors of Protein Tyrosine Phosphatases
Through High-Throughput Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Takeru Hayashi and Masanori Hatakeyama
19 Intravital Imaging of Regulatory T Cells in Inflamed Skin . . . . . . . . . . . . . . . . . . . 247
Michael J. Hickey and M. Ursula Norman
20 Confocal Endomicroscopy Monitoring of Tumor Formation. . . . . . . . . . . . . . . . . 257
Adele Preaudet, Ka Yee Fung, and Tracy L. Putoczki
21 Detection of Free Bioactive IL-18 and IL-18BP in Inflammatory
Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Sébastien Fauteux-Daniel, Charlotte Girard-Guyonvarc’h,
Assunta Caruso, Emiliana Rodriguez, and Cem Gabay
22 MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq
with Image-Based Phenotypic Screening in 2D and 3D Cell Models . . . . . . . . . . 279
Xiang Mark Li, David Yoannidis, Susanne Ramm, Jennii Luu,
Gisela Mir Arnau, Timothy Semple, and Kaylene J. Simpson
23 Primary Intestinal Fibroblasts: Isolation, Cultivation, and Maintenance. . . . . . . . 327
Abhimanu Pandey, Melan Kurera, and Si Ming Man
Contents ix
24 Lipid Nanoparticle-Mediated Delivery of miRNA Mimics

to Myeloid Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Elaine Kang and Marcin Kortylewski
25 Engineering Cell Lines for Specific Human Leukocyte Antigen
Presentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Dongbin Jin, Khai Lee Loh, Tima Shamekhi, Yi Tian Ting,
Terry C. C. Lim Kam Sian, James Roest, Joshua D. Ooi,
Julian P. Vivian, and Pouya Faridi
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Contributors
HELEN E. ABUD • Department of Anatomy and Developmental Biology, Monash University,

Clayton, VIC, Australia; Development and Stem Cells Program, Monash Biomedicine
Discovery Institute, Clayton, VIC, Australia
REYHAN AKHTAR • Department of Anatomy and Developmental Biology, Monash University,
GISELA MIR ARNAU • Sir Peter MacCallum Department of Oncology, University of
Melbourne, Parkville, VIC, Australia; Molecular Genomics Core, Peter MacCallum
Cancer Centre, Melbourne, VIC, Australia
SARAH A. BEST • Personalised Oncology Division, The Walter and Eliza Hall Institute
of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The
University of Melbourne, Parkville, VIC, Australia
BENJAMIN J. BLYTH • Models of Cancer Translational Research Centre, Peter MacCallum
Cancer Centre, Parkville, VIC, Australia; Sir Peter MacCallum Department of Oncology,
The University of Melbourne, Parkville, VIC, Australia
STEVEN BOZINOVSKI • Centre for Respiratory Science and Health, School of Health and
Biomedical Sciences, RMIT University, Bundoora, VIC, Australia
ASSUNTA CARUSO • Department of Pathology and Immunology, University of Geneva, Faculty
of Medicine, Geneva, Switzerland; Division of Rheumatology, Department of Medicine,
Geneva University Hospitals, Geneva, Switzerland
WING HEI CHAN • Department of Anatomy and Developmental Biology, Monash University,
LINDA SHYUE HUEY CHUANG • Cancer Science Institute of Singapore, National University
of Singapore, Singapore, Singapore
DANIEL CROAGH • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of
Medical Research, Clayton, VIC, Australia; Department of Molecular Translational
Science, School of Clinical Sciences, Monash University, Clayton, VIC, Australia;
Department of Surgery, School of Clinical Sciences, Monash University, Clayton, VIC,
Australia
DAISUKE DOUCHI • Cancer Science Institute of Singapore, National University of Singapore,
Singapore, Singapore; Department of Surgery, Tohoku University Graduate School
of Medicine, Sendai City, Japan
SIMON EASTHAM • School of Cellular and Molecular Medicine, Biomedical Sciences Building,
University of Bristol, Bristol, UK
REBEKAH M. ENGEL • Department of Anatomy and Developmental Biology, Monash
University, Clayton, VIC, Australia; Development and Stem Cells Program, Monash
Biomedicine Discovery Institute, Clayton, VIC, Australia; Cabrini Monash University
Department of Surgery, Cabrini Hospital, Melbourne, VIC, Australia
POUYA FARIDI • Department of Medicine, School of Clinical Sciences, Monash Univesity,
Clayton, VIC, Australia; Monash Proteomics & Metabolomics Facility, Department of
Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash
University, Clayton, VIC, Australia
xi
xii Contributors
SÉBASTIEN FAUTEUX-DANIEL • Department of Pathology and Immunology, University of

Geneva, Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology, Department
of Medicine, Geneva University Hospitals, Geneva, Switzerland
JAVIER U. FERNANDEZ • Division of Infection & Immunity, School of Medicine, Cardiff
University, Cardiff, Wales, UK; Systems Immunity University Research Institute, Cardiff
University, Cardiff, Wales, UK
SASKIA FREYTAG • Personalised Oncology Division, The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC, Australia; Department of Medical Biology,
KA YEE FUNG • Personalised Oncology Division, The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC, Australia; Department of Medical Biology,
CEM GABAY • Department of Pathology and Immunology, University of Geneva, Faculty of
Medicine, Geneva, Switzerland; Division of Rheumatology, Department of Medicine,
Geneva University Hospitals, Geneva, Switzerland
MICHAEL P. GANTIER • Centre for Innate Immunity and Infectious Diseases, Hudson
Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and
Translational Science, Monash University, Clayton, VIC, Australia
CHRISTOPH GARBERS • Medical Faculty, Department of Pathology, Otto-von-Guericke-
University Magdeburg, Magdeburg, Germany; Health Campus Immunology, Infectiology
and Inflammation (GC:I3), Otto-von-Guericke-University, Magdeburg, Germany; Center
for Health and Medical Prevention (CHaMP), Otto-von-Guericke-University,
Magdeburg, Germany
CHARLOTTE GIRARD-GUYONVARC’H • Department of Pathology and Immunology, University
of Geneva, Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology,
Department of Medicine, Geneva University Hospitals, Geneva, Switzerland
MASANORI HATAKEYAMA • Laboratory of Microbial Carcinogenesis, Institute of Microbial
Chemistry (BIKAKEN), Microbial Chemistry Research Foundation, Tokyo, Japan; Center
of Infection-Associated Cancer, Institute for Genetic Medicine, Hokkaido University,
Sapporo, Japan
NAOKO HATTORI • Division of Epigenomics, Institute for Advanced Life Sciences, Hoshi
University, Tokyo, Japan
TAKERU HAYASHI • Laboratory of Microbial Carcinogenesis, Institute of Microbial Chemistry
(BIKAKEN), Microbial Chemistry Research Foundation, Tokyo, Japan
MICHAEL J. HICKEY • Centre for Inflammatory Diseases, Department of Medicine, Monash
Medical Centre, Monash University, Clayton, VIC, Australia
CAROLINE L. HOLLEY • Institute for Molecular Bioscience, and Centre for Inflammation and
Disease Research, The University of Queensland, St. Lucia, QLD, Australia
STEVEN HOLLOWAY • Bioservices Department, The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC, Australia
SEBASTIAN A. HUGHES • The Walter and Eliza Hall Institute of Medical Research, Parkville,
VIC, Australia; The Department of Medical Biology, University of Melbourne, Parkville,
VIC, Australia
YOSHIAKI ITO • Cancer Science Institute of Singapore, National University of Singapore,
Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore
THIERRY JARDÉ • Department of Anatomy and Developmental Biology, Monash University,
Contributors xiii
Discovery Institute, Clayton, VIC, Australia; Cancer Program, Monash Biomedicine

BRENDAN J. JENKINS • Centre for Innate Immunity and Infectious Diseases, Hudson Institute
of Medical Research, Clayton, VIC, Australia; Faculty of Medicine, Nursing and Health
Sciences, Department of Molecular and Translational Sciences, Monash University,
Clayton, VIC, Australia; South Australian Immunogenomics Cancer Institute, University
of Adelaide, Adelaide, SA, Australia
MISTY R. JENKINS • Department of Medical Biology, The University of Melbourne, Parkville,
VIC, Australia; Immunology Division, The Walter and Eliza Hall Institute of Medical
Research, Parkville, VIC, Australia
DONGBIN JIN • Department of Medicine, School of Clinical Sciences, Monash Univesity,
GARETH W. JONES • School of Cellular and Molecular Medicine, Biomedical Sciences
Building, University of Bristol, Bristol, UK
SIMON A. JONES • Division of Infection & Immunity, School of Medicine, Cardiff University,
Cardiff, Wales, UK; Systems Immunity University Research Institute, Cardiff University,
Cardiff, Wales, UK
ELAINE KANG • Department of Immuno-Oncology, Beckman Research Institute at City of
Hope, Duarte, CA, USA
GENEVIEVE KERR • Department of Anatomy and Developmental Biology, Monash University,
MARCIN KORTYLEWSKI • Department of Immuno-Oncology, Beckman Research Institute at
City of Hope, Duarte, CA, USA
MELAN KURERA • Division of Immunology and Infectious Disease, The John Curtin School of
Medical Research, The Australian National University, Canberra, ACT, Australia
MAGGIE LAM • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of
Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational
Sciences, Monash University, Clayton, VIC, Australia
XIANG MARK LI • Victorian Centre for Functional Genomics, Peter MacCallum Cancer
Centre, Melbourne, VIC, Australia; Sir Peter MacCallum Department of Oncology,
TERRY C. C. LIM KAM SIAN • Department of Medicine, School of Clinical Sciences, Monash
Univesity, Clayton, VIC, Australia; Monash Proteomics & Metabolomics Facility,
Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery
Institute, Monash University, Clayton, VIC, Australia
YU-YU LIU • Division of Epigenomics, Institute for Advanced Life Sciences, Hoshi University,
Tokyo, Japan
KHAI LEE LOH • Centre for Inflammatory Diseases, Department of Medicine, Monash
University, Monash Medical Centre, Clayton, VIC, Australia
JOANNE LUNDY • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of
Medical Research, Clayton, VIC, Australia; Department of Molecular Translational
Science, School of Clinical Sciences, Monash University, Clayton, VIC, Australia;
Department of Surgery, School of Clinical Sciences, Monash University, Clayton, VIC,
Australia; Peninsula Clinical School, Central Clinical School, Faculty of Medicine
Nursing and Health Sciences, Monash University, Clayton, VIC, Australia
JENNII LUU • Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre,
Melbourne, VIC, Australia
xiv Contributors
SI MING MAN • Division of Immunology and Infectious Disease, The John Curtin School of
Medical Research, The Australian National University, Canberra, ACT, Australia
ASHLEY MANSELL • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of
Sciences, Monash University, Clayton, VIC, Australia
JUNICHI MATSUO • Cancer Science Institute of Singapore, National University of Singapore,
Singapore, Singapore
JACKSON A. MCDONALD • ACRF Cancer Biology and Stem Cells Division, The Walter and
Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of
Medical Biology, The University of Melbourne, Parkville, VIC, Australia
DIANA MICATI • Department of Anatomy and Developmental Biology, Monash University,
DAVID MILLRINE • Medical Research Council Protein Phosphorylation & Ubiquitylation
Unit (MRC-PPU), School of Life Sciences, University of Dundee, Dundee, UK
JOEL J. D. MOFFET • Personalised Oncology Division, The Walter and Eliza Hall Institute of
ZACHERY MOORE • Personalised Oncology Division, The Walter and Eliza Hall Institute of
Medical Research, Parkville, VIC, Australia; Department of Medical Biology, The
ATSUYA MORITA • Division of Genetics, Cancer Research Institute, Kanazawa University,
Kanazawa, Japan
AISLING S. MORRIN • Division of Infection and Immunity, and Systems Immunity University
Research Institute, School of Medicine, Cardiff University, Cardiff, Wales, UK
MIZUHO NAKAYAMA • Division of Genetics, Cancer Research Institute, Kanazawa
University, Kanazawa, Japan; Nano Life Science Institute (WPI-NanoLSI), Kanazawa
University, Kanazawa, Japan
M. URSULA NORMAN • Centre for Inflammatory Diseases, Department of Medicine, Monash
Medical Centre, Monash University, Clayton, VIC, Australia
SHANNON J. OLIVER • Personalised Oncology Division, The Walter and Eliza Hall Institute of
JOSHUA D. OOI • Centre for Inflammatory Diseases, Department of Medicine, Monash
HIROKO OSHIMA • Division of Genetics, Cancer Research Institute, Kanazawa University,
Kanazawa, Japan; Nano Life Science Institute (WPI-NanoLSI), Kanazawa University,
Kanazawa, Japan
MASANOBU OSHIMA • Division of Genetics, Cancer Research Institute, Kanazawa
University, Kanazawa, Japan; Nano Life Science Institute (WPI-NanoLSI), Kanazawa
University, Kanazawa, Japan
ABHIMANU PANDEY • Division of Immunology and Infectious Disease, The John Curtin School
of Medical Research, The Australian National University, Canberra, ACT, Australia
ADELE PREAUDET • Personalised Oncology Division, The Walter and Eliza Hall Institute of
TRACY L. PUTOCZKI • Personalised Oncology Division, The Walter and Eliza Hall Institute of
University of Melbourne, Parkville, VIC, Australia; Department of Surgery, The University
of Melbourne, Parkville, VIC, Australia
Contributors xv
SUSANNE RAMM • Victorian Centre for Functional Genomics, Peter MacCallum Cancer
CHRISTOPHER M. RICE • School of Cellular & Molecular Medicine, University of Bristol,
Bristol, UK
EMILIANA RODRIGUEZ • Department of Pathology and Immunology, University of Geneva,
Faculty of Medicine, Geneva, Switzerland; Division of Rheumatology, Department of
Medicine, Geneva University Hospitals, Geneva, Switzerland
JAMES ROEST • St Vincent’s Institute of Medical Research, Fitzroy, VIC, Australia
STEFAN ROSE-JOHN • Institute of Biochemistry, Kiel University, Kiel, Germany
MOHAMED I. SAAD • Centre for Innate Immunity and Infectious Diseases, Hudson Institute
of Medical Research, Clayton, VIC, Australia; Faculty of Medicine, Nursing and Health
Sciences, Department of Molecular and Translational Sciences, Monash University,
SUNIL SAPKOTA • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of
Science, Monash University, Clayton, VIC, Australia
KATE SCHRODER • Institute for Molecular Bioscience, and Centre for Inflammation and
Disease Research, The University of Queensland, St. Lucia, VIC, Australia
LEANNE SCOTT • ACRF Cancer Biology and Stem Cells Division, The Walter and Eliza Hall
Institute of Medical Research, Parkville, VIC, Australia
TIMOTHY SEMPLE • Sir Peter MacCallum Department of Oncology, University of Melbourne,
Parkville, VIC, Australia; Molecular Genomics Core, Peter MacCallum Cancer Centre,
Melbourne, VIC, Australia
TIMA SHAMEKHI • Department of Medicine, School of Clinical Sciences, Monash Univesity,
KAYLENE J. SIMPSON • Victorian Centre for Functional Genomics, Peter MacCallum Cancer
University of Melbourne, Parkville, VIC, Australia; Department of Biochemistry and
Pharmacology, University of Melbourne, Parkville, VIC, Australia
HARIHARAN SIVARAMAN • Centre for Innate Immunity and Infectious Diseases, Centre for
Cancer Research, Hudson Institute of Medical Research, Melbourne, Clayton, VIC,
Australia; Department of Molecular and Translational Science, School of Clinical Sciences,
Monash University, Clayton, VIC, Australia
KATE D. SUTHERLAND • ACRF Cancer Biology and Stem Cells Division, The Walter and
Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of
Medical Biology, The University of Melbourne, Parkville, VIC, Australia
MICHELLE D. TATE • Centre for Innate Immunity and Infectious Diseases, Hudson Institute
of Medical Research, Clayton, VIC, Australia; Department of Molecular and
Translational Sciences, Monash University, Clayton, VIC, Australia
YI TIAN TING • Centre for Inflammatory Diseases, Department of Medicine, Monash
JASMINE JIE LIN TONG • Cancer Science Institute of Singapore, National University of
Singapore, Singapore, Singapore
TAHNEE TOWERS • Personalised Oncology Division, The Walter and Eliza Hall Institute of
TOSHIKAZU USHIJIMA • Division of Epigenomics, Institute for Advanced Life Sciences, Hoshi
University, Tokyo, Japan
xvi Contributors
JESSICA VAN ZUYLEKOM • Models of Cancer Translational Research Centre, Peter MacCallum
Cancer Centre, Parkville, VIC, Australia
JAMES E. VINCE • The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC,
Australia; The Department of Medical Biology, University of Melbourne, Parkville, VIC,
Australia
JULIAN P. VIVIAN • St Vincent’s Institute of Medical Research, Fitzroy, VIC, Australia;
Department of Medicine, The University of Melbourne, Melbourne, VIC, Australia
ROSS VLAHOS • Centre for Respiratory Science and Health, School of Health and Biomedical
Sciences, RMIT University, Bundoora, VIC, Australia
HAO WANG • Centre for Respiratory Science and Health, School of Health and Biomedical
Sciences, RMIT University, Bundoora, VIC, Australia
JAMES R. WHITTLE • Personalised Oncology Division, The Walter and Eliza Hall Institute of
University of Melbourne, Parkville, VIC, Australia; Department of Medical Oncology,
Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; Sir Peter MacCallum
Department of Oncology, The University of Melbourne, Parkville, VIC, Australia
ANWEN S. WILLIAMS • Division of Infection and Immunity, and Systems Immunity
University Research Institute, School of Medicine, Cardiff University, Cardiff, Wales, UK
WILSON WONG • Centre for Innate Immunity and Infectious Diseases, Centre for Cancer
Research, Hudson Institute of Medical Research, Melbourne, Clayton, VIC, Australia;
Department of Molecular and Translational Science, School of Clinical Sciences, Monash
University, Clayton, VIC, Australia
DAVID YOANNIDIS • Molecular Genomics Core, Peter MacCallum Cancer Centre, Melbourne,
VIC, Australia
Part I
Experimental Model Systems

