Stem Cell Biology and Regenerative Medicine

Series EditorKursad Turksen, Ph.D.kturksen@ohri.ca

For further volumes:http://www.springer.com/series/7896

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Alison L. AllanEditor

Cancer Stem Cells in Solid Tumors

EditorAlison L. Allan Depts. of Oncology and Anat. & Cell BiologySchulich School of Med. and Dent.University of Western OntarioLondon, Ontario, Canadaalison.allan@lhsc.on.ca

ISBN 978-1-61779-245-8 e-ISBN 978-1-61779-246-5DOI 10.1007/978-1-61779-246-5Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011932988

© Springer Science+Business Media, LLC 2011All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

v

Preface

Recently, there has been increasing support for the “cancer stem cell” hypothesis, which postulates that cancer arises from a subpopulation of tumor-initiating cells or cancer stem cells (CSCs). There are currently two conflicting views that attempt to explain tumor formation. The classical stochastic model suggests that every cell within a tumor is a potential tumor-initiator, but that entry into the cell cycle is governed by a low probability of stochastic mutations. According to this model, it would be impossible to tell which cell initiated the tumor since each cell has an equal ability to be malignant. By contrast, the hierarchy theory (upon which the CSC hypothesis is based) proposes that only a subset of cells within a tumor is capable of initiating tumor growth, but that these cells all do so at a high frequency. According to this theory, it should be possible to identify and target the cells responsible for tumor initiation and progression because not all cells have the same phenotypic and func-tional characteristics.

While the idea of CSCs has been around for more than 100 years, evidence from the hematology field has now demonstrated the critical role of stem cells in hemato-logical malignancies and suggested that these same mechanisms could also be cen-tral to the initiation, progression, and treatment of solid cancers. Indeed, several pivotal studies have recently provided compelling evidence that these cells do exist in solid tumors of many types including breast, brain, colorectal, pancreas, prostate, melanoma, lung, ovarian, liver, and head and neck cancer. Furthermore, clinical and experimental studies have demonstrated that CSCs exhibit many classical properties of normal stem cells, including a high self-renewal capacity and the ability to gener-ate heterogeneous lineages; the requirement for a specific “niche”/microenvironment to grow; and an increased capacity for self-protection against harsh environments, toxins, and drugs.

This multi-authored volume focuses specifically on the role of CSCs in solid cancers. The authors are all active investigators with research programs related to oncology and/or stem cell biology, and are leaders in their field. Part I (Chap. 1) serves to introduce the concept of CSCs vs. normal stem cells, including a histori-cal perspective and the contributing lessons from leukemia. Part II (Chaps. 2–11) describes the identification and role of CSCs in various forms of solid cancer,

vi Preface

organized according to disease site. Part III (Chaps. 12–14) elaborates on molecular pathways that are involved in driving CSC function, with a particular focus on the convergence of embryonic and tumorigenic signaling pathways. Part IV (Chaps. 15–18) describes available model systems and modalities for studying CSC biol-ogy and therapeutic development, including in vitro and in vivo model systems and assays and imaging modalities. Part V (Chaps. 19–23) discusses the importance of CSCs for cancer management and treatment, including implications for prognosis, prediction, and treatment resistance. Finally, Part VI (Chap. 24) provides the con-cluding thoughts for the book, including consideration of the controversy sur-rounding the CSC hypothesis. The editor and the authors hope that this work will provide a comprehensive overview of this evolving and important field.

London, ON Alison L. AllanCanada

vii

Acknowledgments

I would like to express my gratitude to all of the authors for their scholarly efforts in summarizing the current literature in this rapidly evolving field. I would also like to thank Mindy Okura-Marszycki and Kursad Turksen for giving me the opportunity to edit this book, and acknowledge Vindra Dass and Renata Hutter for all of their help throughout the editorial and publication process. Finally, I am grateful to mem-bers of my own research group for their patience, contributions, helpful discussion, and continued hard work in this exciting area of research.

