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Stem Cells and Cancer 1st Edition Kimberly E. Foreman
Digital Instant Download
Author(s): Kimberly E. Foreman, Paola Rizzo, Clodia Osipo, Lucio Miele
(auth.), Beverly A. Teicher, Rebecca G. Bagley (eds.)
ISBN(s): 9781603279338, 1603279334
Edition: 1
File Details: PDF, 5.99 MB
Year: 2009
Language: english
Cancer Drug Discovery and Development
Series Editor: Beverly A. Teicher, Genzyme Corporation
Framingham, MA, USA

For other titles published in this series, go to

www.springer.com/series/7625
Stem Cells
and Cancer

Edited by

REBECCA G. BAGLEY, MS
Genzyme Corporation,
Framingham, MA, USA

BEVERLY A. TEICHER, PhD

Genzyme Corporation,
Framingham, MA, USA
Editors
Rebecca G. Bagley Beverly A. Teicher
Genzyme Corporation Genzyme Corporation
Framingham, MA Framingham, MA
USA USA

ISBN: 978-1-60327-932-1 e-ISBN: 978-1-60327-933-8

DOI: 10.1007/978-1-60327-933-8
Springer Dordrecht Heidelberg London New York

Library of Congress Control Number: 2008944031

© Humana Press, a part of Springer Science+Business Media, LLC 2009

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA),
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Preface

The recent surge in stem cell research has ignited a field of discovery into many human
diseases including diabetes, neuropathologies, and cancer. Stem cell therapy is a prom-
ising approach to the treatment of many debilitating diseases to replace specific differ-
entiated cells that have been lost or died. Although stem cells may provide therapeutic
benefit under certain conditions, stem cells are often implicated in the initiation, pro-
gression, and therapeutic resistance of malignant disease.
This first edition of Stem Cells and Cancer is intended to give a current perspective
on the role of stem cells in cancer and strategies for novel therapies directed toward
tumor stem cells. Cancer stem cells remain a controversial topic and the criteria that
define cancer stem cells are continuing to evolve. The current cancer stem cell hypoth-
esis is presented in several chapters with distinctions made between the hierarchical and
stochastic models of tumor cell development. “Stemness,” self-renewal, pluripotency,
clonality, and tumorigenicity are important concepts applied toward defining cancer
stem cells. Signaling pathways such as Wnt, Sonic Hedgehog, Notch, and Bmi-1 that
are involved in differentiation, proliferation, and survival are implicated in the malignant
process. Additional chapters address the identification of cancer stem cell populations
through the evaluation of molecular markers such as CD133, CD44, and CD24, for
example, or by Hoechst dye exclusion to recognize “side populations.” Mesenchymal
and hematopoietic stem cells are described as well as mouse models that are employed
to elucidate the properties and functionality of stem cells in cancer and the stem cell
niche. This book encompasses a wide variety of human cancers that include but are not
limited to leukemia, gliomas, breast, and prostate cancers. Resistance to conventional
therapies, genetic vs. epigenetic changes that affect therapeutic response, and strategies
to prevent disease recurrence are challenges that have been incorporated into this volume.
Stem Cells and Cancer represents a compendium of cutting edge research by experts
in the field and will be instrumental in the study of this intriguing line of investigation
for many years to come.
Framingham, MA Rebecca G. Bagley
Beverly A. Teicher

v
Contents

Part I Introduction to Cancer Stem Cells

1 The Cancer Stem Cell Hypothesis .................................................................. 3
Kimberly E. Foreman, Paola Rizzo, Clodia Osipo, and Lucio Miele
2 Tumor Stem Cells and Malignant Cells, One and the Same ........................... 15
Beverly A. Teicher
Part II The Stem Cell Niche
3 Mesenchymal Stem Cells in Tumor Stroma ................................................... 29
Nicholas J. Sullivan and Brett M. Hall
Part III Molecular Pathways and Gene Expression
4 Wnt Signaling in Cancer: From Embryogenesis
to Stem Cell Self-Renewal .............................................................................. 39
Adam Yagui-Beltrán, Biao He, and David M. Jablons
5 PTEN in Hematopoietic and Intestinal Stem Cells and Cancer ...................... 59
Jason T. Ross, David H. Scoville, Xi He, and Linheng Li
6 Transcription Factors in Cancer Stem Cells
of the Hematopoietic Lineage ......................................................................... 75
Steffen Koschmieder and Daniel. G Tenen
7 Stem Cell Chromatin Patterns and DNA Hypermethylation .......................... 85
Joyce E. Ohm and Stephen B. Baylin
8 Plasticity Underlying Multipotent Tumor Stem Cells .................................... 99
Lynne-Marie Postovit, Naira V. Margaryan, Elisabeth A. Seftor,
Luigi Strizzi, Richard E.B. Seftor, and Mary J.C. Hendrix
Part IV Cancer Stem Cells in Solid Tumors
9 Glioma Stem Cells in the Context of Oncogenesis ......................................... 115
Johan Bengzon, Elisabet Englund, Leif G. Salford,
and Xiaolong Fan

vii
viii Contents

10 Mouse Mammary Tumor Virus: Stem Cells and Mammary Cancer .............. 127
Gilbert H. Smith
11 Tumor Dormancy, Metastasis, and Cancer Stem Cells ................................... 141
Alysha K. Croker, Jason L. Townson, Alison L. Allan,
and Ann F. Chambers
12 Cancer Stem Cells: Gastrointestinal Cancers ................................................. 155
Hideshi Ishii, Naotsugu Haraguchi, Keisuke Ieta,
Koshi Mimori, and Masaki Mori
13 Cancer Stem Cells: Hepatocellular Carcinoma............................................... 165
Thomas Shupe and Bryon E. Petersen
14 Cancer Stem Cells: Lung Cancer .................................................................... 177
Jaclyn Y. Hung
15 Cancer Stem Cells: Pancreatic Cancer ............................................................ 185
Joseph Dosch, Cheong Jun Lee, and Diane M. Simeone
16 Prostate Stem Cells and Cancer in Animals ................................................... 199
Alexander Yu. Nikitin, Melia G. Nafus, Zongxiang Zhou,
Chun-Peng Liao, and Pradip Roy-Burman
17 Prostate Cancer Stem/Progenitor Cells ........................................................... 217
Sofia Honorio, Hangwen Li, and Dean G. Tang
18 Adult Prostate Epithelium Renewal, Stem Cells and Cancer ......................... 231
Chiara Grisanzio and Sabina Signoretti
19 Stem Cells, Angiogenesis, and Neurogenesis in Tumors ............................... 247
Judith A. Varner
Part V Targeting Cancer Stem Cells with Therapy
20 Implications of Cancer Stem Cells for Cancer Therapy ................................. 255
Liang Cheng, Shaobo Zhang, Darrell D. Davidson,
Rodolfo Montironi, and Antonio Lopez-Beltran

21 Targeting Leukemic Stem Cells ...................................................................... 263

Angelika M. Burger
22 Targeting Brain Cancer Stem Cells in the Clinic ............................................ 275
Gentao Liu, Keith L. Black, and John S. Yu
23 Critical Roles of Tumorigenic and Migrating Cancer
Stem/Progenitor Cells in Cancer Progression and their
Therapeutic Implications ................................................................................ 287
Murielle Mimeault and Surinder K. Batra
Contents ix

24 Therapeutic Index and the Cancer Stem Cell Paradigm ................................. 309
Vera S. Donnenberg and Albert D. Donnenberg

Index........................................................................................................................ 327
Contributors

Alison L. Allan, Ph.D. • London Regional Cancer Program, London, ON, Canada
Department of Anatomy and Cell Biology, University of Western Ontario, London,
ON, Canada; Department of Oncology, University of Western Ontario, London,
ON, Canada
Rebecca G. Bagley, M.S. • Genzyme Corporation, Framingham, MA, USA
Surinder K. Batra, Ph.D. • Department of Biochemistry & Molecular Biology,
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska
Medical Center, Omaha, NE, USA
Stephen B. Baylin, MD • Cancer Biology Division, The Sidney Kimmel
Comprehensive Cancer Center, The Johns Hopkins University Medical Institutions,
Baltimore, MD, USA
Johan Bengzon, M.D., Ph.D. • Division of Neurosurgery, Rausing Laboratory,
Department of Clinical Sciences, University Hospital, Lund, Sweden
Lung Strategic Research Center for Stem Cell Biology and Cell Therapy,
University Hospital, Lund, Sweden; Department of Pathology, Division of Neuropa-
thology, University Hospital, Lund, Sweden
Keith L. Black, M.D. • Department of Neurosurgery, Cedars-Sinai Medical Center,
Los Angeles, CA, USA
Angelika M. Burger, Ph.D. • Department of Pharmacology, Wayne State
University, Karmanos Cancer Institute, Hudson-Webber Cancer Research Center,
Detroit, MI, USA
Ann F. Chambers, Ph.D. • London Regional Cancer Program, London, ON,
Canada; Department of Oncology, University of Western Ontario, London, ON,
Canada; Department of Medical Biophysics, University of Western Ontario,
London, ON, Canada
Liang Cheng, M.D. • Department of Pathology and Urology, Indiana School
of Medicine, Indianapolis, IN, USA; School of Medicine, Polytechnic University
of the Marche Region, United Hospitals, Ancona, Italy; Department of Surgery,
Cordoba University School of Medicine, Cordoba, Spain
Alysha K. Croker, B.Sc. • London Regional Cancer Program, London, ON,
Canada; Department of Anatomy and Cell Biology, University of Western
Ontario, London, ON, Canada; Department of Oncology, University of Western