Chapter 1
Identifying Adult Stomach Tissue Stem/Progenitor Cells

Using the Iqgap3-2A-CreERT2 Mouse
Junichi Matsuo, Linda Shyue Huey Chuang, Jasmine Jie Lin Tong,
Daisuke Douchi, and Yoshiaki Ito
Abstract
Identification of unique gene markers of normal and cancer stem cells is an effective strategy to study cells of
origin and understand tumor behavior. Lineage tracing experiments using the Cre recombinase driven by a
stem cell-specific promoter in the CreERT2 reporter mouse model enables identification of adult stem cells
and delineation of stem cell activities in vivo. In our recent research on the mouse stomach, Iqgap3 was
identified as a homeostatic stem cell marker located in the isthmus of the stomach epithelium. Lineage
tracing with the Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato mouse model demonstrated stem cell activity
in Iqgap3-expressing cells. Using the Iqgap3-2A-CreERT2 mouse model to target oncogenic KrasG12D
expression to Iqgap3-expressing cells, we observed the rapid development of precancerous metaplasia in the
stomach and proposed that aberrant Iqgap3-expressing cells may be critical determinants of early carcino-
genesis. In this chapter, we detail a lineage tracing protocol to assess stem cell activity in the murine
stomach. We also describe the procedure of inducing KrasG12D expression in Iqgap3-expressing homeo-
static stem cells to explore their role as cells of origin and to trace the early cellular changes that precede
neoplastic transformation.
Key words Mouse model, Lineage tracing, Stomach, Stem cells, Carcinogenesis, Metaplasia, Iqgap3,
KrasG12D
1 Introduction
Adult tissue stem/progenitor cells maintain organ homeostasis and

effect tissue repair. Because of their proliferative capacity and inher-
ent plasticity, these cells are an ideal candidate for cells of origin of
cancer. The understanding of the molecular underpinnings of adult
tissue stem/progenitor cells advances regenerative medicine and
cancer research through the discovery of novel drug targets and
prognostic biomarkers. The past decade has seen extensive research
on various stem cell markers and potential cells of origin in the
stomach. Two types of stem cells, homeostatic stem cells and
reserve stem cells, have been identified in corpus gastric units,
Brendan J. Jenkins (ed.), Inflammation and Cancer: Methods and Protocols, Methods in Molecular Biology, vol. 2691,
https://doi.org/10.1007/978-1-0716-3331-1_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
3
4 Junichi Matsuo et al.
Fig. 1 Histology of corpus of the mouse stomach. The corpus epithelium of

composed of gastric units. Gastric units can be divided into four regions, pit,
isthmus, neck, and base. Mucin-secreting pit cells reside in the pit. Homeostatic
isthmus stem cells are located in the isthmus. The neck contains two types of
cells, acid-secreting parietal cells and mucus-secreting mucus-neck cells. Chief
cells reside in the base
which are the epithelial structures that line the stomach [1–
6]. Stomach gastric units are primarily divided into pit, isthmus,
neck, and base; of note, homeostatic stem cells reside in the isthmus
of gastric units (Fig. 1) [7–11]. The isthmus stem cells are highly
proliferative—as shown by their expression of proliferation marker
Ki67—and are necessary for maintenance of the stomach epithe-
lium under homeostatic (normal) conditions [1–3]. Reserve stem
cells are a subpopulation of terminally differentiated chief cells
located in the base of the gastric units and are marked by the
expression of Wnt-target gene and stem cell marker Lgr5, as well
as chief cell markers such as Pgc and Gif [5, 6, 12]. Interestingly,
while reserve stem cells are dormant under homeostatic conditions,
the cells acquire stem cell activities to regenerate gastric units after
tissue injury [5, 6, 13, 14]. Although the identification of several
stomach stem cell markers has considerably contributed to our
understanding of the cellular hierarchy in the stomach, the distinct
molecular mechanisms underlying the regulation of homeostatic
and reserve stem cells are not fully understood.
We recently identified cytoskeletal scaffold protein IQ motif con-
taining GTPase activating protein 3 (Iqgap3) as a stem cell marker,
which is expressed specifically in the Ki67+ isthmus cells of gastric
units in mice [3]. Lineage tracing experiments using the Iqgap3-
2A-CreERT2;Rosa26-LSL-tdTomato mouse model uncovered stem
cell activities of Iqgap3-expressing isthmus cells. In this mouse
model, Iqgap3-expressing cells in the isthmus maintain whole gas-
tric units for at least 12 months under homeostatic conditions
[3]. Furthermore, in vitro culture of Iqgap3-expressing cells gen-
erated gastric organoids, suggesting self-renewal and
Lineage Tracing of Stomach Stem Cells 5
differentiation of Iqgap3-expressing cells [3]. On the other hand,

damage induction by using high-dose tamoxifen to stomach tissue
demonstrated the induction of Iqgap3 expression in Lgr5+ reserve
stem cells [3]. In addition, lineage tracing with damage induction
showed that regenerated epithelial cells were derived from Iqgap3-
expressing cells, suggesting the contribution of Iqgap3+/Lgr5+
cells to tissue repair [3].
Iqgap3 is a member of the Iqgap protein family, which interacts
with components of major oncogenic signaling pathways to pro-
mote signal transduction [15, 16]. Of direct relevance to prolifera-
tion and cancer is the interaction of Iqgap protein with the Ras-Erk
pathway, particularly the active form of Ras (e.g., KrasG12D), Erk1/
2, Mek1/2, Raf, Egfr, Rac, and Cdc42 [15, 16]. The lqgap protein
also interacts with key Wnt pathway components β-catenin and Apc
[15, 17]. These protein interactions might contribute to prolifera-
tive capacity and stem cell potential. Gene expression analysis of
human clinical samples revealed elevated IQGAP3 expression in
gastric cancer, compared to normal stomach [3]. Notably, the
depletion of IQGAP3 expression in less differentiated cancer cell
line HGC-27 resulted in the downregulation of key stem cell
factors, including NANOG/OCT4/SOX2, and the upregulation
of stomach differentiation markers pepsinogen C (PGC) [3]. These
findings indicated that Iqgap3 might potentially regulate cancer
stem cell activities.
Lineage tracing is commonly used for adult tissue stem cell
studies and to identify cells of origin. Lineage tracing provides
stagewise in vivo visualization of stem cell behavior, such as self-
renewal and differentiation [18]. In this chapter, we describe how
we use lineage tracing experiments to investigate the stem cell
activity in Iqgap3-expressing cells in the mouse stomach. To con-
duct lineage tracing, two types of mouse models, stem cell marker-
specific CreERT2 mouse model and Rosa26-LSL-tdTomato, are
required. Using stem cell marker Iqgap3, we generated the
Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato mouse model
(Fig. 2). In this mouse model, CreERT2 is selectively expressed in
Iqgap3+ cells. Low-dose tamoxifen treatment activates CreERT2
to remove the Lox-Stop-Lox (LSL) DNA sequence from the
Rosa26 locus to induce tdTomato expression permanently. At
one-day post tamoxifen treatment of Iqgap3-2A-CreERT2;
Rosa26-LSL-tdTomato mouse model, Iqgap3-expressing cells in
the isthmus were labeled by a fluorescent protein tdTomato
(Figs. 3, 4, and 5) [3]. At 12 months post tamoxifen treatment,
whole gastric units were labeled by tdTomato, indicating that
tdTomato-/Iqgap3-expressing cells possess stem cell activities and
maintained gastric units for at least 12 months (Figs. 3, 4, and 5)
[3]. If Iqgap3 were terminally differentiated cells, limited numbers
of tdTomato labeled cells would be observed 12 months post
tamoxifen treatment (Figs. 4 and 5).
Fig. 2 Genetic construct of mouse models. (a) In the Iqgap3-2A-CreERT2 mouse,

2A-CreERT2 was inserted in exon 38 of the Iqgap3 gene. (b) In the Rosa26-LSL-
tdTomato mouse, LSL-tdTomato was inserted in the Rosa26 locus. (c) In the
LSL-KrasG12D mouse, G12D mutation is introduced in the Kras gene and LSL is
placed upstream of the mutation
Fig. 3 Schematic showing experimental procedure of Iqgap3 lineage tracing and

induction of KrasG12D in the isthmus. (a) For lineage tracing, the mice are treated
with tamoxifen and analyzed at 1 day, 6 months, and 12 months postinjection
(p.i.). (b) For induction of KrasG12D, the mice are treated with tamoxifen and
analyzed at 1 month postinjection
KRAS is frequently amplified or mutated in human gastric

cancer [19]. Previous mouse studies have shown that the expression
of an active form of Kras in homeostatic or reserve stem cells
induced metaplasia in stomach epithelium [1, 6, 12, 20,
21]. Mutated Kras might therefore represent a major first “hit”
oncogenic lesion in stem cells and may be used to recapitulate the
contribution of stem cells in early gastric carcinogenesis. In this
chapter, we also describe how the expression of KrasG12D induction
in Iqgap3-expressing cells by using the Iqgap3-2A-CreERT2;LSL-
KrasG12D mouse influences stem cell behavior in early carcinogene-
sis (Fig. 2). Indeed, rapid development of metaplasia was observed
in the stomach at 1-month post tamoxifen treatment (Figs. 3 and
6) [3].
Fig. 4 Determination of stem cell activity in the stomach using lineage tracing.
One day after tamoxifen treatment, CreERT2-expressing cells are labeled by
fluorescent protein tdTomato. If the cells possess stem cell activity, they will
continuously produce daughter cells for a long term (12 months), and whole
gastric units are labeled by tdTomato. If the cells are differentiated cells, the
gastric units will show limited tdTomato expression in certain cells
Fig. 5 Expression of tdTomato (tdTom), E-cadherin (E-cad), and Ki67 on corpus

gastric units. At 1 day post tamoxifen injection (p.i.), tdTomato expression was
observed in Ki67+ isthmus cells. tdTomato-expressing cells were expanded at
6 months after tamoxifen treatment. At 12 months post tamoxifen treatment,
almost whole gastric units were labeled by tdTomato. Scale bar = 100 μM
Fig. 6 Hematoxylin-eosin staining of the stomach from a representative Iqgap3-