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ix

Contents

Part I Introduction to Cancer Stem Cells

1 Cancer Stem Cells: Historical Perspectives and Lessons from Leukemia ................................................................. 3Christopher R. Cogle

Part II Cancer Stem Cells in Solid Tumors

2 Cancer Stem Cells in Breast Cancer .................................................... 15Jenny E. Chu and Alison L. Allan

3 Cancer Stem Cells in Brain Cancer ..................................................... 37Xin Wang, Chitra Venugopal, and Sheila K. Singh

4 Cancer Stem Cells in Colorectal Cancer.............................................. 57Mauro Biffoni, Eros Fabrizi, and Lucia Ricci-Vitiani

5 Cancer Stem Cells in Pancreatic Cancer ............................................. 79Jorge Dorado, Alicia G. Serrano, and Christopher Heeschen

6 Cancer Stem Cells in Prostate Cancer ................................................. 99Paula Kroon, Davide Pellacani, Fiona M. Frame, Norman J. Maitland, and Anne T. Collins

7 Cancer Stem Cells in Melanoma .......................................................... 117Ping Jin, Qiuzhen Liu, Marianna Sabatino, David F. Stroncek, Francesco M. Marincola, and Ena Wang

8 Cancer Stem Cells in Lung Cancer ...................................................... 139Jun Shen and Feng Jiang

9 Cancer Stem Cells in Ovarian Cancer ................................................. 151Fang Fang, Curt Balch, Meng Li, Jay M. Pilrose, and Kenneth P. Nephew

x Contents

10 Cancer Stem Cells in Hepatocellular Cancer ...................................... 177Russell C. Langan and Itzhak Avital

11 Cancer Stem Cells in Head and Neck Cancer ..................................... 197Mark E.P. Prince and Samantha J. Davis

Part III Cancer Stem Cell Gene Expression and Mechanisms: Convergence of Embryonic and Tumorigenic Signaling Pathways

12 Relationship Between Regulatory Pathways in Pluripotent Stem Cells and Human Tumors ................................... 209Olga Gaidarenko and Yang Xu

13 Influence of the Embryonic Microenvironment on Tumor Progression ............................................................................ 223Daniela Quail, Meghan Taylor, Michael Jewer, and Lynne-Marie Postovit

14 The Epithelial-to-Mesenchymal Transition and Cancer Stem Cells ........................................................................... 243Jonas Fuxe

Part IV Model Systems for Studying Cancer Stem Cell Biology and Therapeutic Development

15 Application of Stem Cell Assays for the Characterization of Cancer Stem Cells .............................................................................. 259Pamela M. Willan and Gillian Farnie

16 Zebrafish as a Model to Study Stem Cells in Development, Disease, and Cancer .................................................. 283Viviana Anelli, Cristina Santoriello, and Marina C. Mione

17 Imaging Cancer Stem Cells ................................................................... 297Paula Foster

18 Mouse Models for Studying Normal and Cancer Stem Cells ............ 311David A. Hess

Part V Clinical and Therapeutic Implications of Cancer Stem Cells

19 Cancer Stem Cells and Disease Prognosis ........................................... 329Zeshaan A. Rasheed, Jeanne Kowalski, and William H. Matsui

20 Mechanisms of Radioresistance in Cancer Stem Cells ....................... 345Cleo Y-F Lee and Maximilian Diehn

xiContents

21 The Role of ABC Transporters in Cancer Stem Cell Drug Resistance ...................................................................................... 361Vera S. Donnenberg, Ludovic Zimmerlin, and Albert D. Donnenberg

22 Resistance to Endocrine Therapy in Breast Cancer: Are Breast Cancer Stem Cells Implicated? ......................................... 381Ciara S. O’Brien, Sacha J. Howell, Gillian Farnie, and Robert B. Clarke