xi
xii Contributors

Ontario, London, ON, Canada; Department of Medical Biophysics,

University of Western Ontario, London, ON, Canada
Darrel Davidson, M.D., Ph.D. • Department of Pathology and Urology,
Indiana School of Medicine, Indianapolis, IN, USA
Albert D. Donnenberg, Ph.D. • Hillman Cancer Center, Research Pavillon,
Pittsburgh, PA, USA; Division of Hematology/Oncology, Department of Medicine,
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Vera S. Donnenberg, Ph.D. • Hillman Cancer Center, Research Pavillon,
Pittsburgh, PA, USA; Department of Surgery, Heart, Lung, and Esophageal Surgery
Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Division
of Hematology/Oncology, Department of Medicine, University of Pittsburgh School
of Medicine, Pittsburgh, PA, USA
Joseph Dosch, B.S. • University of Michigan Medical Center, Ann Arbor, MI, USA
Elisabet Englund, M.D., Ph.D. • Department of Pathology, Division of
Neuropathology, University Hospital, Lund, Sweden
Xiaolong Fan, M.D., Ph.D. • Division of Neurosurgery, Rausing Laboratory,
Department of Clinical Sciences, University Hospital, Lund, Sweden; Lung Strategic
Research Center for Stem Cell Biology and Cell Therapy, University Hospital, Lund,
Sweden
Kimberly E. Foreman, Ph.D. • Breast Cancer Research Program, Cardinal
Bernardin Cancer Center, Loyola University Chicago, Maywood, IL, USA
Chiara Grisanzio, M.D. • Department of Pathology, Brigham and Women’s
Hospital, Department of Medical Oncology, Harvard Medical School, Boston,
MA, USA
Brett M. Hall, Ph.D. • Integrated Biomedical Science Graduate Program,
The Ohio State University, Columbus, OH, USA; Center for Childhood Cancer,
The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
Naotsugu Haraguchi, M.D., Ph.D. • Department of Gastroenterological Surgery,
Graduate School of Medicine, Osaka University, Osaka, Japan
Biao He, Ph.D. • Department of Surgery, University of California San Francisco
Helen Diller Family Comprehensive Cancer Center, University of California San
Francisco, San Francisco, CA, USA
Xi He, M.D. • Stowers Institute for Medical Research, Kansas City, MO, USA
Mary J.C. Hendrix, Ph.D. • Children’s Memorial Research Center, Chicago,
IL, USA
Sofia Honorio, Ph.D. • Department of Carcinogenesis, Science Park Research
Division, University of Texas M.D. Anderson Cancer Center, Smithville, TX, USA;
Division of Nutritional Science, Department of Human Ecology, University of Texas
at Austin, Austin, TX, USA; Program in Molecular Carcinogenesis, Graduate School
of Biomedical Sciences, Houston, TX, USA
Contributors xiii

Jaclyn Y. Hung, Ph.D. • Greehey Children’s Cancer Research Institute,

The University of Texas Health Science Center, San Antonio, TX, USA
Keisukke Ieta, M.D. • Department of Surgery, Kyushu University, Medical Institute
of Bioregulation, Beppu, Japan
Hideshi Ishii, M.D., Ph.D. • Department of Gastroenterological Surgery,
Osaka University, Graduate School of Medicine, Osaka, Japan; Department of
Surgery, Kyushu University, Medical Institute of Bioregulation, Beppu, Japan
David M. Jablons, M.D. • Department of Surgery, University of California San
Francisco Cancer Centre, San Francisco, CA, USA
Steffen Koschmieder, M.D. • Department of Medicine, Hematology and Oncology,
University of Munster, Munster, Germany
Cheong Jun Lee, M.D. • University of Michigan Medical Center, Ann Arbor,
MI, USA
Hangwen Li • Department of Carcinogenesis, Science Park Research Division,
University of Texas M.D. Anderson Cancer Center, Smithville, TX, USA; Division
of Nutritional Science, Department of Human Ecology, University of Texas at Austin,
Austin, TX, USA
Linheng Li, Ph.D. • Stowers Institute for Medical Research, Kansas City, MO, USA;
Department of Pathology and Laboratory Medicine, University of Kansas Medical
Center, Kansas City, KS, USA
Chun-Peng Liao, Ph.D. • Department of Pathology, Department of Biochemistry
and Molecular Biology, Keck School of Medicine, University of Southern California,
Los Angeles, CA, USA
Gentao Liu, Ph.D. • Department of Neurosurgery, Cedars-Sinai Medical Center,
Los Angeles, CA, USA; ImmunoCellular Therapeutics, Woodland Hills, CA, USA
Antonio Lopez-Beltra, M.D., Ph.D. • Department of Surgery, Cordoba University
School of Medicine, Cordoba, Spain
Naira V. Margaryan, D.V.M., Ph.D. • Children’s Memorial Research Center,
Chicago, IL, USA
Lucio Miele, M.D., Ph.D. • Breast Cancer Research Program, Cardinal Bernardin
Cancer Center, Loyola University Chicago, Maywood, IL, USA
Murielle Mimeault, Ph.D. • Department of Biochemistry & Molecular Biology,
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska
Medical Center, Omaha, NE, USA
Koshi Mimori, M.D., Ph.D. • Department of Surgery, Kyushu University, Medical
Institute of Bioregulation, Beppu, Japan
Rodolfo Monitroni, M.D. • School of Medicine, Polytechnic University
of the Marche Region, United Hospitals, Ancona, Italy
xiv Contributors

Masaki Mori, M.D., Ph.D., F.A.C.S. • Department of Gastroenterological Surgery,

Graduate School of Medicine, Osaka University, Osaka, Japan; Department of
Surgery, Kyushu University, Medical Institute of Bioregulation, Beppu, Japan
Melia G. Nafus, B.S. • Department of Biomedical Sciences, Cornell University,
Ithaca, NY, USA
Alexander Yu Nikitin, M.D., Ph.D. • Department of Biomedical Sciences,
Cornell University, Ithaca, NY, USA
Joyce E. Ohm, Ph.D. • Cancer Biology Division, The Sidney Kimmel Comprehensive
Cancer Center, The Johns Hopkins University Medical Institutions, Baltimore, MD,
USA
Clodia Osipo, Ph.D. • Breast Cancer Research Program, Cardinal Bernardin
Cancer Center, Loyola University Chicago, Maywood, IL, USA
Bryon E. Petersen, Ph.D. • Department of Pathology/Immunology & Medicine,
University of Florida College of Medicine, Gainesville, FL, USA
Lynne-Marie Postovit, Ph.D. • Children’s Memorial Research Center, Chicago, IL,
USA; Schulich School of Medicine and Dentistry, University of Western Ontario, Lon-
don, ON, Canada
Paola Rizzo, Ph.D. • Breast Cancer Research Program, Cardinal Bernardin Cancer
Center, Loyola University Chicago, Maywood, IL, USA
Jason T. Ross, B.S. • Stowers Institute for Medical Research, Kansas City, MO, USA;
Department of Pathology and Laboratory Medicine, University of Kansas Medical
Center, Kansas City, KS, USA
Pradip Roy-Burman, Ph.D. • Department of Pathology, Department of Biochemistry
and Molecular Biology, Keck School of Medicine, University of Southern California,
Los Angeles, CA, USA
Leif G. Salford, M.D., Ph.D. • Division of Neurosurgery, Rausing Laboratory,
Department of Clinical Sciences, University Hospital, Lund, Sweden
David H. Scoville, B.S. • Stowers Institute for Medical Research, Kansas City,
MO, USA; Department of Pathology and Laboratory Medicine, University of Kansas
Medical Center, Kansas City, KS, USA
Elisabeth A. Seftor, B.S. • Children’s Memorial Research Center, Chicago,
IL, USA
Richard E.B. Seftor, Ph.D. • Children’s Memorial Research Center, Chicago,
IL, USA
Tom Shupe, Ph.D. • Department of Pathology/Immunology & Medicine,
University of Florida College of Medicine, Gainesville, FL, USA
Sabina Signoretti, M.D. • Department of Pathology, Brigham and Women’s
Hospital, Department of Medical Oncology, Harvard Medical School, Boston,
MA, USA
Contributors xv

Diane Simeone, M.D. • University of Michigan Medical Center, Ann Arbor,

MI, USA
Gilbert H. Smith, PhD • Mammary Biology & Tumorigenesis Laboratory,
Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA
Luigi Strizzi, M.D., Ph.D. • Children’s Memorial Research Center, Chicago,
IL, USA
Nicholas J. Sullivan, B.S. • Integrated Biomedical Science Graduate Program,
The Ohio State University, Columbus, OH, USA; Center for Childhood Cancer,
The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
Dean G. Tang, M.D., Ph.D. • Department of Carcinogenesis, Science Park Research
Division, University of Texas M.D. Anderson Cancer Center, Smithville, TX, USA;
Program in Molecular Carcinogenesis, Graduate School of Biomedical Sciences,
Houston, TX, USA
Beverly A. Teicher, Ph.D. • Genzyme Corporation, Framingham, MA, USA
Daniel G. Tenen, M.D. • Center for Life Sciences and Harvard Stem Cell Institute,
Harvard Medical School, Boston, MA 02115, USA
Jason L. Townson, B.Sc. • London Regional Cancer Program, London, ON,
Canada; Department of Medical Biophysics, University of Western Ontario,
London, ON, Canada
Judith A. Varner, Ph.D. • Moores University of California San Diego
Cancer Center, La Jolla, CA, USA
Adam Yagui-Beltran, M.D. • Department of Surgery, University of California San
Francisco Helen Diller Family Comprehensive Cancer Center, University of Califor-
nia San Francisco, San Francisco, CA, USA; Department of Surgery, University of
California San Francisco Cancer Centre, San Francisco, CA, USA
John S. Yu, M.D. • Department of Neurosurgery, Cedars-Sinai Medical Center,
Los Angeles, CA, USA; ImmunoCellular Therapeutics, Woodland Hills, CA, USA
Shaobo Zhang, M.D. • Department of Pathology and Urology, Indiana School
of Medicine, Indianapolis, IN, USA
Zongxiang Zhou, Ph.D. • Department of Biomedical Sciences,
Cornell University, Ithaca, NY, USA
I Introduction to Cancer Stem Cells
 1 The Cancer Stem Cell Hypothesis