2A-CreERT2;LSL-KrasG12D mouse. Metaplastic epithelial structures were
observed 1 month post tamoxifen treatment (see Fig. 1 for normal stomach)
2 Materials
All reagents should be prepared with Milli-Q water.
2.1 Mouse Model All mice should be handled in strict accordance with good animal
practice as defined by the appropriate Institutional Animal Care
Use Committee.
1. Iqgap3-2A-CreERT2 mouse strain: This strain was newly gen-
erated by the National University of Singapore and Cyagen
(Santa Clara, CA) (Fig. 2) [3].
2. Rosa26-lsl-tdTomato strain: This strain (B6.Cg-Gt(ROSA)
26Sortm14(CAG-tdTomato)Hze/J) was obtained from The
Jackson Laboratory (Bar Harbor, ME) (Fig. 2).
3. LSL-KrasG12D strain: The strain (B6.129S4-Krastm4Tyj/J)
was obtained from The Jackson Laboratory (Bar Harbor,
ME) (Fig. 2).
2.2 Animal 1. 10 mg/mL tamoxifen: Dissolve 100 mg of tamoxifen in 1 mL

Treatment of 100% ethanol at 30 °C. Add 9 mL of sunflower oil (auto-
claved) into tamoxifen in ethanol solution. Make aliquots and
store at -20 °C (see Note 1).
2. Syringe (1 mL) and 26-gauge needle (13 mM) (see Note 2).
2.3 Formalin-Fixed 1. Tissue wash buffer: 10% FBS in PBS. Dissolve 10% of fetal
Paraffin Embedded bovine serum (FBS) in phosphate-buffered saline (PBS:
(FFPE) Mouse Tissue 137 mM sodium chloride [NaCl], 2.7 mM potassium chloride
[KCl], 8 mM sodium phosphate dibasic [Na2HPO4], 2 mM
potassium phosphate monobasic [KH2PO4]). Prepare PBS by
dilution of 10× PBS in Milli-Q water.
2. Fixative: 4% paraformaldehyde (PFA) in PBS. Dissolve 4 g of
PFA in 80 mL of Milli-Q water with 25 μL of 10 N sodium
hydroxide (NaOH) at 80 °C. Add 10 mL of 10× PBS in PFA
solution. Add Milli-Q water into PFA in PBS to make volume
of 100 mL (see Note 3).
3. 50%, 70%, 80%, 100% of ethanol: Prepare 50%, 70%, 80% of
ethanol by using 100% of ethanol and Milli-Q water.
4. Butanol/ethanol: Add 100% butanol to 100% ethanol to
obtain the butanol-ethanol ratio of 1:4, 2:3, 3:2, and 4:1.
5. Paraffin: Place paraffin in 60 °C oven (see Note 4).
6. Microtome.
7. Adhesive glass slide.
8. Forceps and scissors.
9. 50-mL tube.
10. 6-well culture plate.
2.4 Antibodies 1. Rabbit anti-RFP antibody (PM005, MBL, 1:500) (see Note 5).
2. Goat anti-RFP antibody (MBS448122, MyBioSource, 1:500)
(see Note 5).
3. Rat anti-Ki67 antibody (14–5698-82, Thermo Fisher Scien-
tific, 1:2000).
4. Mouse anti-E-cadherin Alexa647-conjugated antibody
(560,062, BD Bioscience, 1:200).
5. Goat anti-rabbit IgG Alexa546-conjugated antibody (A11035,
Thermo Fisher Scientific, 1:200) (see Note 5).
6. Donkey anti-goat IgG Alexa555-conjugated antibody
(A31572, Thermo Fisher Scientific, 1:200) (see Note 5).
7. Donkey anti-rat IgG Alexa488-conjugated antibody (A21208,
Thermo Fisher Scientific, 1:200).
2.5 Immuno- 1. Antigen retrieval solution: DAKO target retrieval solution 10×
fluorescence Staining concentrate (pH 6) (see Note 6).
2. Wash buffer: PBST, 0.1% of Tween 20 was added in PBS (see
Note 7).
3. Blocking reagent: DAKO Protein Block Serum-Free (see Note
8).
4. 1 mg/mL DAPI.
5. Antifade mounting media (see Note 9).
6. Coverslip: Size is 24 by 60 mM (#1,5).
7. Autoclave.
8. Coplin jar (staining jar).
9. Pap Pen.
2.6 Hematoxylin- 1. Mayer’s Hematoxylin (Lillie’s Modification).

Eosin Staining 2. Eosin.
3. 0.001% of ammonium hydroxide in Milli-Q water.
4. Eukitt quick-hardening mounting medium.
2.7 Microscope 1. Confocal laser scanning microscope (e.g., Zeiss LSM880

Airyscan).
2. Fluorescence microscope (e.g., Zeiss Axioplan2).
3 Methods
3.1 Mice and 1. Breed stem cell marker-specific CreERT2 strain Iqgap3-2A-
Treatment CreERT2 mice with Rosa26-LSL-tdTomato mice to generate
Iqgap3-2A-CreERT2;Rosa26-LSL-tdTomato (Iqgap3-
CreERT2;Rosa-tdTom) strain (see Note 10).
2. To conduct lineage tracing experiment, perform intraperito-
neal injection of 2 mg/20 g low dose of tamoxifen into
7-week-old Iqgap3-CreERT2;Rosa-tdTom mice. Analyze
mice at 1 day, 3 months, 6 months, and 12 months post-
treatment (Fig. 3) (see Note 11).
3. To investigate the contribution of Iqgap3-expressing cells dur-
ing early carcinogenesis, breed Iqgap3-2A-CreERT2 with LSL-
KrasG12D mice to generate the Iqgap3-2A-CreERT2;
LSL-KrasG12D (Iqgap3-CreERT2;KrasG12D) strain (see Note
12).
4. Perform intraperitoneal injection of 2 mg/20 g of tamoxifen
into 7-week-old mice, and analyze at 1-month posttreatment
(see Note 13).
3.2 Formalin-Fixed 1. Resect the stomach from mice and cut along the greater curva-
Paraffin Embedded ture by scissors.
(FFPE) Stomach at 2. Wash the tissues three times with ice-cold 10% FBS in PBS and
Room Temperature paste on the silicon board (Fig. 7).
Fig. 7 Schematic diagram showing the procedure of tissue fixation for mouse
stomach. After cutting the greater curvature of the stomach, the stomach is
placed and pinned on the silicon board. The stomach pasted on a silicon board is
fixed with 4% PFA/PBS
3. Fix the washed tissues with 4% PFA in PBS in 50-mL tubes at

4 °C for 18–24 h (Fig. 7).
4. Incubate the fixed tissues with 50% and 70% of ethanol at 4 °C
for 1 h in 50-mL tubes, and then incubate tissues with 80% of
ethanol for 18–24 h (see Note 14).
5. Incubate the samples with 100% ethanol in 50-mL tubes at 4 °
C for 1 h, and then incubate with 100% ethanol at room
temperature for 1 h (see Note 15).
6. Incubate the dehydrated samples with 1:4, 2:3, 3:2, and 4:1 of
butanol/ethanol solutions in 50-mL tubes at room tempera-
ture for 15 min, and then incubate with 100% butanol twice at
room temperature for 15 min (see Note 16).
7. Incubate the samples with 1:1 of butanol/paraffin mixture in
6-well plates for 1 h at 60 °C (see Note 17).
8. Incubate the samples with 100% paraffin in 6-well plates for
1 h, and later incubate with 100% paraffin for 2 h, at 60 °C.
9. Embed the samples in paraffin (FFPE sample).
10. Slice FFPE samples at 5-μM thickness using a microtome.
11. Add 37–40 °C warmed water onto adhesive glass slides, and
then place the sliced tissues on top of the warmed water
(Fig. 8) (see Note 18).
12. Remove water from the glass slides, and dry the slides
completely in a 37 °C oven.
3.3 Immuno- 1. Deparaffinize sliced tissues on the glass slides by incubation in

fluorescence (IF) xylene, ethanol, and Milli-Q water at room temperature for
Staining and 3 min.
Visualization 2. Perform antigen retrieval in DAKO target retrieval solution at
100 °C for 20 min in an autoclave, and cool samples at room
temperature for 20 min.
Fig. 8 Schematic showing the procedure of FFPE slide preparation. (a) Glass
slides are covered by 37 °C warmed Milli-Q water. (b) Sliced FFPE samples are
placed on the water. (c) Remove Milli-Q water from slides and completely dry the
slide
3. Wash the slides three times with wash buffer (PBST) in a

Coplin jar.
4. Draw a liquid barrier between the paraffin tissue sample and
glass slide label with a Pap Pen (see Note 19).
5. Block the slides with DAKO Protein Block Serum-Free at
room temperature for 30 min (see Note 20).
6. Dilute rat anti-Ki67 antibody and mouse anti-E-cadherin
Alexa647-conjugated antibody in blocking reagent at 1:2000
and 1:200, respectively. Then add rabbit anti-RFP antibody or
goat anti-RFP antibody at 1:500 dilution (see Note 21).
7. Incubate the slides with primary antibody mixture at 4 °C for
18–24 h (see Note 22).
8. Wash the slides three times with PBST.
9. Dilute donkey anti-rat IgG Alexa488-conjugated antibody in
5% skim milk in PBST at 1:200 dilution. Then add goat anti-
rabbit IgG Alexa546-conjugated antibody or donkey anti-goat
IgG Alexa555-conjugated antibody at 1:200 dilution (see Note
23).
10. Incubate the slides with secondary antibodies at room temper-
ature for 1 hour (see Note 24).
11. Wash the slides three times with PBST. Add 10 μL of 1 mg/mL
DAPI in the first wash (see Note 25).
12. Drop antifade mounting media on the slides, and place cover-
slips on the slides.
13. Visualize stained tissues on the slides by a confocal laser micro-
scope with 405 nM, 488 nM, 561 nM, and 633 nM laser
(Fig. 5) (see Note 26).
3.4 Hematoxylin- 1. Deparaffinize sliced tissues on glass slides by incubation in

Eosin (HE) Staining xylene, ethanol, and Milli-Q water.
and Visualization 2. Stain the sliced tissues with hematoxylin for 1 min, and wash
three times with Milli-Q water at room temperature (see Note
27).
3. Incubate the sliced tissues with 0.001% ammonium hydroxide

for 1 min, and wash three times with Milli-Q water (see Note
28).
4. Stain the sliced tissues with eosin for 1 min, and wash three
times with Milli-Q water (see Note 27).
5. Dehydrate the sliced tissues by incubation in ethanol, and then
incubate in xylene.
6. Drop mounting media on the slides, and place a coverslip on
each slide.
7. Visualize stained tissues by fluorescence microscopy (Fig. 6).
4 Notes
1. It is necessary to dissolve tamoxifen in 30 °C pre-warmed

ethanol before adding sunflower oil; tamoxifen does not dis-
solve easily in sunflower oil.
2. Two different lengths, 25 mM and 13 mM, are available for the
26-gauge needle. A shorter needle eases the injection of tamox-
ifen in mice. Moreover, although a higher needle gauge
reduces stress on mice, a 26-gauge needle is recommended
due to the viscosity of sunflower oil.
3. Adding 10 N NaOH to Milli-Q water helps to dissolve PFA.
After dissolved PFA, 10× PBS is added to make the pH neutral.
Alternatively, neutralized buffered formalin can be used for
fixation instead of PFA.
4. Paraffin solidifies at room temperature and liquid paraffin is
required to process and embed tissue. To melt paraffin, solid
paraffin is placed in a 60 °C oven overnight.
5. Two types of antibodies, rabbit anti-RFP antibody (PM005)
and goat anti-RFP antibody (MBS448122), can be used for
tdTomato staining. Secondary antibody for rabbit anti-RFP
antibody is goat anti-rabbit IgG Alexa546-conjugated anti-
body (A11035). Secondary antibody for goat anti-RFP anti-
body (MBS448122) is donkey anti-goat IgG Alexa555-
conjugated antibody (A31572).
6. Alternatively, DAKO target retrieval solution pH 9 (10×) or
sodium citrate buffer (10 mM sodium citrate, 0.05% Tween
20, pH 6.0) can be used. In order to prepare sodium citrate
buffer, trisodium citrate (dehydrate) is dissolved in Milli-Q
water. After adjusting pH to 6.0 with HCl, Tween 20 is
added in the buffer. The choice of antigen retrieval solution
depends on the choice of antibody. Some antibodies work with
all antigen retrieval solutions, but some only work with DAKO
target retrieval solution pH 9.0.
7. Alternatively, TBST (140 mM NaCl, 2.7 mM KCl, 25 mM