Kimberly E. Foreman, Paola Rizzo,

Clodia Osipo, and Lucio Miele

Abstract
The “cancer stem cell” hypothesis is receiving increasing interest and has become the object of considerable
debate among cancer biologists and clinicians. This ongoing debate is focusing attention on the very
definition of stemness and its significance in the context of a malignancy. From a therapeutic standpoint,
the cancer stem cell hypothesis emphasizes the cellular heterogeneity in cancers, and the need to specifi-
cally target small cell populations that resemble tissue stem cells and are phenotypically different from the
majority of cancer cells. Regardless of their origin, these cells divide slowly, have the ability to undergo
asymmetric cell division and are highly resistant to conventional chemotherapeutics. These characteristics
make them prime suspects as potential causes of disease recurrence and metastasis, which are the main
causes of morbidity and mortality in oncology. This chapter provides an introduction to the cancer stem
cell hypothesis, briefly summarizes the evidence supporting this theory and the aspects that remain contro-
versial. Finally, we present a brief discussion of the possible therapeutic significance of cancer stem cells
and the current efforts to target developmental pathways on which these cells depend.

Key Words: Cancer stem cells, Tumor-initiating cells, Stem cell niche, Targeted therapies

THE CANCER STEM CELL MODEL OF CARCINOGENESIS

For decades, the prevailing theory of cancer initiation and progression has been that cancers derive
from the serial acquisition of genetic mutations by normal somatic cells. These mutations resulted in
enhanced proliferation, inhibition of differentiation, and reduced capacity to undergo apoptosis. Each
mutation would result in progressive “dedifferentiation” so that the tumor cells would continually lose
their mature, tissue-specific attributes, and regress to a more primitive phenotype. As differentiated
cells have limited life spans, it would be difficult for any given cell to acquire all the mutations neces-
sary to become transformed, thus explaining the relatively uncommon occurrence of transformation.
However, if initial mutations led to unrestrained proliferation, this would generate more cells that could
potentially be affected by further oncogenic mutations. Once transformed, cancer cells would prolifer-
ate indefinitely and form a tumor where each viable tumor cell was in principle equally capable of
forming a new tumor.
Recent findings suggest that this model may be overly simplistic. The “cancer stem cell hypothe-
sis” has gained considerable interest in recent years (1–3). This theory states that cells in a tumor are
organized as a hierarchy similar to that of normal tissues, and are maintained by a small subset of

From: Cancer Drug Discovery and Development: Stem Cells and Cancer,
Edited by: R.G. Bagley and B.A. Teicher, DOI: 10.1007/978-1-60327-933-8_1,
© Humana Press, a part of Springer Science + Business Media, LLC 2009
3
4 Part I / Introduction to Cancer Stem Cells

“Differentiation”
Self-replication
Proliferation

Asymmetric
Cell
Division
CSC

“Progenitor”
CSC

“Progenitor”

Bulk
Cancer cells

Fig. 1. The CSC hypothesis. CSCs are thought to maintain their numbers by slow self-replication, and produce other
tumor cells by asymmetric cell division. In this process, cell division of a CSC generates a CSC and a transformed
“progenitor-like” cell, which has limited self-renewal ability but are highly proliferative, similar to a transit-amplifying
population in normal tissue. These progenitors give rise to more or less partially differentiated bulk tumor cells through
a combination of proliferation and abortive differentiation.

tumor cells that are ultimately responsible for tumor formation and growth. These cells, defined as
“cancer stem cells” (CSCs) or tumor initiating cells (TICs), possess several key properties of normal
tissue stem cells including self-renewal (i.e., the ability of a cell to renew itself indefinitely in an
undifferentiated state), unlimited proliferative potential, infrequent or slow replication, resistance to toxic
xenobiotics, high DNA repair capacity, and the ability to give rise to daughter cells that differentiate.
However, unlike highly regulated tissue stem cells, CSCs demonstrate dysregulated self-renewal/dif-
ferentiation programs and produce daughter cells that arrest at various stages of differentiation. The
daughter cells make up the bulk of the tumor and are characterized by rapid replication, limited pro-
liferative potential, and the inability to form a new tumor. Only the CSC is able to initiate tumor
formation as it is solely capable of self-renewal. A diagrammatic representation of the CSC hypoth-
esis is shown in Fig. 1.
The strongest evidence for the CSC theory comes from studies in acute myelogenous leukemia
(AML). Landmark studies by Dick and colleagues demonstrated that only rare cells in AML were able
to initiate leukemia in murine models, and serial transplantation studies revealed these cells had a high
self-renewal capacity (4, 5). The cell responsible for tumor initiation was identified as having a
CD34+CD38− phenotype, which was particularly interesting as bulk AML samples tend to be CD34−.
Furthermore, CD34+CD38− is a phenotype characteristic of normal hematopoietic stem cells (HSC)
indicating the putative CSCs may have a primitive phenotype. Bonnet et al. found that as few as 5 ×
103 CD34+CD38− cells could engraft an immunocompromised mouse, while 100 times more CD34− or
CD34+CD38+ cells from the same donor could not (5). Importantly, the tumors derived from injection of
the CD34+CD38− cells was heterogeneous and composed of a mixture of tumorigenic and nontumori-
genic cells similar to the donor sample (5). Since then, stem-like cells have been identified in a variety
Chapter 1 / The Cancer Stem Cell Hypothesis 5

of human malignancies including other leukemias and solid tumors such as breast, colon, brain, head
and neck, lung, pancreatic, nasopharyngeal cancers, and melanomas (4–18). In many cases, a tumori-
genic subset of cells could be reproducibly identified and isolated based on a distinct set of cell mark-
ers separating it from the nontumorigenic subset (19). Attempts to isolate CSCs from other malignancies
are underway in laboratories worldwide, and this list is likely to grow. Remarkably, even established
cancer cell lines that have been grown in vitro for many years appear to contain CSC-like populations
that can be isolated and are highly tumorigenic (20, 21). The surface markers of CSCs from different
tumor types are diverse, suggesting that their biological behaviors may be different as well.
One reason the CSC theory has generated such enthusiasm is that it may help explain long-standing
problems in cancer biology. It is well-recognized that tumors are heterogeneous in terms of both
functional heterogeneity and cellular composition. Functional heterogeneity refers to the observation
that only a small portion of tumor cells can give rise to colonies in clonogenic assays in vitro or
tumors in vivo. Under the traditional theory of tumor formation (also called the stochastic model),
every tumor cell should be equally capable of forming a tumor. As tens to hundreds of thousands of
tumor cells are needed to reproducibly initiate tumors in animal models, investigators concluded that
the process was inefficient. However, with the CSC theory, the number of cells needed to form a
tumor would simply be determined by the relative frequency of CSC in the tumor population. A suf-
ficient number of CSCs must be present in the inoculum, since most cells in the line are proliferative
but nontumorigenic. The phenotypic heterogeneity of tumors is also more easily explained by the CSC
theory. Mutations in the CSC would be passed on to each daughter cell, and as the daughter cell dif-
ferentiates, it may arrest at any one of numerous points prior to full maturation. In the stochastic
model, the tumor cell would need to dedifferentiate to different degrees to form a phenotypically
heterogeneous but genetically clonal population. Although the genomic instability associated with
cancer clearly makes this possible, it is easier to envision an abortive version of the normal hierarchi-
cal differentiation program in a tissue as opposed to a random back-differentiation process affecting
individual cells to different extents.
It has also been postulated that the CSC theory may explain why it is so difficult to treat cancer. If
this model is correct, then directing cancer therapeutics at the bulk of rapidly replicating tumor cells
is not likely to achieve tumor eradication, unless the CSCs are eliminated. This could explain the vex-
ing problem faced by oncologists worldwide, who often can achieve complete clinical and pathologi-
cal remissions of cancers with chemotherapy, only to see the cancers recur, often in metastatic and
ultimately lethal forms. This clinical phenomenon implies that very small numbers of cells, some-
times undetectable even by sophisticated molecular diagnostic tools, are capable of causing tumor
relapses. Standard chemotherapeutic strategies using mitotic poisons, DNA-damaging agents, antime-
tabolites, or even modern “targeted” agents such as growth factor receptor kinase inhibitors often are
aimed at actively proliferating cells resulting in growth arrest and/or cell death. This strategy effi-
ciently kills the daughter cells, but is much less effective against CSCs, which can remain quiescent
for extended periods of time. Thus, tumors may shrink in response to traditional chemotherapy, even
to the point where they are undetectable, yet CSCs often persist and eventually cause relapsed and
metastatic disease (2). Furthermore, when CSCs are exposed to and escape from chemotherapy-in-
duced death, they may become more resistant to these insults and pass this on to their daughter cells.
This may explain why recurrent cancers are often more resistant to treatment than primary disease.
An additional characteristic of CSCs that makes them more difficult to eradicate than “bulk” cancer
cells is their high level expression of ABC family transporters, which catalyze the ATP-dependent
transport of toxic chemicals from the cell (22). These molecules were originally identified as one of
the main cause of multidrug resistance in cancers (23). Evolutionarily, it is plausible that normal tissue
stem cells would be particularly well protected against toxic insults, because of their fundamental role
6 Part I / Introduction to Cancer Stem Cells

in tissue regeneration. Unfortunately, this property also makes the neoplastic counterparts of tissue
stem cells highly resistant to many common chemotherapeutic drugs. Indeed, one of the most popular
ways of isolating putative CSC population takes advantage of their ability to rapidly efflux DNA-
binding fluorescent dyes such as Hoechst or 7-AAD, which is due to high level expression of ABC
transporters. Cells that retain less dye appear as a “side population” (SP) in flow cytometry experi-
ments. In several cases, SP cells have been shown to be enriched in putative CSCs (24–26).