Tris, 0.1% Tween 20, pH 7.5) can be used as wash buffer
instead of PBST.
8. DAKO Protein Block Serum-Free was used in this chapter. Five
percent skim milk in PBST, 3% bovine serum albumin (BSA) in
PBST, or 2% normal donkey serum in PBST can be used for
blocking instead of DAKO protein block. The choice of block-
ing reagent is highly dependent on the choice of antibody.
9. Confocal laser microscope induces photobleaching to immu-
nofluorescent stained samples due to the high energy level of
the laser. Antifade mounting media helps to prevent
photobleaching.
10. Iqgap3-2A-CreERT2 mice express CreERT2 in Iqgap3-
expressing cells, while leaving Iqgap3 expression intact
[3]. Tamoxifen treatment activates CreERT2 specifically in
Iqgap3-expressing cells. In the Rosa26-LSL-tdTomato mouse
strain, the lox-stop-lox (LSL) DNA sequence inhibits tdTo-
mato expression. Thus, tamoxifen treatment in the Iqgap3-2A-
CreERT2;Rosa26-LSL-tdTomato mouse induces CreERT2
activity in Iqgap3-expressing cells, and this active CreERT2
removes LSL sequence to induce tdTomato expression.
11. Previous studies demonstrated that tamoxifen treatment
damages the stomach epithelium, particularly in parietal cells
[22–24]. In order to minimize tissue damage, a low dosage of
tamoxifen treatment is required, and 0.5–1 mg/20 g dosage is
recommended. By contrast, 5 mg/20 g high-dose tamoxifen
treatment has been used for tissue damage repair studies.
High-dose tamoxifen treatment concurrently induces tissue
damage and CreERT2 activation. Lineage tracing studies
through monitoring tdTomato expression are used to deter-
mine the nature and behavior of marker-derived CreERT2-
expressing cells. If the CreERT2-expressing cells were fully
differentiated, localized and limited numbers of tdTomato-
expressing cells are observed. In the case of the CreERT2-
expressing cells possessing stem cell properties, the whole gas-
tric unit is labeled by tdTomato at 1-year post tamoxifen
treatment.
12. KrasG12D is a constitutively active form of Kras, and this muta-
tion has been frequently detected in various cancers [25]. In
the LSL-KrasG12D mouse strain, the LSL sequence inhibits
KrasG12D expression. Tamoxifen administration to Iqgap3-
2A-CreERT2; LSL-KrasG12D mouse induces expression of
KrasG12D in Iqgap3-expressing cells.
13. KrasG12D expression in stomach stem cells, including homeo-
static stem cells and reserve stem cells, induces metaplasia
which represents the early step of gastric carcinogenesis [1, 6,
12, 20, 21]. In Iqgap3-2A-CreERT2; LSL-KrasG12D mice, a U-

shape-like structure of metaplasia is expected at 1–2 months
post tamoxifen treatment [3].
14. This ethanol incubation series is required to gradually remove
water from the tissues. Prolonged incubation of tissues with
80% ethanol promotes effective dehydration. The tissues can
be stored in 4 °C of 80% ethanol for a maximum of 1 year.
15. This step helps to remove water in tissue completely.
16. Compared to ethanol, an organic compound, butanol, has
better compatibility with paraffin. This step helps to replace
ethanol in tissues with butanol.
17. This step helps to replace butanol in tissues with paraffin. To
maintain paraffin in its liquid state, this procedure is required to
be performed at 60 °C. We perform this step inside a 60 °
C oven.
18. Sliced paraffin tissue unfolds and spreads on 37–40 °C warm
water. If the temperature exceeds 40 °C, the tissue disperses in
the water. We placed none sliced tissues on a glass slide.
19. A liquid barrier separating the paraffin tissue sample and label is
required to prevent the backflow and contain any reagents used
in tissue staining.
20. The slides were rocked at least ten times to agitate the blocking
reagent. This rocking helps uniform staining.
21. Concentration of antibodies used is determined by optimiza-
tion. RFP, Ki67, and E-cadherin antibodies used in this chapter
can also be used with another blocking reagent, including 5%
skim-milk in PBST and 3% BSA in PBST.
22. The slides were rocked at least ten times to agitate the primary
antibody solution. This rocking helps uniform staining.
23. Five percent skim milk in PBST is used for all secondary anti-
body diluents. The concentration of secondary antibodies is
fixed at 1:200.
24. The slides were rocked at least ten times to agitate the second-
ary antibody solution. This rocking helps uniform staining.
25. DAPI integrates into DNA and helps to visualize the nuclei in
cells.
26. Instead of the confocal laser microscope, the fluorescent micro-
scope can be used to visualize immunofluorescence staining.
However, using the confocal laser microscope is highly recom-
mended to obtain an enhanced high-resolution image.
27. Hematoxylin stains the nucleus while Eosin stains the cyto-
plasm of the cell.
28. Ammonium hydroxide enhances the blue color of hematoxylin
staining.
Acknowledgements
This work is supported by the National Research Foundation Sin-

gapore and the Singapore Ministry of Education under its Research
Centre of Excellence initiative. The work is also supported by the
Singapore Ministry of Health’s National Medical Research Coun-
cil’s Open Fund Large Collaborative Grant (OFLCG18May-
0023), the Singapore Ministry of Health’s National Medical
Research Council under its Clinician-Scientist Individual Research
Grant (MOH-CIRG21jun-0003), and the National University of
Singapore School of Medicine (NUSMed) Internal Grant Funding
(NUHSRO/2019/086/StomachStemCell and NUHSRO/
2022/043/NUSMed/25/LOA).
References
1. Matsuo J, Kimura S, Yamamura A et al (2017). 11. Karam SM, Leblond CP (1993) Dynamics of
Identification of stem cells in the epithelium of epithelial cells in the corpus of the mouse
the stomach corpus and antrum of mice. Gas- stomach. V. Anat Rec 236:333–340
troenterology 152:218–231. e14 12. Douchi D, Yamamura A, Matsuo J et al (2021)
2. Han S, Fink J, Jorg DJ et al (2019). Defining Induction of gastric cancer by successive onco-
the identity and dynamics of adult gastric isth- genic activation in the corpus. Gastroenterol-
mus stem cells. Cell Stem Cell 25:342–356. e7 ogy 161:1907–1923
3. Matsuo J, Douchi D, Myint K et al (2021) 13. Miao ZF, Adkins-Threats M, Burclaff JR et al
Iqgap3-Ras axis drives stem cell proliferation (2020). A metformin-responsive metabolic
in the stomach corpus during homoeostasis pathway controls distinct steps in gastric pro-
and repair. Gut 70:1833–1846 genitor fate decisions and maturation. Cell
4. Hata M, Kinoshita H, Hayakawa Y et al (2020). Stem Cell 26:910–925. e6
GPR30-expressing gastric chief cells do not 14. Miao ZF, Lewis MA, Cho CJ et al (2020). A
dedifferentiate but are eliminated via dedicated evolutionarily conserved molecular
PDK-dependent cell competition during network licenses differentiated cells to return
development of metaplasia. Gastroenterology to the cell cycle. Dev Cell 55:178–194. e7
158:1650–1666. e15 15. Hedman AC, Smith JM, Sacks DB (2015) The
5. Stange DE, Koo BK, Huch M et al (2013) biology of IQGAP proteins: beyond the cyto-
Differentiated Troy+ chief cells act as reserve skeleton. EMBO Rep 16:427–446
stem cells to generate all lineages of the stom- 16. Nojima H, Adachi M, Matsui T et al (2008)
ach epithelium. Cell 155:357–368 IQGAP3 regulates cell proliferation through
6. Leushacke M, Tan SH, Wong A et al (2017) the Ras/ERK signalling cascade. Nat Cell Biol
Lgr5-expressing chief cells drive epithelial 10:971–978
regeneration and cancer in the oxyntic stom- 17. Briggs MW, Sacks DB (2003) IQGAP proteins
ach. Nat Cell Biol 19:774–786 are integral components of cytoskeletal regula-
7. Karam SM, Leblond CP (1993) Dynamics of tion. EMBO Rep 4:571–574
epithelial cells in the corpus of the mouse 18. Hsu YC (2015) Theory and practice of lineage
stomach. I. Anat Rec 236:259–279 tracing. Stem Cells 33:3197–3204
8. Karam SM, Leblond CP (1993) Dynamics of 19. Deng N, Goh LK, Wang H et al (2012) A
epithelial cells in the corpus of the mouse stom- comprehensive survey of genomic alterations
ach. II. Anat Rec 236:280–296 in gastric cancer reveals systematic patterns of
9. Karam SM, Leblond CP (1993) Dynamics of molecular exclusivity and co-occurrence
epithelial cells in the corpus of the mouse stom- among distinct therapeutic targets. Gut 61:
ach. III. Ten Anat Recd 236:297–313 673–684
10. Karam SM, Leblond CP (1993) Dynamics of 20. Choi E, Hendley AM, Bailey JM et al (2016).
epithelial cells in the corpus of the mouse stom- Expression of activated Ras in gastric chief cells
ach. IV. Anat Rec 236:314–332 of mice leads to the full Spectrum of
metaplastic lineage transitions. Gastroenterol- coordinates normal and metaplastic gastric epi-
ogy 150:918–930. e13 thelial progenitor cell proliferation. J Biol
21. Min J, Vega PN, Engevik AC et al (2019) Chem 288:16085–16097
Heterogeneity and dynamics of active Kras- 24. Manning EH, Lapierre LA, Mills JC et al
induced dysplastic lineages from mouse corpus (2020). Tamoxifen acts as a parietal cell proto-
stomach. Nat Commun 10:5549 nophore. Cell Mol Gastroenterol Hepatol 10:
22. Huh WJ, Khurana SS, Geahlen JH et al (2012). 655–657. e1
Tamoxifen induces rapid, reversible atrophy, 25. Huang L, Guo Z, Wang F et al (2021) KRAS
and metaplasia in mouse stomach. Gastroenter- mutation: from undruggable to druggable in
ology 142:21–24. e7 cancer. Signal Transduct Target Ther 6:386
23. Khurana SS, Riehl TE, Moore BD et al (2013)
The hyaluronic acid receptor CD44
Chapter 2
In Vitro and In Vivo Models for Metastatic Intestinal Tumors

Using Genotype-Defined Organoids
Atsuya Morita, Mizuho Nakayama, Hiroko Oshima, and Masanobu Oshima
Abstract
It has been established that the accumulation of driver gene mutations causes malignant progression of
colorectal cancer (CRC) through positive selection and clonal expansion, similar to Darwin’s evolution.
Following this multistep tumorigenesis concept, we previously showed the specific mutation patterns for
each process of malignant progression, including submucosal invasion, epithelial mesenchymal transition
(EMT), intravasation, and metastasis, using genetically engineered mouse and organoid models. However,
we also found that certain populations of cancer-derived organoid cells lost malignant characteristics of
metastatic ability, although driver mutations were not impaired, and such subpopulations were eliminated
from the tumor tissues by negative selection. These organoid model studies have contributed to our
understanding of the cancer evolution mechanism. We herein report the in vitro and in vivo experimental
protocols to investigate the survival, growth, and metastatic ability of intestinal tumor-derived organoids.
The model system will be useful for basic research as well as the development of clinical strategies.
Key words Colon cancer, Organoids, Collagen gel, Metastasis, Imaging
1 Introduction
The accumulation of driver gene mutations causes malignant pro-

gression of cancer, a well-known concept of multistep tumorigen-
esis [1, 2]. This model is based on the positive selection of tumor
cells that acquire growth advantages, similar to Darwin’s evolution
theory. We previously established genetically engineered mouse
models that carried driver gene mutations in various combinations
and examined the genotype-phenotype relationships [3–5]. Nota-
bly, intestinal tumor-derived organoids carrying quadruple muta-
tions in ApcΔ716, KrasG12D, Tgfbr2–/–, and Trp53R270H, hereafter
“AKTP organoids,” showed a metastatic ability with a high inci-
dence after loss of wild-type Trp53, and the loss of heterogeneity
(LOH) cells achieved dominance in the liver metastatic foci
through positive selection [6].
https://doi.org/10.1007/978-1-0716-3331-1_2,
19
20 Atsuya Morita et al.
We previously examined the cancer evolution mechanism based

on the belief that cancer cells always progress toward a malignant
state. However, controversy remains concerning the reverse evolu-
tion of cancer cells, namely, the loss of malignant characteristics and
elimination of these cells from tumors by negative selection [7–
9]. We therefore established several subclones from metastatic
AKTP organoid cells after long passage. Although cell proliferation
was not altered under standard two-dimensional (2D) culture con-
ditions, some subclones showed a significantly suppressed survival
and growth in collagen gel culture conditions. Furthermore, these
subclones showed complete loss of their metastatic ability, as con-
firmed by in vivo imaging [10].
These in vitro and in vivo analyses using genetically defined
organoids can aid in the analysis of the survival, stemness, prolifer-
ation, and metastatic ability of cancer cells. We herein report in vitro
and in vivo experimental methods for evaluating the stemness as
well as the metastatic ability of (colorectal) cancer cells using AKTP
and AKTP-derived subcloned organoids.
2 Materials
2.1 Mouse Intestinal 1. AKTP organoids: Established from ApcΔ716 (A), KrasG12D (K),
Tumor-Derived Tgfbr2 –/– (T), and Trp53R270H (P) quadruple mutant mouse
Organoids intestinal tumors [5]. The wild-type Trp53 gene was lost by
LOH [6]. AKTP organoids develop liver metastasis at a high
incidence when transplanted to the spleen.
2. tdTomato- or Venus-labeled AKTP organoids: These lines can
be used for in vitro growth assay as well as in vivo metastatic
analyses. tdTomato and Venus can be detected directly by
immunofluorescent microscopy or indirectly by immunohisto-
chemistry using anti-green fluorescent protein (GFP) and anti-
red fluorescent protein (RFP) antibody, respectively. tdTomato
and Venus are expressed under transcriptional regulation of the
CAG promoter [11].
3. Luciferase-expressing AKTP organoids: These lines can be used
for in vitro proliferation and in vivo imaging of liver metastatic
foci. The luciferase gene is expressed under transcriptional
regulation of the CAG promoter [10].
4. These organoid lines can be maintained in either 2D or three-
dimensional (3D) culture conditions. All organoids will be
provided as a collaboration effort upon request.
2.2 Standard Culture 1. CO2 incubator.

(2D) 2. Cell culture treated 6-well plates and dishes.
3. 1.5-mL microtube.
Tumor-Derived Organoid Models 21
4. Phosphate-buffered saline (PBS), pH 7.4, and PBS-EDTA.

5. 0.25% trypsin-EDTA (Gibco).
6. Cell freezing media (e.g., Bambanker, Nippon Genetics)
7. 1.8 mL cryotube.
8. 35 μm cell strainer with round-bottom polystyrene tube.
2.3 Collagen Gel 1. Auto cell counter (e.g., Bio-Rad).

Culture (2D or 3D) 2. 96-well black/clear bottom plates for 3D collagen gel culture.
3. Cellmatrix Type I-A (collagen, Type I, 3 mg/mL, pH 3.0;
Nitta Gelatin).
4. Reconstitution buffer (Nitta Gelatin): 50 mM sodium hydrox-
ide, 260 mM sodium bicarbonate, 200 mM HEPES.
5. Concentrated culture solution, DF culture solution (Nitta
Gelatin).
6. Collagenase, type I.
7. 15 mL conical tube.
8. Fluorescence inverted microscope.
2.4 Culture Medium 1. Standard culture medium: Advanced Dulbecco’s Modified

Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)
medium (Gibco), 10% fetal bovine serum (FBS), supplemented
with 5 μM A83-01 (Sigma), 5 μM CHIR99201 (Tocris Biosci-
ence), and 10 μM Y27632 (Wako) (see Note 1).
2. Collagen gel culture medium (2D or 3D): Advanced DMEM/
F-12 medium, 10 mM HEPES (Gibco), supplemented with
2 mM Glutamax, 1× B27, 1× N2 (Gibco), 100 ng/mL recom-
binant murine Noggin (Peprotech), and 1 μM N-acetylcysteine
(Sigma) (see Note 2).
2.5 Spleen 1. 27-gauge needle syringe.