WHERE DO CANCER STEM CELLS COME FROM?

While the CSC theory has offered possible new explanations for several key aspects of tumor biol-
ogy, it has also raised new questions. Perhaps one of the most interesting, and yet difficult to answer,
is what is the origin of the CSC? The answer to this question depends on our understanding of the
stem cell differentiation process in normal tissues. If tissue stem cell differentiation is a “one way
only” process, and partially differentiated cells cannot return to a “stem-like” program even when
transformed, then the most obvious candidate precursor of the CSC is the tissue-specific stem cell that
normally functions to replace dead and injured cells in tissues. Several points support this possibility.
First, normal stem cells are already capable of indefinite self-renewal and generate more differentiated
progenitors, most likely through asymmetric cell division. Even slightly more differentiated progeni-
tor cells would have lost this ability and would have to reacquire self-renewal through mutations – a
potentially complicated process. Second, tissue stem cells are long-lived and would be capable of
accumulating the serial mutations necessary for transformation over the lifetime of the cell. Acquisition
of multiple mutations would be more difficult for a short-lived cell. Finally, CSCs isolated from
tumors tend to possess a primitive phenotype. As already mentioned, the putative CSC in AML has a
CD34+CD38− phenotype, which is the same as the HSC, while more differentiated cells (CD34+CD38+)
could not initiate tumor formation (4, 5). Similarly, CSCs derived from various primary tumors or
cultured cell lines routinely express other markers of normal tissue stem cells including CD133,
nestin, c-kit, sox2, oct4, and musashi-1 (7, 8, 11, 27, 28). Clearly, it is simpler to conclude that CSCs
derived from stem cells continue to express stem cell markers than to envision a more mature cell
specifically regaining the ability to express these markers as a consequence of a random dedifferentia-
tion event.
Nevertheless, formal proof that CSCs can only derive from normal tissue stem cells has yet to be
obtained. At least theoretically, it is conceivable that the process of transformation puts a strong selec-
tive pressure on differentiated cells so that only cells that undergo the epigenetic changes necessary
to restore “stemness” are capable of surviving transformation. In this model, reversion to a stem-like
state is part of the transformation process. This is essentially a modified restatement of classical trans-
formation models in which loss of differentiation results from a process of selection in a population
of genomically unstable cells.
The feasibility of cloning organisms from somatic cell nuclei shows that under some circumstances
the nucleus of a somatic cell can be reprogrammed all the way back to totipotency, generating a viable
embryo and a complete organism. In fact, the recent demonstration that cells equivalent to human
embryonic stem cells can be obtained from normal fibroblasts by transduction of specific factors
supports the hypothesis that achieving stemness through dedifferentiation is possible, at least under
some circumstances. Yu et al. recently showed that expression of oct4, sox2, nanog, and LIN28 in
human dermal fibroblasts converts them into pluripotent cells with a phenotype virtually indistin-
guishable from embryonic stem cells (29). In another report, Takahashi et al. (30) showed that expres-
sion of Oct3/4, sox2, Klf4, and c-Myc can achieve the same result. The fact that the protooncogene
c-Myc can be part of the reprogramming mix of genes supports the idea that under some conditions,
Chapter 1 / The Cancer Stem Cell Hypothesis 7

the transformation process could reprogram a cell to a stem-like phenotype. It is important to note that
these studies were conducted in fibroblasts and not epithelial cells. Can a similar process of reprogram-
ming occur in common epithelial malignancies? A process of partial dedifferentiation has been known
for years in epithelial cancers as epithelial-mesenchymal transition (EMT) (31–34). This consists in
loss of epithelial markers, such as tissue-specific cytokeratins, and adhesion molecules, such as
E-cadherin, and acquisition of markers typical of mesenchymal cells, such as vimentin and N-cadherin.
The process of EMT is thought to contribute to the ability of transformed epithelial cells to metasta-
size. In this model, cancer cells need to undergo EMT to migrate through the body, and once they seed
distant metastatic sites, they can revert to a more or less “epithelial” phenotype through a process of
mesenchymal-epithelial transition (MET). Several transcription factors such as Twist, Snail, or Slug
and secretory proteins of the TGF-β family, including some bone morphogenetic proteins (BMPs), can
induce the EMT program (34). Vascular mimicry is thought to be a specialized form of EMT in which
tumor cells can acquire an endothelial phenotype (35, 36).
Thus, the question seems to be not whether or not differentiation plasticity is possible in epithelial
cancer cells, but whether this process can go as far as generating a cell that has the functional
characteristics of a stem cell. What a simple dedifferentiation model does not immediately explain is
the hierarchical organization of cells in malignancies. If dedifferentiation is a secondary event that
arises through selection and confers a selective advantage to less differentiated cells, why is there a
hierarchical organization among neoplastic cells with a highly tumorigenic, dededifferentiated popula-
tion capable of generating less tumorigenic, more differentiated cells? One possible explanation is that
dedifferentiation is a highly improbable event, which produces a cell fate program that includes func-
tional “stemness.” Thus, only a few cells or even a single cell would have to undergo this process to
generate a small population of CSCs. These then give rise to the rest of the cancer cell population
through a process of hierarchical abortive differentiation that imperfectly recapitulates that of a
normal tissue.
An intermediate possibility is that the CSC could originate not exclusively from tissue stem cells,
but from a restricted number of cell populations including tissue stem cells and immature progenitor
cells, which are immediately below tissue stem cells in the differentiation hierarchy and are capable
of short-term self-replication. Experimental support for this hypothesis comes from several studies in
leukemia where the introduction of oncogenic fusion gene products into hematopoietic progenitor cells
resulted in AML in animal models. Cozzio et al. found that expression of the MLL-ENL fusion gene
product in hematopoietic progenitor cells resulted in leukemia, albeit with less efficiency than when it
was expressed in true hematopoietic stem cells (37). Similar results were also found with the MOZ-
TIF2 fusion gene product (38). More recently, Somervaille and Cleary enforced MLL-AF9 expression
in normal murine HSC and progenitor cells (39). Using serial transplantation in mice, they discovered
that the functional CSC expressed MAC-1 and Gr1, two markers associated with more mature cells
(39). Interestingly, the cells also expressed the stem cell marker c-kit, suggesting CSCs may express an
unusual combination of cell markers (39). Taken together, these studies clearly support the notion that
AML may arise from either stem or progenitor cells in a mouse model; however, caution should be
used in interpreting this data. Murine cells are generally easier to transform than human cells; hence,
it is unclear if these findings are relevant to human disease (40). A similar theory has been proposed
for breast cancer (41, 42). According to Dontu et al. (41), the existence of ERa-negative breast cancers
and ERa-positive breast cancers of variable biological aggressiveness may be explained by postulating
that CSCs in these cancers originate from different cell populations. The most aggressive, undifferenti-
ated ERa-negative cancers and poor-prognosis ERa-positive cancers would arise from the most primi-
tive mammary stem cells, which are ERa-negative, while less aggressive ERa-positive cancers would
arise from CSCs derived from intermediate progenitors that are ERa-positive. These can generate
8 Part I / Introduction to Cancer Stem Cells

Differentiation
Self-replication

Proliferation

Asymmetric
Cell
TSC
Division

TSC Progenitor

1 2

“De - differentiation”
CSC CSC

Fig. 2. Possible origins of CSCs. Three different but not mutually exclusive models are schematically presented.
“Lightning” symbols indicate transforming mutations. CSCs may originate exclusively from the transformation of
primitive tissue stem cells (TSC, model 1), or from the transformation of either TSC or progenitor cells (model 2).
Alternatively, CSCs may originate from the transformation and dedifferentiation of more mature cells, which reac-
quire stem cell properties as a consequence of transforming mutations (model 3) (see Color Plates).

ERa-negative, rapidly proliferating “transit-amplifying” cells. This is a conceptually plausible model.

However, experimentally it is difficult to distinguish it from a scenario in which all breast cancer arise
from primitive, ERa-negative mammary stem cells, which lose their differentiation ability to variable
degrees depending on the transforming mutations they undergo. Figure 2 represents three different,
nonmutually exclusive models for the origin of CSCs.