Transplantation for 2. Immunodeficient mice (Crl:SHO-PrkdcscidHrhr (SCID hairless
Liver Metastasis Model outbred, SHO) or immunocompetent mice (C57BL/6)
(Charles River) (see Note 3).
3. Isoflurane.
4. Inhalation anesthesia device and induction chamber (e.g.,
NARCOBIT-E; Natsume Seisakusho, Japan).
5. Hair clippers for small animals.
6. Hair depilatory cream.
7. 70% ethanol for disinfection.
8. Surgical instruments (forceps, scissors, sutures, clips).
2.6 Bioimaging of 1. 15 mg/mL D-luciferin potassium solution dissolved in PBS.

Tumor Growth by 2. IVIS Lumina LT in vivo imaging system (Perkin Elmer) (see
Luciferase Activity Note 4)
3. Living image software program (Perkin Elmer).
4. Isoflurane anesthesia device (e.g., MK-AT210; Muromachi
Kikai, Japan).
3 Methods
3.1 AKTP Organoid 1. Wash the AKTP 3D organoids or semi-confluent cultured

Standard Culture on organoid cells in a 6-well plate with PBS-EDTA, and treat the
2D Culture Plates (Fig. cells with 100 μL of 0.25% trypsin-EDTA for 5–10 min at 37 °
1) C.
2. Add 1 mL of Advanced DMEM/F-12 with 10% FBS to stop
the trypsin reaction, and suspend the dissociated AKTP cells by
pipetting.
3. Spin down the cells in a 1.5-mL tube at 300 × g for 5 min,
suspend the cells in 2 mL of standard culture medium, and seed
them onto a new 6-well plate.
4. Culture the AKTP cells at 37 °C in a 5% CO2 incubator.
5. Passage cells on reaching 70–80% confluence (repeat Subhead-
ing 3.1, steps 1–4).
6. To make AKTP cell freeze stocks, suspend the cell pellets in
Bambanker cell freezing media (Subheading 3.1, step 3), and
transfer to a cryotube (Fig. 1). Store the tubes at -80 °C in a
deep freezer overnight, and then move them to liquid nitrogen
for long-term storage.
Fig. 1 Schematic drawing of AKTP organoid standard culture on 6-well plates with preparation of frozen stocks
and subcloning
7. To establish AKTP-derived subclones, dissociate AKTP orga-

noid cells to single cells by trypsinization and a cell strainer.
Plate single cells onto a 96-well plate at 1 cell/well (Fig. 1; see
Note 5). After confirmation of single colony formation in each
96-well plate, expand cells by passaging to establish subclones.
3.2 Organoid Growth 1. Prepare collagen gel by mixing Cellmatrix Type I-A, concen-
Analyses in 3D trated culture solution DF culture solution, and reconstitution
collagen Gel (See Note buffer at an 8:1:1 volume ratio according to the manufacturer’s
6) instructions.
2. Prepare the dissociated parental AKTP cells or luciferase-
expressing AKTP-derived subcloned cells by trypsinization
(Subheading 3.1, steps 1–3).
3. Count the number of cells with an auto cell counter, and then
suspend them in ice-cold collagen gel at 2 × 102 cells/20 μL.
4. Fill 96-well black/clear bottom plates with ice-cold collagen
gel (see Note 7) as the bottom gel (70 μL/well), and then pour
20 μL of cell suspension onto the bottom gel (Fig. 2a).
Fig. 2 Organoid culture experiment in 3D collagen gel. (a) Schematic drawing of AKTP organoid culture in
collagen gel. (b, c) Example data showing cell proliferation of stemness-high and stemness-low subclones
examined by the luciferase activity (b) and the size and number of organoids (c). Bars in the photographs,
1 mm
5. Leave the plate at 37 °C to polymerize the collagen gel, and

then add 100 μL of warm collagen gel culture medium to
each well.
6. When analyzing luciferase-expressing AKTP cells, the prolifer-
ation rate can be determined by calculating the luciferase activ-
ity relative to the initial luciferase measurement (2 × 102 cells at
day 0). Add D-luciferin/PBS to each well at 150 μg/mL (see
Note 8), and then analyze luciferase activity using IVIS Lumina
LT and the Living Image software program. An example of cell
proliferation assay data for stemness-high and stemness-low
cells by measuring the relative luciferase activity is provided
[10] (Fig. 2b).
7. To examine the size and numbers of developed organoids in
collagen gels, take bright-field microscopic photographs under
a dissection microscope and measure the values in the photo-
graphs. An example of an organoid growth assay of stemness-
high and stemness-low cells is provided [10] (Fig. 2c).
3.3 Competition Co- 1. Fill a 6-well plate with 1 mL of ice-cold collagen gel (Subhead-
culture Analysis on 2D ing 3.2, step 1), and leave it at 37 °C to polymerize as the
Collagen Gel (See bottom gel.
Notes 9 and 10) 2. Prepare trypsin-dissociated Venus-labeled AKTP-derived sub-
cloned cells and tdTomato-labeled parental AKTP cells from
organoids or a 2D standard culture plate (Subheading 3.1,
steps 1–3, 7).
3. Spin down cells in the 1.5-mL tube at 300 × g for 5 min,
suspend the cells in collagen culture medium, and count the
number of cells with an auto cell counter.
4. Mix Venus-labeled AKTP-derived subcloned cells and
tdTomato-labeled parental AKTP cells at a 1:1 ratio (2 × 105
cells for each), and then seed the cell mixture onto the bottom
gel (step 1). Add 2 mL of collagen gel culture medium to each
6-well plate, and culture the cells in an incubator at 37 °C
(Fig. 3a).
5. When the cells reach semiconfluency at approximately
90–100%, examine the cell ratio by counting the Venus- and
tdTomato-expressing cells using a fluorescent microscope
(at least 3 fields/well) (see Note 11). Imaging examples of a
competition co-culture analysis using stemness-low subclones
(green) and parental cells (red) are provided [10] (Fig. 3b).
6. For passage of the cells cultured on a collagen gel, treat the
cultured well with 0.1% collagenase type I/PBS at 37 °C for
30 min to digest the collagen gel. Wash the cells twice with
10 mL PBS in a 15-mL tube, and spin down the cells at 300 × g
for 5 min.
Fig. 3 Competition co-culture experiment. (a) Schematic drawing of the competition co-culture analysis of
parental AKTP cells (red) and subcloned cells (green) on 2D collagen gel culture. (b) Representative photo-
graphs of co-culture of Venus-labeled stemness-low subclone cells and tdTomato-labeled parental AKTP
cells. Note that the subcloned cells are gradually eliminated from culture. Bars in the photographs, 500 μm
7. Treat the cell pellets with Trypsin-EDTA at 37 °C for 5 min.

8. Add Advanced DMEM/F-12 with 10% FBS, and suspend cells
by pipetting to stop the trypsin reaction.
9. Spin down dissociated cells in a 1.5-mL tube at 300 × g for
5 min, suspend the cells in collagen gel culture medium, seed
the cells onto a new collagen bottom gel, and continue the
competition co-culture analysis (Subheading 3.3, steps 4
and 5).
3.4 Liver Metastasis 1. Prepare the dissociated AKTP organoid cells from 2D standard
Model (See Note 12) culture (Subheading 3.1, steps 1–3).
2. Count the number of cells with an automatic cell counter, and
suspend cells in PBS at 5 × 105 cells/40 μL (see Note 13).
3. Leave the cell-containing tube on ice until transplantation
(Fig. 4a).
4. Anesthetize immunodeficient SHO mice or isogenic C57BL/6
mice by isoflurane inhalation, and place the mice in the right
lateral recumbent position (see Notes 3 and 14).
5. Scrub the surgical site of the skin (left lateral side) with 70%
ethanol for disinfection, cut the skin and abdominal wall
<10 mm with surgical scissors, and exteriorize the spleen
gently with surgical forceps (Fig. 4b).
6. Inject tumor cells into the spleen (5 × 105 cells/40 μL/mouse)
using a 27-gauge needle syringe (Fig. 4b).
Fig. 4 Liver metastasis models by spleen transplantation of AKTP cells. (a) Schematic drawing of the protocol
for liver metastasis model generation. Trypsin-dissociated AKTP cells are prepared from 2D standard cultures.
(b) Tumor cell suspensions are transplanted into the spleen using a syringe
7. Put the spleen back in the abdominal cavity, close the perito-
neum with surgical sutures, and close the skin with a surgical
clip (Fig. 4b).
8. If parental AKTP organoid cells are used for transplantation,
multiple liver metastases will develop 2–4 weeks after
transplantation.
3.5 In Vivo Imaging 1. In vivo imaging analyses of metastatic tumor growth should be
Analyses conducted as follows: Inject D-luciferin potassium into the
organoid-transplanted mice at 150 mg/kg intraperitoneally
(see Note 8), and anesthetize the mice with isoflurane.
2. If using C57BL/6 mice as recipients, the hair at the scanning
site should be removed (see Note 14).
3. Set the mice on the stage of the IVIS Lumina LT under
continuous isoflurane inhalation, and measure the lumines-
cence signals from luciferase-expressing tumor cells by scan-
ning the mice (Fig. 5a).
4. Analyze the luminescence signals of the liver and spleen area
with the Living Image software program at day 0 (immediately
after transplantation) and at 1–4 weeks after transplantation.
Tumor growth in the liver can be examined by calculating the
bioluminescence intensity relative to that at day 0 (see Note
15). Examples of bioimaging and tumor growth analyses are
provided (Fig. 5b).
Fig. 5 In vivo luciferase imaging analyses of metastatic tumor growth. (a) Schematic drawing of luciferase
imaging analyses of the transplanted model mice. Mice are injected with D-luciferin, anesthetized, and set on
the IVIS Lumina LT device. (b) Representative bioluminescent imaging photographs of mice transplanted with
luciferase-expressing AKTP cells at day 0, week 1, and week 2 (left) and a bar graph of the relative
luminescence of liver tumors at each time point with photographs of liver and spleen tumors (right). Arrow-
heads indicate tumor foci
4 Notes
1. A83-01 is a TGF-β type I receptor (ALK5) inhibitor, which

suppresses TGF-β-induced differentiation. CHIR99201 is a
GSK3 inhibitor, which activates the Wnt signaling pathway
through suppression of β-catenin phosphorylation. These inhi-
bitors are supplemented in the standard culture medium to
keep cells in undifferentiated status. Y27632 is a ROCK inhib-
itor, which suppresses apoptosis or anoikis of dissociated orga-
noid cells.
2. Glutamax, B27, N2, Noggin, and N-acetylcysteine are supple-
mented in collagen gel culture medium according to the origi-
nal protocol for intestinal epithelia-derived organoid culture in
Matrigel [12].
3. Although AKTP organoids were established from a C57BL/6

background mouse tumor, the efficiency of liver metastasis is
higher in immunodeficient mice than in C57BL/6 mice. We
used SHO mice that were homozygous for the PrkdcSCID and
the Hrhr mutations, resulting in severe immunodeficiency and
are also hairless. When examining the host immune response to
tumors, C57BL/6 mice should be used for transplantation. If
using C57BL/6 mice as the host, use male mice, as they will be
the same sex as the AKTP organoids.
4. IVIS Lumina LT in vivo imaging system is one of the biolumi-
nescence imaging systems designed by Perkin Elmer. Other
imaging systems that have standard function for the measure-
ment of bioluminescence signals may be used for the detection
of liver metastasis.
5. Suspend the trypsin-treated and strainer-filtrated single-disso-
ciated AKTP organoid cells at 1 cell/100 μL, and add 100 μL
of the cell suspension to each well of a 96-well plate.
6. Organoid cells that have lost their stemness and metastatic
ability showed significant growth suppression in 3D collagen
gel culture, resulting in decreased numbers and sizes of orga-
noids [10]. This effect is more prominent than with Matrigel
culture. For this assay, a ROCK inhibitor (Y27632), GSK3
inhibitor (CHIR99021), and ALK5 inhibitor (A83-01) should
not be supplemented.
7. Use of a 96-well plate with black-colored walls and clear bot-
toms is recommended when examining both the luciferase
activity and observing tumor organoids. The black-colored
walls block interference with the luminescent signal from adja-
cent wells, which is required to ensure the exact measurement
of the luciferase activity.
8. For luciferase bioimaging analyses, prepare 100× stock solution
of D-luciferin potassium in PBS (15 mg/mL), and store it at
-30 °C.
9. Organoid subclones with decreased stemness are gradually
eliminated from the whole tumor cell population by negative
selection when co-cultured on 2D collagen gel. A competition
co-culture assay on a collagen gel can be used to examine the
negative selection of subclones.
10. This assay can be used to examine the stemness of organoid
cells, as organoids with the decreased expression of the
stemness-related gene Lgr5 are outcompeted by Lgr5-high
cell populations over several passages [10].
11. Photographs should be taken of several independent fields to
calculate the mean cell ratio. A fluorescence-activated cell
sorting (FACS) analysis is recommended to confirm the