OPEN QUESTIONS: LIMITATIONS OF THE EXPERIMENTAL EVIDENCE

An important issue that remains to be addressed is that almost all of the experimental evidence for
the cancer stem cell model comes from studies in which human CSCs are transplanted into immuno-
compromised mice (43, 44). Thus, a possible objection to the model is that selection protocols for
CSCs could simply identify cells that are more adept at forming tumors in the xenogeneic microenvi-
ronment of an immunocompromised mouse. Given the limitations of current experimental models,
this cannot be ruled out. However, if CSCs are essentially an artifact of xenograft models, it is not
clear why human cancer cell lines of diverse tissue origins that have been grown in vitro for decades
retain cell populations that exhibit stem-like characteristics very similar to CSCs isolated from primary
tumors including increased expression of ABC transporters and asymmetric cell division, and are
highly tumorigenic in mice. Specifically, it is not clear what selection pressure could explain the
remarkable persistence of these CSC-like populations outside of the mouse microenvironment, if they
Chapter 1 / The Cancer Stem Cell Hypothesis 9

are not necessary for continued in vitro propagation of the cell line. Strasser and colleagues have
proposed that the reason so many human cancer cells are needed to initiate tumor formation is that
the murine microenvironment is not appropriate for development of human cancers, and only a few
cells are capable of overcoming this hostile environment (43). These authors have taken the approach
of genetically engineered mouse cells to develop lymphoma (primary Em-myc lymphomas), isolating
subpopulations of the tumor cells based on the murine stem cell markers Sca-1 and AA4.1 (CD93),
and examining tumor formation in syngeneic naïve, immunocompetent mice (43). They report iden-
tify a small subpopulation (2–5%) of cells with stem-like characteristics, but found that Sca-1+AA4.1hi
and Sca-1+AA4.1lo cells were equally capable of forming tumors (43). These data have been inter-
preted as evidence against the universal validity of the cancer stem cell model. It should be pointed
out that although xenograft models are certainly artificial, transgenic mouse models of carcinogenesis
have important limitations of their own, and may or may not faithfully recapitulate human carcino-
genesis. Typically, in these models a very potent oncogene is overexpressed in a target cell population,
and the whole process of carcinogenesis and tumor progression is dramatically accelerated compared
with human disease. Mouse cells are far more susceptible to transformation than human cells, and
may be able to more easily reacquire functional “stemness.” It is interesting to notice that the oncogene
used in this particular experimental model, c-Myc, is also one of the stemness-inducing genes that can
reprogram human fibroblasts to an embryonic stem cell-like phenotype. Thus, an alternate explana-
tion for these data is that both Sca-1+AA4.1hi and Sca-1+AA4.1lo cells in this transgenic model have
acquired functional “stemness” through a process of dedifferentiation, and can behave as CSCs. More
sophisticated animal models will be required to gain further insights into this issue. These models
should be based on human cells, but attempt to recapitulate as much as possible the human microen-
vironment. Such a humanized xenograft model has been generated for the mammary gland (45), and
should provide valuable information on putative breast cancer stem cells.
The controversy on the human relevance of CSC data obtained in xenograft models underscores
the importance of tumor microenvironment in the biology of CSCs. Tumor-stroma interactions may
indeed be critical in reprogramming cancer cell developmental pathways. Transforming growth factor
(TGF)-b and bone morphogenetic proteins (BMPs) can be produced by tumor stroma, as can several
other mediators of intercellular communication such as Wnt, Hedgehog, and Notch ligands. There is
growing interest in studying the CSC “niche” as a potential therapeutic target. Normal stem cells are
well known to require signals from their immediate environment, including stromal cells, microvas-
cular endothelial cells, and extracellular matrix for their long-term survival and self-renewal. This
specialized microenvironment is commonly referred to as the stem cell niche, and it is best understood
in the hematopoietic system (46). There is increasing evidence that CSCs also require microenviron-
mental signals from specialized niches (47–49) (Fig. 3). Autocrine and paracrine mediators secreted
by the CSCs themselves or by other tumor cells may also play an important role, at least in some
malignancies (50, 51). How much autocrine or paracrine interactions contribute to the CSC niche is
still unclear. However, at least under some circumstances CSCs can recreate a niche-like environment
in the absence of other cell types. Putative breast cancer stem cells can form spheroids called “mam-
mospheres” in suspension culture (52, 53). Other putative CSCs can also form similar spheroids.
Mammospheres contain few CSCs, and mostly consist of precursors and partially differentiated cells.
Mammospheres can propagate in vitro and form secondary and tertiary mammospheres, which retain
the original cellular composition. This implies that at least under some culture conditions, the CSCs
themselves and their immediate progeny can form a functional niche that is capable of sustaining
self-renewal, asymmetric cell division and partial differentiation.
Undoubtedly, much remains to be clarified and further studies are needed. These may well reveal
that the origin of the functional CSC may vary based on the cell type involved and the specific nature
of the oncogenic events leading to transformation.
10 Part I / Introduction to Cancer Stem Cells

Endothelial
PG cells

SC CSC

ECM

Fig. 3. The CSC “niche”. In vivo, CSCs may require signals from their microenvironment to maintain their properties,
as is the case for normal tissue stem cells. Microenvironmental signals may be received from endothelial cells, from
various types of stromal cells (SC), such as fibroblasts, bone marrow stromal cells, or immunocytes infiltrating the
tumor, from progenitor cells (PG) derived from the CSCs themselves, and/or from the extracellular matrix (ECM). It
is likely that the cross-talk between CSCs and other cells is bidirectional. These signals may be therapeutically tar-
geted to deprive CSCs of indispensable microenvironmental signals.

CLINICAL IMPLICATIONS: TARGETING CANCER STEM CELLS

FOR THERAPY
Regardless of the origin of CSCs, perhaps the most important aspect of the cancer stem cell model
is that it has drawn increasing attention to the hierarchical organization of malignancies. Cancers have
been known for decades to be heterogeneous, but until recently the idea that the most abundant cancer
cells derive from a much smaller and often elusive pool of stem-like cells was commonly accepted only
in the field of leukemia. This model appears to apply to many solid tumors, and the list is growing by
the day. Whatever their genesis, if human cancers do contain a small population of cells that proliferate
slowly, are highly resistant to current chemotherapeutic regimens and could cause disease recurrence
and metastasis, eradication of these cells may be necessary to achieve a long-lasting remission or cure.
Thus, new therapies targeting the CSC must be developed if we hope to prevent or eliminate recurrent
and metastatic disease. It has been proposed that identification of signaling pathways that are involved
in self-renewal and are deregulated in CSCs may be an effective approach for novel target discovery (2).
Alternatively, the identification of proteins expressed preferentially by CSCs, such as CD96 in leukemia,
could provide targets for antibody-based therapies or to modulate cell signaling and promote differen-
tiation (54). Yet another possibility is disrupting the interactions between CSCs and their niche
(48, 49). Several signaling pathways have been identified as playing critical roles in stem cell self-renewal
including among others Notch, Wnt, and Hedgehog (55). These pathways are evolutionarily ancient
and have fundamental roles during development, when they control multiple cell fate decisions. They
are primarily used for short-range intercellular communication utilizing secreted factors such as
Hedgehog (56) or the Wnts (57, 58) or cell membrane-associated ligands such as Notch ligands Jagged
and Delta (59). Importantly, these pathways are involved in several of the phenomena we described
above, from EMT (60) to CSC-niche communication (46, 49, 51). Drugs that inhibit Notch signaling
are in early clinical development and others are in the pipeline (61), and Hedgehog inhibitors are not
Chapter 1 / The Cancer Stem Cell Hypothesis 11

far behind. Interest in using Notch inhibitors to target CSCs is growing. In glioblastomas, elevated
Notch expression has been associated with high nestin levels and is linked to a poor prognosis (62, 63).
Furthermore, Notch inhibition reduced the ability of brain CSCs to form tumors (64). In breast cancer,
Notch expression and activation has been associated with a poor prognosis, and studies indicate that
Notch inhibitors can kill breast cancer cells in vitro (65–67). As CSCs have been identified in primary
breast cancers, there has been much interest in Notch signaling in breast CSCs (6). Farnie et al. recently
compared mammospheres derived from normal mammary tissue and human ductal carcinoma in situ
(DCIS) and reported that activated Notch-1, Notch-4, and the downstream target Hes-1 were expressed
in mammospheres from DCIS samples, but not those derived from normal breast tissue (68). Notch
inhibition with a g-secretase inhibitor or a neutralizing Notch-4 antibody significantly reduced the ability
of DCIS derived cells to form mammospheres (68). These results suggest that Notch inhibition may be
able to preferentially target breast CSCs, while sparing normal mammary stem cells. Laboratories
around the world, including ours, are exploring the development of therapeutic regimens including
Notch, Hedgehog, or Wnt inhibitors to target CSCs. Figure 4 shows a simplified representation of
pathways that have been associated with CSC maintenance.

Notch
ligands

Hh Wnt

BMPs ECM

TGF-β CD133 Met GFs

DNA repair

ABC
xenobiotics Slow replication
Self-renewal
Pluripotency
Bmi-1
Musashi
Sox2
Oct4

Fig. 4. Molecular pathways affecting CSCs. The figure shows a list, not meant to be all-inclusive, of pathways that have
been shown to modulate the CSC phenotype. Extracellular signals delivered through the Hedgehog (Hh), Notch, Wnt
pathways or through TGF-b and the related BMPs, or from ECM proteins and from growth factors such as hepatocyte
growth factor (Met ligand) may all participate in regulating the maintenance, self-renewal, and differentiation of CSCs.
Slow replication, ability to generate partially differentiated progenies (pluripotency) highly effective DNA repair, abil-
ity to eliminate xenobiotics through ABC family transporters (ABC), and expression of primitive membrane markers
(CD133, Met) have been documented in many putative CSC populations isolated from tumors or cell lines. Transcription
factors such as Bmi-1, Musashi, Sox2, Oct4, and others have been shown to be commonly expressed in putative CSCs
and participate in controlling their phenotype.
12 Part I / Introduction to Cancer Stem Cells

CONCLUSIONS
The CSC hypothesis has sparked a tremendous increase in scientific interest in the hierarchical
organization of cancer cells, the isolation of rare cellular subpopulations that may be responsible for
treatment failures, and the role of microenvironmental niches in the maintenance of these populations.
There are still many questions that remain unanswered, particularly surrounding the origin of CSC
populations in human tumors and the interpretation of data generated by current experimental models.
Yet, looking at cancers from the perspective offered by the CSC hypothesis may answer fundamental
questions in tumor biology and open the way to paradigm shifts in our therapeutic approach to malig-
nancies. Thus, it is reasonable to take the view that studying the mechanisms regulating the survival,
self-renewal, and differentiation of normal and transformed stem cells could potentially lead to tre-
mendous advances in the treatment of neoplastic diseases.