Venus-/tdTomato-positive cell ratio after digestion of the col-
lagen gel and dissociation of cells.
12. We found that approximately 30% of AKTP-derived subclones
lost their metastatic ability to the liver with the decreased
expression of the stem cell-related gene Lgr5 [10]. It is possible
that decreased stemness causes an impaired survival of
disseminated cells, resulting in the suppression of metastasis.
13. To avoid leakage of the transplanted cells from the spleen,
AKTP cells can be suspended with Matrigel for injection.
However, to minimize the effect of Matrigel on metastasis
development, suspension of cells in PBS is recommended for
transplantation.
14. When using C57BL/6 mice for luciferase imaging, remove the
hair with clippers and hair removal cream before transplanta-
tion surgery.
15. The luminescence signal from the liver tumors increases imme-
diately after intraperitoneal injection of D-luciferin. To detect
the optimal luminescence signal, it is recommended to measure
the luminescence with an auto exposure time mode of every
2 min until 20 min after D-luciferin injection, subsequently
selecting the highest value as the optimal signal.
Acknowledgements
We thank Ayako Tsuda, Yoshie Jomen, and Manami Watanabe for

their valuable technical assistance in the organoid culture and his-
tological analyses.
References
1. Fearon ER, Vogelstein B (1990) A genetic effectively drives metastasis of intestinal cancer.
model for colorectal tumorigenesis. Cell 61: Cancer Res 78:1334–1346
759–767 6. Nakayama M, Hong CP, Oshima H et al
2. Markowitz SD, Bertagnolli MM (2009) Molec- (2020) Loss of wild-type p53 promotes mutant
ular origins of cancer: molecular basis of colo- p53-driven metastasis through acquisition of
rectal cancer. N Eng J Med 361:2449–2460 survival and tumor-initiating properties. Nat
3. Oshima H, Nakayama M, Han TS et al (2015) Commun 11:2333
Suppressing TGFβ signaling in regenerating 7. Weghorn D, Sunyaev S (2017) Bayesian infer-
epithelia in an inflammatory microenviron- ence of negative and positive selection in
ment is sufficient to cause invasive intestinal human cancers. Nat Genet 49:1785–1788
cancer. Cancer Res 75:766–776 8. Zapata L, Pich O, Serrano L et al (2018) Neg-
4. Nakayama M, Sakai E, Echizen K et al (2017) ative selection in tumor genome evolution acts
Intestinal cancer regression by mutant p53 on essential cellular functions and the immu-
through the acquisition of invasiveness asso- nopeptidome. Genome Biol 19:67
ciated with complex glandular formation. 9. Banyai L, Trexler M, Kerekes K et al (2021)
Oncogene 36:5885–5896 Use of signals of positive and negative selection
5. Sakai E, Nakayama M, Oshima H et al (2018) to distinguish cancer genes and passenger
Combined mutation of Apc, Kras, and Tgfbr2 genes. eLife 10:e59629
10. Morita A, Nakayama M, Wang D et al (2023) clusters through fibrotic niche generation.
Frequent loss of metastatic ability in subclones Nat Commun 12:863
of Apc, Kras, Tgfbr2, and Trp53 mutant intes- 12. Sato T, Stange DE, Ferrante M et al (2011)
tinal tumor organoids. Cancer Sci 114:1437– Long-term expansion of epithelial organoids
1450 from human colon, adenoma, adenocarci-
11. Kok SY, Oshima H, Takahashi K et al (2021) noma, and Barrett’s epithelium. Gastroenterol-
Malignant subclone drives metastasis of genet- ogy 141:1762–1772
ically and phenotypically heterogenous cell
Chapter 3
On Target: An Intrapulmonary Transplantation Method