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 2 Tumor Stem Cells and Malignant Cells,
One and the Same

Beverly A. Teicher

Abstract
Cancer is a proliferative, invasive, and metastatic disease often caused by repeated tissue insults result-
ing in accumulation of genetic abnormalities that rarely produce malignant cells. The survival of mouse
L1210 leukemia was determined for inoculations of 1 cell up to 106 cells. The survival times varied in
a log-linear manner with the inoculum cell number from 19 days with 1 cell to 7 days with 106 cells
implanted. In preclinical tumor models or in patients, tumor nodules of 108–109 cells are advanced can-
cer. Malignant cells frequently secrete growth modulatory substances that regulate their growth and alter
growth of normal cells. Whether the metastatic malignant cell is the same or significantly different from
the primary lesion malignant cell remains a topic of active investigation. Reaching a detectable lesion takes
10 years. Genetic instability produces variants in the primary tumor and metastases that are more hetero-
geneous than the early disease. The argument that cancer arises only from the tissue stem cell populations
and that cancer stem cells comprise perhaps 1 in 100,000 or 1 in 10,000 cells within the tumor leads to
the notion that agents that selectively kill cancer stem cells will not decrease the tumor mass. The cells
that initiate, sustain, and populate cancers are malignant cells. Cancer stem cell notion is useful if it leads
to important research questions and to better therapeutics.

Key Words: Colony forming units, Malignant cells, Genetic instability, Metastasis, L1210 leukemia

INTRODUCTION
Cancer is a proliferative, invasive, and metastatic disease that is frequently caused by repeated
insults to a tissue resulting in accumulation of genetic abnormalities that, by rare chance, produce a
malignant cell. Cancer cells are genetically aberrant and instable. Some cancers begin as a single
clone (and a few remain clonal) and other arise from a field of repeatedly damaged cells. The search
for an understanding of cancer and for the key as to how to control and ablate malignant disease often
returns to the remarkable processes of normal tissue/embryo development and normal tissue repair.
The “well-behaved” proliferative and self-limiting biology of wound repair, gut lining replacement,
liver regeneration, skin renewal, and bone marrow generation of hematopoietic cells has taught us that
cell proliferation and differentiation are a constant process in complex organisms and are well-con-
trolled under normal circumstances.
The concept of a stem cell was put forth by Till and McCulloch to describe the ability of a single
mouse bone marrow cell to produce a colony of cells in the mouse spleen and later to describe the

From: Cancer Drug Discovery and Development: Stem Cells and Cancer,
Edited by: R.G. Bagley and B.A. Teicher, DOI: 10.1007/978-1-60327-933-8_2,
© Humana Press, a part of Springer Science + Business Media, LLC 2009
15
16 Part I / Introduction to Cancer Stem Cells

ability of similar single bone marrow cells to give to colonies of varied types in cell culture (1, 2). A
colony-forming unit (CFU) is an individual cell that is able to clone itself into a colony of identical
cells. A CFU is a measure of viable bacterial numbers or a measure of viable mammalian malignant
cells in a culture. In reconstituting the immune system of lethally irradiated mice, bone marrow cells
from syngeneic donors are intravenously injected into the recipient animals and colonies form in the
spleen. Each colony is the progeny of a pluripotent stem cell; therefore, the number of colonies is a
measure of the number of stem cells. These findings led to the notion that cancer can arise from mul-
tiply insulted cells that by rare chance have aberrantly turned on genes that normally are expressed only
by normal tissue “stem” cells. Thus, cancer cells have aberrantly reverted to a dedifferentiated prolif-
erative state. These malignant cells are trying to build a tissue but they are abnormal and lethal.
Indeed, an area of therapeutic investigation has a goal to discover agents that can terminally dif-
ferentiate malignant cells to a quiescent nonproliferative state.

EARLY OBSERVATIONS
An interesting aspect of the current cancer stem cell debate regards the number of human tumor
cells required to initiate the growth of a subcutaneous nodule in immunodeficient mice. A very large
number of variables would need to be optimized to achieve reliable data from such observations. A
historical perspective looking at syngeneic mouse tumors may help. The L1210 and P388 mouse
leukemias were developed in 1948 and 1955, respectively (3–5). L1210 and P388 leukemias were
both chemically induced in a DBA/2 mouse by painting the skin with methylcholanthrene. The leuke-
mias have been propagated in DBA/2 mice by implanting intraperitoneally 0.1 mL of a diluted ascetic
fluid containing either 105 L1210 cells or 106 P388 cells. These mouse leukemias were the first tumors
used for large-scale drug discovery screening programs by the national drug development program
instituted in 1954 by Congress, which directed the National Cancer Institute to start a program. The
Cancer Chemotherapy National Service Center (CCNSC) screen consisted of three mouse tumors:
L1201 leukemia, SA-180 sarcoma, and mammary adenocarcinoma 755 (6). Over the years, the pri-
mary screen varied from the original three tumors to L1210 plus two arbitrarily selected tumors to
L1210 plus Walker 256 carcinosarcoma to L1210 plus P388 leukemia to L1210 plus B16 melanoma
or Lewis lung carcinoma. In 1976, a change occurred in the NCI primary screen. The new screen
included a panel of colon, breast, and lung tumor models (mouse and human); however, compounds
were initially screened in P388 leukemia (7).
Skipper and Schabel and colleagues explored the growth characteristics of the L1210 and P388 leuke-
mias in mice (8, 9). The testing was conducted in a hybrid of DBA/2 hosts. Tumor cell implant sites
were intraperitoneal injection, subcutaneous implant, intravenous injection, and intracranial injection.
For L1210 leukemia with an inoculum of 105 cells, the mean days of survival and tumor cell doubling
times for these implant sites were 8.8, 9.9, 6.4, and 7.0 days and 0.34, 0.46, 0.45, and 0.37 days, respec-
tively (Fig. 1). The mean survival times of mice implanted with L1210 cells by these various routes was
determined for inoculations of 1 cell up to 106 cells. The survival times varied in a log-linear manner
with the inoculum cell number. Thus, when the mice were implanted with 1 L1210 cell by intraperito-
neal injection, they survived 19 days and when the mice were implanted with 106 L1210 cells intraperi-
toneally, they survived 7 days (Fig. 1). Similar studies were conducted with P388 leukemia. For P388
leukemia (106 cells), the mean days of survival and the tumor doubling times for the same implant sites
were 10.3, 13.0, 8.0, and 8.0 days and 0.44, 0.52, 0.68, and 0.63 days, respectively. From these studies,
it must be concluded that every L1210 and P388 cell is a cancer stem cell.
Skipper and Schabel applied similar analyses to solid tumors especially the mouse Ridgway osteo-
genic sarcoma (10). In preclinical tumor models or in patients, tumor nodules of 108–109 cells are
Chapter 2 / Tumor Stem Cells and Malignant Cells, One and the Same 17

1e+6

NUMBER OF L1210 LEUKEMIA CELLS IMPLANTED

1e+5

1e+4

1e+3

1e+2

1e+1 IP IMPLANT
IV IMPLANT
IC IMPLANT

1e+0
0 2 4 6 8 10 12 14 16 18 20

MEAN SURVIVAL TIME, Days

Fig. 1. Mean survival times of mice inoculated with various numbers of murine L1210 leukemia cells injected intra-
peritoneally, intravenously, or intracranially. These data form the basis for the in vivo bioassay method for determin-
ing the number of L1210 cells surviving after treatment of L1210 tumor-bearing mice with therapy. From these
survival curves, it was determined that from: (1) intraperitoneal inoculation of L1210 cell-generation time = 0.55
days; the lethal number of L1210 cells = 1.5 × 109; (2) intravenous inoculation the L1210 cell-generation time = 0.43
days; and (3) intracranial inoculation the L1210 cell-generation time = 0.46 days (8,9).

advanced cancer (Fig. 2) (10, 11). One source of variability in the response of drug-sensitive tumor
cells to a drug is the heterogeneity of the blood supply such that the drug does not reach the tumor
cells distal from the blood supply in sufficient concentration to be lethal. Thus, the pharmacokinetics
and concentration of a drug required to kill tumor cells distal from vasculature should be documented.
In addition, the physiologic heterogeneity of tumor masses as a source of varied treatment response,
Skipper and Schabel considered the heterogeneity of tumor stem cells, defined as cells capable of unlim-
ited proliferative thrust, caused by the inherent genetic instability of malignant cells to be a source of variable
treatment response. Skipper and Schabel considered various types of tumor stem cells that might account
for fluctuation in response to chemotherapy in similarly treated individuals bearing a specific cancer, and
classifications of cancers by chemotherapeutic effect. Fluctuating ratios of treatment responsive to treat-
ment resistant stem cells, as predicted by the mutation theory, could account for one patient responding
to a drug and the next not responding. Differences in tumor growth fraction and differences in tumor
distribution into pharmacologic sanctuaries could also strongly influence a patient’s response to therapy.
Treatment resistant stem cells are primarily responsible for the failure of the best available chemotherapy
to cure responsive, refractory, and very refractory experimental neoplasms. These data examined suggest
that differences in the resistant to responsive stem cell ratios in different types of cancer may account for
their being classified as responsive, refractory, or very refractory (12).
18 Part I / Introduction to Cancer Stem Cells

1.00E+14
Tumor Mass
1.00E+13
1012 = 1 kg
1.00E+12
1.00E+11
1010 = 10 g
1.00E+10
Number of Tumor Cells
109 = 1 g
1.00E+09
1.00E+08 108 = 130 mg

1.00E+07
106 = 1 mg
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
0 5 10 15 20 25 30 35 40 45 50
Number of Population Doublings

Fig. 2. Tumor cell numbers and weight of the tumor mass are shown. In patients, tumors are advanced at first
presentation or at recurrence after initial noncurative therapy (10,11).

TUMOR CELL HETEROGENEITY

Experience with the heterogeneous response of well-controlled preclinical tumor models grown in
inbred strain of mice led investigators in the mid-1980s to believe that the assumption that there
should be a common pattern of cellular heterogeneity for histologically identical types of cancer was
not warranted (13). Although malignant disease may develop from a single transformed cell, even in
tumors where the single cell has diversified to heterogeneous cell phenotypes, evidence of a clonal
origin still exists (14). Although Foulds concluded that tumor evolution (progression) is characterized by
permanent, irreversible changes, we recognize today that cell remain very plastic and adaptable and
can often modulate their biology to changes in the microenvironment (15). During molecular progres-
sion of tumors, neoplastic cells accumulate increasing genetic alterations that are generated by muta-
tional events, genetic instability (16, 17). Tumor cell genetic instability ensures that malignant disease
contains heterogeneous, phenotypically diverse tumor subpopulations (14). Tumor cell diversification
mechanisms may be similar or identical to normal development during embryonic and postembryonic
diversification and development. Tumor cell subpopulations can influence the properties of other
subpopulations in the tumor including proliferation, sensitivity to drugs, immunogenicity, and meta-
static potential (14, 18–20).
Understanding the biology of malignant cells (cancer stem cells) that allows them to escape the
constraints that normally regulate cell growth and differentiate is critical. Malignant cells frequently
secrete growth modulatory substances that regulate their own growth (autocrine) and/or alter the
growth of normal cells (paracrine) in the vicinity of the malignancy (Fig. 3) (21). A malignant tumor
whose growth depends upon the release of autocrine and paracrine growth factors may be vulnerable
to treatment with specific receptor antagonists or growth factor neutralizing antibodies.
Chapter 2 / Tumor Stem Cells and Malignant Cells, One and the Same 19

Autocrine Factors

Paracrine Factors

Malignant Cell
Cancer Stem Cell

Blood Vessel
Cells mobilized
from distal sites

Vascular Cells

Stromal Cells

Immune System Cells

Fig. 3. Malignant cells can have abscopal effects on the host through secretion of paracrine factors and can produced
autocrine factors that can sustain proliferation of the malignancy.

HEMATOPOIESIS AS A MODEL
Analogies for the development of malignancy have been sought in the processes of normal aging
and in the differentiation of cells in the hematopoietic system (22, 23). The incidence of many cancers
increase with age because of increased probability of DNA changes that may allow occurrence of a
malignant cell and because some of the alterations associated with normal aging increase the suscep-
tibility of cells to carcinogenic events. In normal aging, there is a decrease in DNA repair capacity
and a decline in cellular immune reactivity that could contribute to permitting malignant growth (22).
Normal hematopoiesis, the formation of the many cell types in blood, is a process of development,
self-renewal through mitosis, and differentiation of hematopoietic stem cells, the source cell of all
blood cell lineages (24). Because most blood cells have relatively short lifespans, hematopoietic stem
cells continuously replicate themselves through self-renewal to prevent depletion of the stem cell
pool while simultaneously differentiating into multiple lineages of the varied blood cell types. The
fate choice of hematopoietic cells to either self-renew or differentiate is controlled by intrinsic mecha-
nisms and extrinsic signals from the environment or the stem cell niche (25). In adults, the hematopoi-
etic stem cell number is relatively constant under normal conditions. Bone marrow hematopoietic
stem cells appear quiescent; however, the majority divide regularly as shown by their slow constant
incorporation of radio-labeled nucleotides (26, 27). There are two proposed mechanisms by which
asymmetric cell division may be achieved called divisional asymmetry and environmental asymmetry.
In divisional asymmetry, specific cell fate determinants in the genome, RNA, and proteins are distrib-
uted unequally during cell division. After cell division, only one daughter cell receives the deter-
minants, thus retaining the hematopoietic stem cell fate while the other daughter differentiates.
20 Part I / Introduction to Cancer Stem Cells

In environmental asymmetry, one hematopoietic stem cell niche and retains the stem cell identity,
while the other enters a different environment favoring its differentiation (24).
The first human leukemia-lymphoma cell lines were Burkitt’s lymphoma lines developed in 1963
(28). The most widely used of these lines is the Raji Burkitt’s lymphoma (29). The term “cell line”
indicates that the population of cells grew continuously in culture (to this day); therefore, the cells were
“immortal” or capable of continuous self-renewal. Some the leukemia-lymphoma cell lines were pre-
sumed to be monoclonal that is derived from one malignant cell and that the current multiclonal lines
emerged during extended culture. The differentiation of the cells arrested at a discrete stage during
maturation of the lineage. The early cell lines were grown autonomously in basic nutrient media sup-
plemented with fetal calf (or other serum) serum independent of external growth factors, although, in
some cases, exogenously added growth factors could stimulate proliferation. Later cell lines were
selected to be growth factor dependent (30). Nearly all of the established leukemia and lymphoma cell
lines are genetically abnormal. Of 429 lines analyzed only two had normal karyotypes (31). In addition
to gross cytogenetic alterations, many of the lines harbor point mutations deletions and amplifications
of specific genes (32). However, despite the evidence of genetic instability and abnormality, these cell
lines have remained “stable” in long-term culture for nearly 50 years.
An interesting case is the impact of stem cell “dose” on hematopoietic recovery in autologous
blood stem cell recipients (23). Hematopoietic cells collected from blood and reinfused into patients
following high-dose chemotherapy are generally termed “stem cells,” even though the population of
cells contains true stem cells and differentiated progenitor cells (33, 34). Mobilized blood stem cell
quantity, identified by expression of CD34, may be the strongest predictor of days to hematopoietic
recovery (i.e., platelets and neutrophils) in autologous blood stem cell recipients (35). The majority
of patients will recover if a stem cell dose of ³5 × 106/kg stem cells are infused, which corresponds
to 3.5 × 108 cells (36). Bone marrow engraftment is the result of early undifferentiated stem and pro-
genitor cell self-renewal and differentiation. The rate of engraftment is dependent, in part, on the
number and type of stem cells infused. Using a genetically engineered model cell to represent human
leukemia stem cells, Hope et al. tracked these human leukemia stem cells in SCID mice and found
that the leukemia stem cells were not functionally homogeneous (37). Like normal hematopoietic
stem cells, some human leukemia stem cells divided rarely and underwent self-renewal rather than
commitment after cell division and others demonstrated heterogeneity in self-renewal potential.

METASTASIS
The detection of circulating tumor cells in the blood and lymph system and micrometastases has
been investigated through multiple eras of cancer research because of the potential importance of
these cells to prognosis and therapeutic approaches (38). The first publication of circulating tumor
cells was in 1869 when Ashworth reported cancer cells in the blood of a patient at autopsy (39). In
the decade 1955–1965, 5,000 cancer patients were tested for circulating tumor cells using 20 different
cytological methods; however, it was finally realized that the tests used were not sufficiently discrimi-
natory (40). When immunohistochemical detection methods were developed and more recently with
the development of PCR techniques, interest in documenting a connection between circulating tumor
cell numbers and stage of disease and in detecting occult metastases renewed (41–43). Although the
accuracy of methods for detecting tumor cells in circulation has markedly improved over the years,
the application of these data to determination of the most appropriate treatment regimen for individual
patients has yet to occur. It is not clear whether circulating tumor cells are enriched for cancer “stem
cells,” malignant cells that can initiate metastasis or whether circulating tumor cells reflect the quality
of the primary lesion vasculature or other properties of the malignant disease (44, 45). There is no
doubt that malignant cells must be plastic and adaptive to altering environments and stress. The capacity
Chapter 2 / Tumor Stem Cells and Malignant Cells, One and the Same 21

Clonal Selection

Parallel Evolution

Dynamic
Heterogeneity

Clonal Dominance

Stem Cell

Fig. 4. The various patterns of cell division that can lead to the cellular heterogeneity found in most malignant disease.

of some malignant cells to make epithelial to mesenchymal transition and then return to an epithelial
phenotype is an example of the plasticity of malignant cells (44). During wound healing processes
normal cells can undergo these changes as well.
Metastasis is a critical characteristic of malignant disease (45) (Fig. 4). The placement of a cancer stem
cell in the metastatic process and understanding whether the metastatic malignant cell is the same or
significantly different from the malignant cell of the primary lesion remains a topic of active investigation.
The clonal selection hypothesis is that cell populations with metastatic capacity are subpopulations within
the primary lesion. The parallel evolution model theorizes that metastasis occurs early in malignant dis-
ease and evolves along a different path than the primary lesion. The dynamic heterogeneity hypothesis
indicates that metastatic variants arise within the primary tumor, and these variants spread and evolve
further at secondary sites. The notion of clonal dominance is that more virulent metastatic subclones
occur within the primary tumor and outgrow the original malignant cells and eventually dominate the
primary lesion and metastases. The stem cell model indicates that only cancer stem cells and not the
majority population of the tumor have the capacity to metastasize and establish distant lesions (46–48).
Gatenby and Gillies proposed a model for the somatic evolution of invasive cancer as overcoming
a series of barriers to proliferation (49). Tumor development and the genotypic and phenotypic hetero-
geneity of cancer cell populations is described using an equivalence principle such that multiple
alterations in the cell may allow to successfully adapt to and overcome a critical barrier moving
toward malignancy. During tumor initiation, progression and metastasis, approximately 30 generation
22 Part I / Introduction to Cancer Stem Cells

Metastasis Metastasis Metastases

Primary
Tumor

Doublings 0 3 30 40
Tumor cells 1 8 109 1012
Time 0 1yr 10yr 13yr

Metastases
Metastasis Primary
Tumor

1 cm tumor

Fig. 5. Two possible patterns of malignant tumor growth and spread of metastatic disease over time and malignant
cell doublings is shown.

times or about 10 years are required for a single malignant cell to produce a palpable mass of about
1 cm3 composed of about 109 tumor cells (45) (Fig. 5). Over the course of these 10 years to reach a
clinically detectable lesion, genetic instability produces variants in the primary tumor and metastases
that are more heterogeneous than the early disease. A lethal tumor burden of 1012 tumor cells could
be expected after 10 cell doublings or 3 years from detection of the disease without treatment.