for Modelling Lung Tumor Development in its Native
Microenvironment
Jackson A. McDonald, Leanne Scott, Jessica Van Zuylekom,
Steven Holloway, Benjamin J. Blyth, and Kate D. Sutherland
Abstract
The development of in vivo lung cancer models that faithfully mimic the human disease is a crucial research
tool for understanding the molecular mechanisms driving tumorigenesis. Subcutaneous transplantation
assays are commonly employed, likely due to their amenability to easily monitor tumor growth and the
simplistic nature of the technique to deliver tumor cells. Importantly however, subcutaneous tumors grow
in a microenvironment that differs from that resident within the lung. To circumvent this limitation, here
we describe the development of an intrapulmonary (iPUL) orthotopic transplantation method that enables
the delivery of lung cancer cells, with precision, to the left lung lobe of recipient mice. Critically, this allows
for the growth of lung cancer cells within their native microenvironment. The coupling of iPUL transplan-
tation with position emission tomography (PET) imaging permits the serial detection of tumors in vivo and
serves as a powerful tool to trace lung tumor growth and dissemination over time in mouse disease models.
Key words Lung cancer, Intrapulmonary injection, Orthotopic transplantation, Tumor microenvi-
ronment, Preclinical imaging, PET/CT imaging
1 Introduction
The use of in vivo lung cancer mouse models that recapitulate the
diverse characteristics of the human disease are imperative for
understanding tumor cell intrinsic and extrinsic mechanisms that
influence tumor cell behavior. Genetically engineered mouse mod-
els (GEMMs), whereby cancer-associated mutations are created in a
spatial and temporal manner in lung epithelial cells, serve as power-
ful model systems due to the development of autochthonous
tumors [1]. However, the long latencies associated with several of
Jackson A. McDonald and Leanne Scott contributed equally to this chapter.
https://doi.org/10.1007/978-1-0716-3331-1_3,
31
32 Jackson A. McDonald et al.
the models and the need to assess disease postmortem are restrictive
for assessing new therapeutic interventions and/or evaluating the
antitumor role of distinct immune cell subsets on tumorigenesis.
To overcome this, cell lines established from lung tumors that
develop in GEMMs have been generated and are amendable for
such studies [2–4].
Syngeneic transplantation models bypass the requirement for
conditional mouse strains, specialized reagents, and techniques
afforded when working with GEMMs. Subcutaneous transplants
are by far the most common method employed, largely due to the
simplicity of the technique and ease by which tumor cell growth can
be monitored by successive caliper measurements over time.
Indeed, transplantation experiments into immune-competent and
immune-deficient recipient mice have proven a powerful method-
ology to dissect the molecular mechanisms that regulate antitumor
immunity [5–7]. The greatest limitation of this transplantation
model, however, is its inability to replicate the tissue-specific micro-
environment of the lung. Orthotopic transplantation models,
whereby tumors grow within the organ of interest, are therefore a
more relevant model to study human disease.
The delivery of cancer cells into the lungs of recipient mice can
be achieved following intravenous (IV) injection via the tail vein.
This transplantation method, however, is routinely used as an
“experimental metastasis” model, as the colonization of tumor
cells in the lung is reliant on the ability of cells to survive in the
circulatory system [8]. While lung adenocarcinoma cell lines
derived from KrasLSL-G12D/+;p53f/f mice result in the development
of lung metastasis following IV injection [9], murine small cell lung
cancer (SCLC) cell lines fail to seed metastatic lesions in the lung
but instead result in the formation of liver metastasis
[10, 11]. Thus, orthotopic transplant methodologies, such intra-
tracheal [12, 13] and intrathoracic [14] injections, involving the
direct delivery of tumor cells into the lung are advantageous and
overcome possible organ tropism effects encountered following IV
injection.
One challenge associated with orthotopic transplants is the
ability to readily detect and monitor in vivo tumor growth with
accuracy in a live animal. Bioluminescent imaging can be utilized
for cell lines engineered to stably express luciferase. Stable expres-
sion of luciferase may alter the behavior of transduced cells in vivo,
due to the immunogenicity of the reporter presented by the tumor
cells when transplanted in an immunocompetent host [15]. Alter-
natively, imaging modalities based on inherent tumor-specific char-
acteristics serve as powerful tools to monitor tumor growth and
avoid the need for additional genetic modifications. Positron emis-
sion tomography (PET) imaging is one of these tools, allowing
visualization of disease by way of injection of a radiolabelled tracer
that is internalized by or bound to receptors on the tumor cells. In
Another random document with
no related content on Scribd:
the Senate or the House make amendments to which the other
chamber does not agree? That is just what very frequently occurs. In
such cases a conference committee is appointed, made up as a rule
of three members from each chamber. These conferees meet and try
to reach a common ground by compromise. Then, when they have
agreed, they report to their respective chambers and the latter must
accept or reject the conference report without further amendment.
Some Tricks of the Lawmaker’s Trade.— The value of
Lawmaking is a skilled profession; it takes the experience in
average congressman most of his first term to Congress.
learn just how it is done. He must acquire a knowledge of the rules,
written and unwritten, the traditions, and what may rightly be called
the “tricks of the trade”. Ability as an orator does not count for much,
particularly in the House. The house chamber is a big, noisy place
where only a leather-lunged speaker can make himself clearly heard.
Congressmen, moreover, do not take kindly to long speeches; they
expect members of the House to say what they have to say in five or
ten minutes. If a congressman desires to make an impression upon
the voters of his home district by sending them accounts of his able
speeches in their behalf, he can usually obtain from the House, by
unanimous consent, permission to have his speech printed at the
public expense and distributed without its ever having been delivered
on the floor of the chamber at all. When long speeches are made it is
usually to waste the time of Congress and prevent the passage of
some measure to which the speakers are opposed.[118]
Attempts to talk a measure to death are Filibusters.
known as “filibusters”. Both chambers are now
able to put an end to filibusters by applying rules which shut off
further debate when a specified majority of the members so desire;
but in the old days, before these rules were adopted, senators
sometimes kept the floor hour after hour all day and all night long,
talking on every irrelevant matter, reading long extracts from books,
and employing all their ingenuity to lengthen the debate. The
proceedings in the Senate are often interesting; but the visitor to the
House gallery is likely to be disappointed if he goes with the
expectation of hearing some good speech-making. The real work of
the House is done in committee.
The Powers of Congress.—The powers of Classification of
Congress, as the lawmaking branch of the congressional
national government, are set forth in eighteen powers.
clauses of the federal constitution. Hence it is customary to speak of
the “eighteen powers of Congress”, although there are in fact more
than eighteen separate powers, as anyone will find if he takes the
trouble to count them. These powers may be conveniently grouped
together under eight heads: (1) financial, the power to levy taxes and
to borrow money; (2) commercial, the power to regulate commerce
with foreign nations and among the several states; (3) military, the
power to declare war, to raise and support armies, to maintain a
navy, and to provide for organizing, arming, and calling forth the
state militia; (4) monetary, the power to coin money, to regulate the
value thereof, and to protect the currency against counterfeiting; (5)
postal, the power to establish post-offices and post roads; (6)
judicial, the power to constitute tribunals subordinate to the Supreme
Court; (7) miscellaneous, including powers in relation to bankruptcy,
naturalization, patents, copyrights, and the government of the
national capital; (8) supplementary, the power to make all laws which
may be found “necessary and proper for carrying into execution the
foregoing powers”. This is a rather tedious classification of
congressional powers, but the section which enumerates these
powers is, by all odds, the most important part of the whole
constitution and no one can claim to know much about the
government of the United States unless he understands, at least in a
general way, what these eighteen clauses express and imply.
It will be noted that all the powers except the Express and implied
last are express powers, that is, they are powers.
conveyed to Congress in so many words. The last is a grant of
implied authority, in other words it is a provision for supplementing
the express powers. Where Congress has the express right to tax, to
borrow, to regulate interstate commerce, to raise and support
armies, or to coin money, it has the implied right to make whatever
laws may be “necessary and proper” to carry its express powers into
full operation. Having the express power to borrow money, Congress
may therefore establish a system of banks if this is needed to render
more easy the operations of borrowing. Having the express power to
support armies, it may place almost any sort of restriction upon
industry in war time. By the implied powers clause of the constitution
the authority of Congress is given great elasticity.
Are the Powers of Congress Broad Enough?—If the words of
the constitution had been strictly interpreted, the powers of Congress
would now be too narrow for the work which a strong national
government must perform. It is easy to understand why the framers
of the constitution were cautious about conferring broad powers
upon the new government. They were anxious that no legislative
despotism should be built up in America. But as time passed the
express powers of Congress have been steadily widened by the
process of interpreting them broadly so that today the real authority
of Congress is much greater than one would suspect from a mere
reading of the constitution. For all practical purposes they are broad
enough although it is probably true that if the constitution were to be
redrawn tomorrow, the authority of the national government would be
increased. Nearly all the amendments proposed in recent years have
been in the direction of expanding the powers of Congress.
The Efficiency of Congress.—In comparison The handicaps to
with the other great parliaments and legislatures good work.
of the world, the Congress of the United States does its work fairly
well. It is rather too large in membership, and the House of
Representatives would probably gain in efficiency if it were reduced
in size. Another handicap to good work arises from the enormous
grist of measures which comes forward at every session. Congress
is always under constant pressure for time. Many millions are often
voted in a single hour and it is impossible for the congressmen to go
carefully through the long list of financial items. Until very recently,
the absence of a budget system afforded an incentive to
extravagance; but this defect has now been remedied.[119]
Congress also lacks leadership. In European The lack of
countries every parliament and legislature has a leadership.
recognized leader, usually called the prime minister. He or his
colleagues present the business and carry it through.[120] There is
nothing of this sort in either the Senate or the House of
Representatives. It is true that each political party has a floor leader,
but he has not effective control over his followers. The chairmen of
the various committees also supply a certain measure of leadership,
but their work is not unified. Mention should also The lobby.
be made of the pressure which is applied to
individual members of Congress by the lobbyists. These lobbyists
are hired workers, usually lawyers, who are paid to help get
measures through, or in some cases to prevent the passage of
certain laws. They are employed by corporations, or by labor
organizations, or by anyone who is deeply concerned in measures
pending before Congress. They use every form of persuasion in their
efforts to have congressmen see their side of the case. The “lobby”
has been placed under various restrictions in recent years, but it is
still an influential factor.
The Congressional Oligarchies.—We are in The influence of
the habit of assuming that the power in national small groups in
lawmaking rests with the 531 men who Congress.
constitute the Senate and the House of Representatives; but the
dominating influence is in reality exercised by a relatively small
group of men in both chambers. The chairmen of important
committees and certain others of long congressional experience are
the men whose influence counts. The rest follow their lead for the
most part. Important measures, moreover, are often discussed in a
caucus of the majority party, and the action of the caucus is
considered binding on all who attend it. A member in either chamber,
especially a new member, who displays a disposition to be wholly
independent, and to disregard the advice of his party leaders or the
decisions of his party caucus is not likely to get many favors for
himself or for his district. The senator or representative who desires
to be effective finds it necessary, therefore, to help others with their
plans whether he approves them heartily or not, in order that he may
be, in turn, helped with his own. It is almost always true that a group
of thirty or forty members, on the majority side, can secure the
passage of measures which they desire and can prevent the
passage of measures to which they are opposed.[121] In this respect
the Congress of the United States does not differ much from
legislative bodies the world over. Large deliberative bodies are
invariably prone to follow the lead of some relatively small group in
their own membership; otherwise they would never make headway,
and the larger the chamber the more likely is this to be true.
General References
James Bryce, American Commonwealth, Vol. I, pp. 97-208;
Woodrow Wilson, Congressional Government, pp. 58-129;
C. A. Beard, American Government and Politics, pp. 231-293; Ibid., Readings
on American Government and Politics, pp. 214-271;
Everett Kimball, National Government of the United States, pp. 308-378;
W. B. Munro, Government of the United States, pp. 146-218;
S. W. McCall, The Business of Congress, pp. 43-84;
Lynn Haines, Your Congress, pp. 67-109.
Group Problems
1. Is it desirable to restrict the present powers of the Senate in relation to
treaties? Reasons for giving the Senate special powers in relation to treaties. The
meaning of “advice and consent”. Washington’s attitude and experience. The
action of the Senate on important treaties during the past hundred years. The
practical difficulty of obtaining a two-thirds majority. Confirmation as a barrier to
secret diplomacy. References: Ralston Hayden, The Senate and Treaties, pp.
169-195; H. C. Lodge, The Senate of the United States, pp. 1-31; Everett
Kimball, National Government of the United States, pp. 549-551; 573; S. B.
Crandall, Treaties: Their Making and Enforcement, pp. 67-92; Congressional
Record, 1919-1922.
2. The personnel of Congress. References: Types of men elected. Their
occupations at home. Their legislative experience. Are there too many lawyers?
Length of service. How the personnel might be improved. (Material for this study
may be had in the Congressional Directory, and in the various autobiographical
works such as James G. Blaine’s Twenty Years in Congress; Champ Clark’s
Autobiography, etc.)
3. The merits and faults of the committee system. References: James
Bryce, American Commonwealth, Vol. I, pp. 156-166; S. W. McCall, The
Business of Congress, pp. 43-60; L. G. McConachie, Congressional Committees,
pp. 58-86; Everett Kimball, National Government of the United States, pp. 344-
356; P. S. Reinsch, Readings on American Federal Government, pp. 257-264.
Short Studies
1. The old and the new method of choosing Senators. George H. Haynes,
The Election of Senators, pp. 36-129.
2. The procedure in impeachments. W. B. Munro, Government of the United
States, pp. 168-173.
3. The Speaker of the House. C. A. Beard, American Government and
Politics, pp. 280-289; M. P. Follett, The Speaker of the House of
Representatives, pp. 296-330.
4. The rights of minorities. F. A. Cleveland and Joseph Schafer,
Democracy in Reconstruction, pp. 446-467.
5. The general powers of Congress. W. B. Munro, Government of the United
States, pp. 208-218.
6. How Congress legislates. Everett Kimball, National Government of the
United States, pp. 350-356; P. S. Reinsch, Readings on American Federal
Government, pp. 290-296.
7. An Englishman’s observation on the work of Congress. James Bryce,
American Commonwealth, Vol. I, pp. 191-208.
8. The rules of the House and Senate. Everett Kimball, National
Government of the United States, pp. 333-344.
9. Obstruction in Congress. S. W. McCall, The Business of Congress, pp. 85-
92.
10. Party organizations in Congress. W. W. Willoughby and Lindsay
Rogers, Introduction to the Problem of Government, pp. 334-351.
11. The lobby. P. S. Reinsch, American Legislatures and Legislative Methods,
pp. 228-298.
12. The Library of Congress. F. J. Haskin, American Government, pp. 287-
288.
Questions
1. Do the merits of the double-chamber system outweigh the objections? Why
should the members of the two chambers be chosen by different methods? Name
at least three different methods of selecting representatives.
2. What is the present value of equal representation of the states in the Senate?
What legal and practical obstacles are there to changing this system?
3. Look up and explain the following terms which are commonly used in
Congress: executive session; morning hour; union calendar; ranking member;
filibuster; leave to print; pigeon-holing a bill; pork barrel; rider.
4. What are the practical difficulties which arise when the Senate declines to
confirm appointments proposed by the President?
5. Explain the difference between an impeachment and a bill of attainder.
6. The Senate usually exercises more influence than the House in matters of
lawmaking. Can you give reasons why this should be so?
7. Tell how congressional districts are mapped out. Mark on an outline map the
districts in your state. Have any of them been gerrymandered?
8. The chairmanships of committees usually go to senior members. Do you think
this a wise or unwise practice?
9. What would be gained, and what would be lost by lengthening to four years
the term for which representatives are chosen?
10. Two women, Miss Rankin of Montana and Miss Robertson of Oklahoma,
have sat in Congress. What are the arguments for and against electing women in
future?
11. Members of the House of Representatives receive salaries of $7500 per
year. Is this too much or too little? Give your reasons.
12. Congressmen are entitled to the free use of the mails. (This is called the
franking privilege.) Some years ago one senator sent nearly 750,000 copies of his
speeches through the mails free. Do you believe this privilege should be withdrawn
or retained?
13. Should the rules of the House provide for unlimited debate?
14. Can you suggest any practical way in which the work of Congress might be
improved?
Topics for Debate
1. The English practice of choosing non-resident representatives is
advantageous and should be adopted in the United States.
2. The states should be represented in the Senate according to their respective
populations.
3. The provision relating to a reduction in representation, whenever citizens are
excluded from voting (see Amendment XIV) should be enforced.
CHAPTER XV
THE PRESIDENT AND HIS CABINET
The purpose of this chapter is to explain how the President of the United
States is chosen, what his powers are, and what functions his cabinet
performs.
The President
The Man and the Office.—Forty years ago, The notable
an eminent English writer on American Presidents.
government spoke of the presidency as the greatest secular office in
the world “to which anyone can rise by his own merits”.[122] In view of
this fact, he asked, how does it come that the position is not more
frequently filled by great and striking men? There have been twenty-
nine presidents since the constitution went into force in 1788. Of
these at least three, Washington, Jefferson, and Lincoln have won
an assured place in world history. Five or six (including Adams,
Jackson, Grant, Cleveland, and Roosevelt) displayed during their
respective terms of office some qualities which marked them as men
of uncommon force or ability. Three others are still living and their
achievements cannot yet be fairly estimated. But taking all these
together, and even adding a few more for good measure, would it not
still be a fair statement to say that at least half the presidents have
been men whose names would be entirely forgotten nowadays were
it not for the fact that they occupied the presidential chair?
Alexander Hamilton, Daniel Webster, John Marshall, Henry Clay,
and John C. Calhoun are great and striking figures in American
history although they never reached the presidency; on the other
hand the nation has, at various times, bestowed its highest honor
upon men of commonplace qualities. This, of course, was not what
the Fathers of the Republic expected. It was their anticipation that
the presidential office would always be filled by men of “pre-eminent
ability and virtue”.
Why has this expectation been in part disappointed? That is a
question which can only be answered by a study of the methods by
which presidents are chosen, the relations between the office and
the party system, and the duties that presidents are required to
perform.
How the President is Chosen.—The Articles Why the plan of
of Confederation did not provide for a President; indirect election was
adopted.
executive functions were performed by committees of the Congress.
But this plan was found to be altogether unsatisfactory and the
framers of the constitution decided in 1787 that the new federal
government ought to have a single executive head. How to choose
this head, however, was a problem which gave them great difficulty
and they debated it for a long time. They did not approve a plan of
election by direct popular vote, for they feared that this might result
in the choice of men who were personally popular but had no other
qualifications. Their study of ancient and mediaeval republics made
them averse to choosing the head of the nation by direct popular
vote. They were not prepared to trust the people; in those days the
risk seemed too great. On the other hand they did not desire to have
the President chosen by Congress because this would give
Congress control of the office, whereas their aim was to make the
presidency a check upon Congress. So they finally decided upon the
expedient of direct election by means of an electoral college.[123]
The Original Plan of Election.—Stated The presidential
briefly the plan which they agreed upon and electors.
inserted in the constitution was as follows: Each state shall choose,
in such manner as its legislature may determine, a number of
electors equal to the state’s combined quota of senators and
representatives in Congress. A state having, for example, two
senators and twelve representatives, is entitled to fourteen electors.
On a definite date, once in four years, the electors meet in their
respective states and give in writing their votes for President and
Vice President. These votes are sealed up, sent to Washington,
counted, and announced. This plan did not contemplate that
nominations should be made in advance, or that political parties
should have anything to do with the election, or that the various
states, in choosing their electors, should pledge them to vote for any
particular candidate. It was expected that the electors would meet,
discuss the merits of all the available men for the position, and give
their votes accordingly.
The Actual Methods of Election Today.—At How the plan
the first two elections this plan was followed. worked in the earlier
There were no nominations and no campaign elections.
preceding the election. But at the election of 1796 it was well
understood, even before the electors met, that the contest would be
between John Adams and Thomas Jefferson. And as time went on
the actual practice drifted further away from the original plan of free
choice by unpledged electors. Political parties grew up; the electors
were chosen with the definite understanding that they would vote for
a particular party candidate, and their share in the election became
purely nominal. In 1804 some changes were made in the method of
election but they did not affect the general plan or the current
practice. Gradually the people took into their own hands the function
of choosing the President; everywhere the state legislatures turned
the work of choosing the electors over to them, so that the
presidential elections became, in everything but name and form,
direct elections by the people.[124]
In the choice of a President there are now five Five steps in the
steps, but only two of these are of any practical choosing of a
importance. First, each political party nominates President:
its candidate at a national convention, as already described.[125]
Second, in each state the political parties 1. The nomination
nominate, either by primaries or state of candidates.
conventions, their respective slates or groups of electors. Third, the
voters on election day decide which group of 2. The nomination
electors shall be given the formal function of of electors.
electing the President. This the voters do on the Tuesday following
the first Monday in November every fourth year. 3. The polling.
Each voter marks his ballot for a group of
electors but what he really does is to indicate his preference for one
of the candidates already nominated at the party conventions. This
means, of course, that one or the other group of electors is chosen
as a whole and the state’s vote cast solidly. It rarely happens, for
example, that a state casts ten electoral votes for one candidate and
five for another; if it has fifteen votes they all go to one candidate.
For this reason it sometimes happens that a candidate receives a
majority of the electoral votes although not a majority of the popular
votes, taking the country as a whole. Fourth, the 4. The action of the
electors meet in their respective states and cast electors.
their votes. Fifth, these votes are opened in Washington and counted
in the presence of Congress. Among these five steps the first and
third are the important ones. The last step is 5. Counting the
nothing but a formality unless it appears that no votes.
candidate has received a majority. In case this happens the House of
Representatives proceeds to choose a President from among the
three candidates who have stood highest. In the case of the Vice
President the choice rests with the Senate.[126]
Factors which Influence Presidential The “availability” of
Nominations and Elections.—As matters have candidates.
worked out it is not possible for anyone to be elected President
without first obtaining a nomination from one of the two leading
political parties. The party organizations and the party conventions
are influenced by groups of political leaders and these leaders are
often more interested in a man’s strength as a candidate than in his
personal qualifications for the work which a President has to do. The
consequence is that candidates have sometimes been nominated by
party conventions because they were compromises on whom
opposing factions of the party could agree, or because they could be
counted upon to carry some important state at the polls, or for some
other reason having nothing to do with the executive capacity of the
individual concerned.
A big national convention, comprising more “Dark horses”
than a thousand delegates, cannot be expected
to do its work with calm deliberation or to weigh carefully the
personal qualifications of all those who seek to be nominated. If
there is a prolonged contest between two or three strong candidates,
no one of whom can obtain the requisite number of votes in the
convention, the delegates in their impatience are likely to turn to a
“dark horse”, that is to someone less prominent on whom there is a
chance of agreement.[127] This has often happened.
The real work of nominating candidates is not done on the floor of
the convention. The plans are laid and put into operation by groups
of leaders in private conferences, the delegates following these
leaders when called upon. And the fact that a candidate possesses
“great and striking qualities” does not always commend him to these
party leaders. On most occasions they are likely to prefer a man
who, if elected, will work in harmony with the party organization
rather than take the reins of office wholly into his own hands.[128] By
various combinations of circumstances, therefore, men of mediocre
quality have sometimes been nominated.
Narrowness of the People’s Choice.—A The election may
nomination by one of the two leading parties is turn upon various
in some cases almost equivalent to election. things.
There are times, of course, when the election turns chiefly upon the
merits of the two leading candidates; but more often the result is
determined by other factors entirely. Each candidate embodies the
strength of his party as well as his own, and each political party is for
various reasons stronger in some years than in others. When a party
has been in power for a term of years the people usually grow
disgruntled with its policy and refuse to support the candidate of that
party at the next election no matter how capable he may be. There is
every reason to believe that the Democratic candidate was doomed
to defeat in 1920 no matter who he might have been. When one
political party remains in power for eight or twelve years it makes
many enemies; people find fault on one score or another and decide
that they will vote for a change. Even a strong candidate in such
circumstances has very little hope of winning.
Public opinion is a very fickle thing. It exalts a public man as a
hero today and execrates him tomorrow. It is strong for one policy
this year and often veers around to something quite different a year
or two later. Men are borne into the presidential office on this surging
tide, sometimes without much reference to their individual
qualifications. They are nominated because they are acceptable to
the party leaders, or because they come from some strong and
doubtful state, or because they are agreed upon by compromise, or
for any one of a dozen other reasons. The capacity of the man is not
always, and indeed not usually, the chief factor in determining a
presidential nomination.[129] Under the circumstances the wonder is
that the country has obtained, in the presidential office, such a high
general level of personal capacity and character.
Powers of the President.—The actual Presidential
powers of the President are greater than those powers:
of any other ruler in the world, whether hereditary or elective. He is
the chief engineer of a great mechanism which controls an army,
raises several billion dollars a year in taxes, enforces laws, regulates
commerce, and employs the full time of more than half a million
public officials. Congress makes the laws, it is true; but were it not
for the President and those whom he appoints, the laws would not
be enforced. Congress decides what taxes shall be levied; but the
President and his subordinates collect them. Congress appropriates
money out of the treasury; but the executive branch of the
government, of which the President is the head, spends the money.
The President, in other words, is the nation’s chief executive—he is
charged with the duty of executing the laws. This is a large
responsibility and a good deal of the work is necessarily entrusted to
subordinates whom the President appoints.
The appointing power is, then, an important 1. Appointments.
phase of the President’s authority. He names all
the higher officials of the Government subject to confirmation by the
Senate as has already been explained. He has the power to remove
any national official. In the case of minor officials he may, and usually
does, depend upon the advice of senators or congressmen both as
regards appointments and removals; but in the case of all high
officers these things must have the President’s personal attention.
Naturally they take a great deal of his time.
In relation to Congress the President has the 2. The executive
right to make recommendations and to veto any veto.
measure which he does not approve. These recommendations he
may make either by written message or by appearing before
Congress in person. The veto power places a powerful weapon in
the President’s hands. Every bill or resolution which passes both
Houses of Congress must be laid before the President. If he
approves, he signs it. If not, he is entitled, at any time within ten
days, to return the bill or resolution without his signature, giving his
reasons for the refusal to sign. When the Scope of the veto
President vetoes a measure in this way power.
Congress reconsiders it and a vote is then taken to determine
whether the action of the President shall be sustained or overridden.
If two-thirds of the members present in both the Senate and the
House vote to override the veto, the measure becomes effective; if
less than two-thirds so vote, the measure becomes null.
The “pocket veto”.
But suppose the President neither signs nor vetoes the measure
within ten days after it is sent to him, what then? The constitution
provides that in such case the measure shall become a law. If
Congress adjourns before the ten-day period has expired, however,
the bill does not become a law. It is not necessary for a President to
veto any measure that may come to him during the ten days
immediately preceding the adjournment of Congress. If he does not
approve the measure, he merely withholds his signature and it dies
on his table. This is known as the “pocket veto”.
The veto power has been used very little by Its use and abuse.
some presidents and a great deal by others.
During the first forty years of the Republic only nine bills were
vetoed. But during the past forty years presidential vetoes have been
very common. When a measure has been vetoed there is great
difficulty, as a rule, in obtaining the necessary two-thirds vote to
override the veto; but vetoes, nevertheless, are occasionally
overcome. The use of the veto, although it is an exercise of
executive power, makes the President a vital factor in legislation.
Under ordinary circumstances he can defeat any measure that is not
acceptable to him.[130] There are exceptions to this rule, to be sure,
but it is valid in the main.
Although the power of appointment and the 3. The conduct of
veto power in normal times the two chief foreign relations.
sources of the President’s authority, he has others of considerable
importance. He conducts relations with foreign governments and
negotiates all treaties. Treaties do not become valid, however, until
ratified by the Senate. He decides whether ambassadors and other
diplomats sent to Washington from other countries shall be formally
recognized. He has power to pardon offenders sentenced in the
federal courts. He is commander-in-chief of the military and naval
forces. All these functions are vested in the President by the
constitution and the laws.
Other powers have been acquired by usage, 4. Other powers.
for example, the right to have a large voice in
controlling the policy of the political party to which the President
belongs. The President is a party man, a party leader. He is elected
on a party platform. The people expect the President to carry out the
pledges which this platform contains. To do this the President finds it
necessary at times to take the initiative in securing the passage of
laws by Congress and also to bring influence to bear upon the
members of both Houses. Strictly speaking, the President has no
formal share in the making of the laws; but as a matter of usage he
has a highly-important influence upon legislation.
Succession to the Presidency.—In case the President should
die, or resign, or be removed by impeachment, or be otherwise
incapable of performing his duties, the Vice President succeeds. In
the absence of the Vice President it has been provided by law that
the members of the cabinet, beginning with the Secretary of State,
have the right of succession according to the seniority of their
offices.[131] No President has ever resigned or been removed from
office. On several occasions, however, a Vice President has
succeeded by reason of a President’s death. Some presidents have
been seriously ill during their terms of office, and President Wilson
was absent in France for several months during 1918-1919; but in no
case has the Vice President been called upon to exercise the
presidential functions.
The office of Vice President, apart from the The Vice President.
right of succession which it carries, is not of
much importance. In selecting their candidates for the office the two
leading political parties have usually given very little thought to the
problem of getting the most capable man. By the time the great task
of nominating a candidate for the presidency has been finished, the
delegates are in a mood to get home. They will not spend hours and
days taking ballot after ballot for the second place on the ticket. Apart
from presiding in the Senate the Vice President has no regular
official duties, but there is the ever-present chance that he may have
to step into the chief executive position. For that reason the work of
selecting candidates ought to be done more carefully than has
usually been the case.
The Cabinet
The Cabinet.—The constitution makes no The whole cabinet
definite provision for a cabinet. Its framers system rests on
expected that the President would appoint usage.
subordinates to assist him in the performance of his numerous
functions and they made allusion to these officials; but there was no
anticipation that the officials in charge of the various departments
would be formed into an organized branch of the government. So the
cabinet rests upon usage, not upon the constitution or the laws. The
same is true of the cabinet in England. It has no legal status,
exercises no formal powers, keeps no records, and has no fixed
membership. The prime minister selects, for membership in the
cabinet, whomsoever he pleases, the only restrictions being that
they shall have seats in parliament and that the cabinet as a whole
shall have the support of a majority in the House of Commons. The
President of the United States has an even wider range of choice in
the selection of his cabinet. He is not bound to choose a group of
men who control a majority in either branch of Congress. His cabinet
may be as large or as small as he chooses to make it. By usage,
however, the American cabinet consists of the heads of the national
administrative departments, these departments having been at
various times established by law.[132] There are now ten such
departments and hence ten members of the cabinet. The ten
departments are as follows: State, Treasury, War, Navy, Post-Office,
Interior, Justice, Agriculture, Commerce, and Labor. The head of
each is appointed by the President with the confirmation of the
Senate; but for more than eighty years this confirmation has never
been refused. The heads of departments are responsible to the
President alone and may be dismissed by him at any time. They are
not permitted to have seats in either the Senate or House of
Representatives.
The Functions of the Cabinet.—In The cabinet’s
describing the functions of the cabinet it is functions:
advisable to make, at the outset, a distinction between those duties
which are performed by the cabinet as a whole, and those which
pertain to the members of the cabinet individually, as heads of their
own departments.
The cabinet as a whole has no legal authority. 1. As a body.
[133]
It is merely a group of high officials which the
President calls together once or twice a week to discuss such
matters as he chooses to lay before it, or matters which he permits
individual members to bring up. The President may follow its advice
or he may not. He does not need the approval of the cabinet for any
of his actions. At the same time it has become the custom to consult
the cabinet on practically all important questions of general policy
and to give considerable weight to the cabinet’s advice. How much
this weight will be depends, in large measure, upon the temperament
and attitude of the President himself.[134]
Meetings of the cabinet are not public; no records are kept or
printed. Nobody knows what goes on at the meetings of the cabinet
except those who are present. It is a point of honor among the
members that no one will disclose the proceedings to outsiders.
Thus the cabinet always presents an outward appearance of being
unanimous. If any member cannot work in harmony with the
President or with his fellow-members, he is expected to resign.
More vital than the functions of the cabinet as 2. As individuals.
a whole are those which its members perform,
as individuals, as heads of their departments. Every member of the
cabinet, as has been mentioned, is the head of a department, and as
such is given charge of some branch of the government’s work,
subject at all times, however, to the direction of the President. The
functions of each department are indicated, in a general way, by their
respective titles.[135] These duties are so numerous and so varied
that the various departments are divided into bureaus, each bureau
having charge of a certain division of the work. On all routine matters
the head of the department has practically independent authority, but
questions of general policy and those which affect more than one
department are either discussed at cabinet meetings or taken to the
President for his decision.[136]
Should the Cabinet be Enlarged?—Proposals are now under
consideration for enlarging the cabinet by the creation of a
department of education and a department of public health. It is
contended, and perhaps rightly, that the work of the national
government in these two fields is sufficiently important to warrant
their being placed upon the same footing as agriculture, labor, and
commerce. As an alternative it has been suggested that education
and public health might be combined into a single department of
public welfare; but the objection to this is that the two things have no
close relation to each other. There is a feeling, moreover, that the
cabinet should not be made much larger than it now is. If every
request for the creation of a new department were granted, the
cabinet would soon become too cumbrous for the effective
performance of its advisory functions.
American and English Cabinet Systems Compared.—The
cabinet system in the United States is like that of England in some
respects and different in others. These similarities and contrasts may
be made clear by putting them in parallel columns.
Similarities
1. The American cabinet 1. The English cabinet system