CARCINOGENESIS
Chemical carcinogen-induced rat mammary tumors provided an in vivo model for the early cancer
stem cell hypothesis (50). In the rat mammary gland, epithelial stem cells insulted by a chemical
targeting DNA can give rise to more-rapidly proliferating benign stem cells, which retain some
response to normal growth-controlling factors. These altered but benign stem cells can also differenti-
ate to a degree. The genetic instability of these cells results in the generation of malignant cells in
subsequent generations with loss of response to growth-controlling factors, decreased capacity for
differentiation and escape from immune surveillance. The cells which then compose the malignant
disease have departed in critical ways from the normal epithelial stem cells of origin and continue to
suffer from genetic instability and to evolve.
Colony formation is a demonstration of self-renewal capacity. It was noted early that colony-
forming human epidermal cells were heterogeneous in capacity for sustained growth and that the
potential for continued growth from a single cell could be predicted from the phenotype of the colony
produced (51). Three types of colonies were described: (1) holocolonies characterized as large with a
smooth perimeter containing mainly small cells that may be considered the most undifferentiated
population; (2) paracolonies characterized as being small with a highly irregular perimeter containing
cells that are large and flattened and appear terminally differentiated; and (3) merocolonies characterized
Chapter 2 / Tumor Stem Cells and Malignant Cells, One and the Same 23

by medium to large size and a wrinkled perimeter with heterogeneous cells. In the case of normal cells,
the transitions from holoclone (stem-like) to meroclone to paraclone (terminally differentiated) are
thought to be unidirectional and result in progressively restricted growth potential. However, malig-
nant cells have increased plasticity and may dedifferentiate toward holoclone states or abnormally
differentiate forming giant multinucleated cells that no longer proliferate. Locke et al. studied seven
well-established human cancer cell lines from varied histology and found that cell lines generated
from carcinomas produced cell culture colony patterns similar to those produced by the stem and
amplifying cells of normal epithelia as described earlier (52). The cancer cell lines appeared to main-
tain a subpopulation of “stem cells” during passage of the line. The heterogeneity of the colonies
produced from the cancer cell lines indicates that the stem cell property of asymmetrical division
persists in cancer cell lines but is shifted toward stem cell self-renewal. Thus, malignant cells attempt
to recapitulate the behavior of normal tissue cells; however, their behavior is aberrant and detrimental.
Malignant cells are damaged cells that have dedifferentiated, returned to the behavior of embryonic
cells rather than dying or being recognized as abnormal by the immune system.
The skin epidermis is a normal tissue that is continuously confronted with physiochemical traumas
from the environment (53). The skin undergoes continual rejuvenation through homeostasis and
is primed to undergo wound repair in response to injury. The epidermis has tissue “stem cells,”
which both self-perpetuate and give rise to the differentiating cells that constitute the tissue. The
TGFb pathway is involved in the control of proliferation of many cells. The loss of response to TGFb
proliferation control is a necessary step in malignant transformation (54–56). In the absence of
TGFb, proliferation control, squamous cell carcinomas spontaneously develop in mice (57). These
tumors have many of the characteristics of invasive squamous cell carcinomas.
Although some may argue that the embryological, stem-like properties of tumors and tumor cell
lines indicate that the malignant cell originates only from tissue stem cells, this argument seems
unnecessary to explain the dedifferentiated properties of the malignant cell (58). The argument that
cancer arises only from the stem cell populations of tumor that comprise perhaps 1 in 100,000 or 1 in
10,000 cells within the tumor leads to the notion that treatment agents that selectively kill the cancer
stem cell will not result in decrease in the tumor mass. Classic studies and recent studies show that
with some tumor cell lines inoculation of mice with few cells from 1 to 10 can produce tumors that
are lethal to the host (59). Most transplantable tumor models are carried out with the implant for
105–107 tumor cells primarily due to the impatience of the experimentalist, who would like to see a
palpable nodule in 1 or 2 weeks and not in several months or years. Generally, if a tumor nodule is
not palpable in 2 or 3 weeks, the tumor is considered a no take. The timeline for these studies is that
of the experimentalist not reflective of the nature of cell proliferation. Although it appears that only a
minute proportion of human tumor cells can grow readily and rapidly in mice, syngeneic tumor mod-
els indicate that inoculums of many fewer cells that divide quite rapidly in the murine host yield lethal
malignant disease in a short time (60).

CONCLUSIONS
In most incarnations, the cancer stem cell is very similar to a normal tissue stem cell. The cancer
stem hypothesis has been advanced to describe how a cancer originates, how it is sustained, and what
makes it drug resistant. Among the properties of tissue stem cells in the adult are the relative quies-
cence manifested by infrequent cell divisions, resistance to drugs/toxins mediated by ABC transport-
ers, active DNA repair pathways and resistance to apoptosis. The cancer stem cell has been defined
as a cell involved in malignant disease process that possess the capacity to self-renew and give rise to
the heterogeneous lineages of cancer cells that comprise the tumor. Whether the malignant cells with
24 Part I / Introduction to Cancer Stem Cells

long term self-renewal capacity that sustain the cancer arose from tissue stem cells that acquired
mutations leading to a differentiation block and malignancy or whether more differentiated cells
acquired mutations that dedifferentiated them and resulted in a malignant cell capable of self-renewal
has not been determined. Thus, the use of the term “stem cell” may not be accurate. The cells that
initiate, sustain, and populate cancers are malignant cells (61, 62). The notion of the cancer stem cell
is useful, if it leads important research questions being addressed and to better therapeutics being
developed. The self-renewal, pluripotency, and drug resistance of malignant cells is a product of their
abnormality and genetic instability.

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96–105.
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II The Stem Cell Niche
 3 Mesenchymal Stem Cells in Tumor
Stroma

Nicholas J. Sullivan and Brett M. Hall

Abstract
Mesenchymal stem cells (MSC) are defined, minimally, as cells that display a fibroblastic morphology in
cell culture, exhibit a robust self-renewal capacity, and retain the ability to undergo trilineage differentia-
tion into adipocytes, chondrocytes, and osteoblasts. MSC can be isolated from diverse tissues but are most
commonly isolated from red bone marrow. Accumulating evidence suggests that bone marrow MSC can
be mobilized into the periphery to serve as regenerative stem cells at sites of injury and inflammation.
Although the in vivo biology of MSC is poorly understood, several studies have demonstrated that MSC
can be selectively recruited into tumors. Following engraftment within tumor stroma, MSC proliferate
and acquire an activated phenotype similar tumor-associated fibroblasts (TAF). Tumor-homing properties
of MSC have lead to their utility as therapeutic cell-based antitumor protein delivery vehicles. However,
with a greater appreciation for the influential role that the tumor microenvironment can serve during tumor
initiation, promotion, and progression, MSC may enhance tumor progression following acquisition of
TAF-like characteristics. A more comprehensive delineation of the biological role of MSC within tumor
stroma will improve our understanding pf tumor-stroma interactions and facilitate future development of
MSC-based clinical therapies.

Key Words: Mesenchymal stem cell (MSC), Tumor microenvironment, Tumor-associated fibroblast
(TAF), Stromagenesis, Myofibroblast, Desmoplasia, Tumor stroma

INTRODUCTION
Carcinomas are solid tumors that arise from malignant epithelial cells and represent the most com-
mon type of cancer in humans. However, it is becoming increasingly clear that malignant cells are
supported by nonepithelial tumor stroma that shape a given tumor microenvironment. Tumor stroma
can be generally divided into four main components: (1) tumor vasculature, (2) inflammatory immune
cells, (3) extracellular matrix (ECM)/soluble growth factors, and (4) tumor-associated fibroblasts
(TAF) (Fig. 1). Bidirectional paracrine communications between connective tissue fibroblasts and
epithelial cells are vital for normal tissue homeostasis, and a comparable but progressive intimacy
continues throughout tumor development. Although mesenchymal cell fibroblasts (herein referred to
as fibroblasts) can enhance tumor growth and metastasis by means of promoting angiogenesis (1),
epithelial–mesenchymal transition (2), and progressive genetic instability (3, 4), there is also evidence
that healthy tumor microenvironments can act in a dominant manner to inhibit tumor growth (5).

From: Cancer Drug Discovery and Development: Stem Cells and Cancer,
Edited by: R.G. Bagley and B.A. Teicher, DOI: 10.1007/978-1-60327-933-8_3,
© Humana Press, a part of Springer Science + Business Media, LLC 2009
29
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