system rests on custom or also rests on usage, having no
usage. basis in the laws of England.
2. Members of the American 2. Members of the English

cabinet are chosen by the cabinet are selected in the
chief executive—the name of the nominal chief
President. executive—the king, by the
actual chief executive—the
prime minister.

cabinet are heads of cabinet are also heads of
departments. departments; but in England
not all heads of departments
become members of the
cabinet.
4. The American cabinet 4. The English cabinet, through

advises the President. the prime minister, advises the
king.
Contrasts

cabinet are not permitted to cabinet must be members of
sit in Congress. parliament.

cabinet are responsible to cabinet are responsible to the
the President only; they do House of Commons and must
not have to resign if they fail resign whenever they lose the
to retain the confidence of support of a majority of that
Congress. chamber.
3. The American cabinet does 3. The English cabinet is the

not prepare business for “great standing committee” of
Congress nor assume any parliament, preparing all
formal initiative in law- important measures for its
making. consideration and assuming a
definite leadership in the
making of laws.
Which is the Better Plan?—The relative Merits and defects
merits of the American and English cabinet of each plan.
systems have been much discussed by writers in both countries. The
American plan enables the executive branch of the government to
retain its independence and thus prevents the lodging of too much
power in the hands of Congress. The English system makes the
House of Commons the supreme governing organ of the realm, with
no legal checks upon its omnipotence. It affords, moreover, a degree

Inflammation and Cancer Methods and Protocols Methods in Molecular Biology 2691 2nd Edition Brendan J. Jenkins (Editor)

Uploaded by

Copyright:

Available Formats

You might also like

Inflammation and Cancer Methods and Protocols Methods in Molecular Biology 2691 2nd Edition Brendan J. Jenkins (Editor)

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Inflammation and Cancer Methods and Protocols Methods in Molecular Biology 2691 2nd Edition Brendan J. Jenkins (Editor)

Uploaded by

Copyright:

Available Formats

Inflammation and Cancer Methods and

Protocols Methods in Molecular

Cancer Cell Biology Methods and Protocols Methods in

Cancer Cell Culture Methods and Protocols Methods in

Cancer Drug Resistance Methods and Protocols Methods in

Ovarian Cancer Methods and Protocols Methods in

Whole-Body Regeneration: Methods and Protocols (Methods

Circadian Regulation Methods and Protocols Methods in

Rhodopsin Methods and Protocols Methods in Molecular

Ferroptosis Methods and Protocols Methods in Molecular

Brendan J. Jenkins Editor

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Clayton, VIC, Australia Brendan J. Jenkins

PART I EXPERIMENTAL MODEL SYSTEMS

1 Identifying Adult Stomach Tissue Stem/Progenitor Cells

PART II EXPERIMENTAL TECHNIQUES

10 Tracking Cardiovascular Comorbidity in Models of Chronic

24 Lipid Nanoparticle-Mediated Delivery of miRNA Mimics

HELEN E. ABUD • Department of Anatomy and Developmental Biology, Monash University,

SÉBASTIEN FAUTEUX-DANIEL • Department of Pathology and Immunology, University of

Discovery Institute, Clayton, VIC, Australia; Cancer Program, Monash Biomedicine

Experimental Model Systems

Identifying Adult Stomach Tissue Stem/Progenitor Cells

Adult tissue stem/progenitor cells maintain organ homeostasis and

Fig. 1 Histology of corpus of the mouse stomach. The corpus epithelium of

differentiation of Iqgap3-expressing cells [3]. On the other hand,

Fig. 2 Genetic construct of mouse models. (a) In the Iqgap3-2A-CreERT2 mouse,

Fig. 3 Schematic showing experimental procedure of Iqgap3 lineage tracing and

KRAS is frequently amplified or mutated in human gastric

Fig. 5 Expression of tdTomato (tdTom), E-cadherin (E-cad), and Ki67 on corpus

Fig. 6 Hematoxylin-eosin staining of the stomach from a representative Iqgap3-

All reagents should be prepared with Milli-Q water.

2.2 Animal 1. 10 mg/mL tamoxifen: Dissolve 100 mg of tamoxifen in 1 mL

2.6 Hematoxylin- 1. Mayer’s Hematoxylin (Lillie’s Modification).

2.7 Microscope 1. Confocal laser scanning microscope (e.g., Zeiss LSM880

3. Fix the washed tissues with 4% PFA in PBS in 50-mL tubes at

3.3 Immuno- 1. Deparaffinize sliced tissues on the glass slides by incubation in

3. Wash the slides three times with wash buffer (PBST) in a

3.4 Hematoxylin- 1. Deparaffinize sliced tissues on glass slides by incubation in

3. Incubate the sliced tissues with 0.001% ammonium hydroxide

1. It is necessary to dissolve tamoxifen in 30 °C pre-warmed

7. Alternatively, TBST (140 mM NaCl, 2.7 mM KCl, 25 mM

12, 20, 21]. In Iqgap3-2A-CreERT2; LSL-KrasG12D mice, a U-

This work is supported by the National Research Foundation Sin-

In Vitro and In Vivo Models for Metastatic Intestinal Tumors

Key words Colon cancer, Organoids, Collagen gel, Metastasis, Imaging

The accumulation of driver gene mutations causes malignant pro-

We previously examined the cancer evolution mechanism based

2.2 Standard Culture 1. CO2 incubator.

4. Phosphate-buffered saline (PBS), pH 7.4, and PBS-EDTA.

2.3 Collagen Gel 1. Auto cell counter (e.g., Bio-Rad).

2.4 Culture Medium 1. Standard culture medium: Advanced Dulbecco’s Modified

2.5 Spleen 1. 27-gauge needle syringe.

2.6 Bioimaging of 1. 15 mg/mL D-luciferin potassium solution dissolved in PBS.

3.1 AKTP Organoid 1. Wash the AKTP 3D organoids or semi-confluent cultured

7. To establish AKTP-derived subclones, dissociate AKTP orga-