23 Future Directions: Cancer Stem Cells as Therapeutic Targets .......................................................................... 403Alysha K. Croker and Alison L. Allan

Part VI Final Thoughts

24 Final Thoughts: Complexity and Controversy Surrounding the “Cancer Stem Cell” Paradigm ....................................................... 433Craig Gedye, Richard P. Hill, and Laurie Ailles

Index ................................................................................................................ 465

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xiii

Contributors

Laurie Ailles Ontario Cancer Institute, Campbell Family Institute of Cancer Research; and Departments of Medical Biophysics, University of Toronto, Toronto, ON, Canada

Alison L. Allan Departments of Oncology and Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

London Regional Cancer Program, London Health Sciences Centre, London, ON, Canada

Viviana Anelli IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy

Itzhak Avital National Cancer Institute (NIH), Surgery Branch, Bethesda, MD, USA

Curt Balch Medical Sciences Program, Indiana University, Bloomington, IN, USA

Indiana University Simon Cancer Center, Indianapolis, IN, USA

Mauro Biffoni Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

Jenny E. Chu Department of Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

Robert B. Clarke Breast Biology Group, School of Cancer and Enabling Sciences, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK

Christopher R. Cogle Department of Medicine, Division of Hematology/ Oncology, University of Florida, Gainesville, FL, USA

Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, FL, USA

xiv Contributors

Anne T. Collins Cancer Research Unit, Department of Biology, University of York, York, UK

Alysha K. Croker Department of Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

Samantha J. Davis Department of Otolaryngology-Head and Neck Surgery, University of Michigan, Ann Arbor, MI, USA

Maximilian Diehn Stanford Cancer Center, CA, USA

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA, USA

Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA

Albert D. Donnenberg Department of Medicine, Division of Hematology/ Oncology, University of Pittsburgh Cancer Institute and University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Vera S. Donnenberg Department of Cardiovascular Surgery, Division of Hematology/Oncology, University of Pittsburgh Cancer Institute and University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Jorge Dorado Clinical Research Programme, Stem Cells & Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Eros Fabrizi Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

Fang Fang Medical Sciences Program, Indiana University, Bloomington, IN, USA

Gillian Farnie Cancer Stem Cell Research, University of Manchester, School of Cancer and Enabling Sciences, Paterson Institute for Cancer Research, Manchester, UK

Paula Foster Robarts Research Institute, London, ON, Canada

Department of Medical Biophysics, University of Western Ontario, London, ON, Canada

Fiona M. Frame Cancer Research Unit, Department of Biology, University of York, York, UK

Jonas Fuxe Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

Olga Gaidarenko Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA

xvContributors

Craig Gedye Ontario Cancer Institute, Campbell Family Institute of Cancer Research, Toronto, ON, Canada

Christopher Heeschen Clinical Research Programme, Stem Cells & Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

David A. Hess Department of Physiology & Pharmacology, The University of Western Ontario, London, ON, Canada

Vascular Biology Group, Krembil Centre for Stem Cell Biology, Robarts Research Institute, London, ON, Canada

Richard P. Hill Ontario Cancer Institute, Campbell Family Institute of Cancer Research, Departments of Medical Biophysics and Radiation Oncology, University of Toronto, Toronto, ON, Canada

Sacha J. Howell Department of Medical Oncology, The Christie NHS Foundation Trust, University of Manchester, Manchester, UK

Michael Jewer Department of Anatomy & Cell Biology, University of Western Ontario, London, ON, Canada

Feng Jiang Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA

Ping Jin Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA

Jeanne Kowalski Winship Cancer Institute, Emory University, Atlanta, GA, USA

Paula Kroon Cancer Research Unit, Department of Biology, University of York, York, UK

Russell C. Langan Surgery Branch, National Cancer Institute, National Institute of Health, Bethesda, MD, USA

Cleo Y-F Lee Stanford Cancer Center, Stanford University School of Medicine, Stanford, CA, USA

Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA

Meng Li Medical Sciences Program, Indiana University, Bloomington, IN, USA

Qiuzhen Liu Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA

Norman J. Maitland Cancer Research Unit, Department of Biology, University of York, York, UK

Francesco M. Marincola Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA

xvi Contributors

William H. Matsui The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Marina C. Mione IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy

Kenneth P. Nephew Medical Sciences Program, Indiana University, Bloomington, IN, USA

Indiana University Simon Cancer Center, Indianapolis, IN, USA

Ciara S. O’Brien Department of Medical Oncology, The Christie NHS Foundation Trust, University of Manchester, Manchester, UK

Davide Pellacani Cancer Research Unit, Department of Biology, University of York, York, UK

Jay M. Pilrose Medical Sciences Program, Indiana University, Bloomington, IN, USA

Lynne-Marie Postovit Department of Anatomy & Cell Biology, University of Western Ontario, London, ON, Canada

Mark E.P. Prince Department of Otolaryngology-Head and Neck Surgery, University of Michigan, Ann Arbor, MI, USA

Daniela Quail Department of Anatomy & Cell Biology, University of Western Ontario, London, ON, Canada

Zeshaan A. Rasheed The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Lucia Ricci-Vitiani Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy

Marianna Sabatino Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA

Cristina Santoriello IFOM, The FIRC Institute of Molecular Oncology, Milan, Italy

Alicia G. Serrano Clinical Research Programme, Stem Cells & Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Jun Shen Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA

Sheila K. Singh McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ON, Canada

Departments of Surgery, Biochemistry & Biomedical Sciences, and Neuroscience, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada

xviiContributors

David F. Stroncek Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA

Meghan Taylor Department of Anatomy & Cell Biology, University of Western Ontario, London, ON, Canada

Chitra Venugopal McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ON, Canada

Ena Wang Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, MD, USA

Xin Wang McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ON, Canada

Pamela M. Willan Cancer Stem Cell Research, University of Manchester, School of Cancer and Enabling Sciences, Paterson Institute for Cancer Research, Manchester, UK

Yang Xu Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA

Ludovic Zimmerlin Department of Cardiovascular Surgery, Division of Hematology/Oncology, University of Pittsburgh Cancer Institute Pittsburgh, Pittsburgh, PA, USA

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Part IIntroduction to Cancer Stem Cells

3A.L. Allan (ed.), Cancer Stem Cells in Solid Tumors, Stem Cell Biology and Regenerative Medicine, DOI 10.1007/978-1-61779-246-5_1, © Springer Science+Business Media, LLC 2011

Abstract Cancer has a long history rooted in developmental biology. Early scientists regarded cancer as remnant embryonal tissues waiting to be provoked into a malignant state. Whereas this embryonal rest theory fits well with certain childhood cancers like teratocarcinomas, acquired cancers in adulthood require more explana-tion. Because of early advances in hematology and immunology, investigations of hematologic malignancies like leukemias have benefited from translated tech-nology. Seminal discoveries in leukemia stem cell biology are reviewed in this chapter. Some of these discoveries translate to novel opportunities for improved diagnostics and therapeutics. Importantly, several lessons in the leukemia stem cell experience are applicable to ongoing cancer stem cell investigations. These lessons are discussed relative to leukemia stem cells and with an eye toward defining and testing cancer stem cells in solid tumors.

Abbreviations

ABC ATP binding cassetteABL AblesonALDH Aldehyde dehydrogenaseALL Acute lymphoblastic leukemiaAML Acute myeloid leukemia

C.R. Cogle (*)Department of Medicine, Division of Hematology/Oncology, University of Florida, Gainesville, FL, USA

Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, FL, USA e-mail: c@ufl.edu

Chapter 1Cancer Stem Cells: Historical Perspectives and Lessons from Leukemia

Christopher R. Cogle

4 C.R. Cogle

ATP Adenosine triphosphateBCR Breakpoint cluster regionCD Cluster of differentiationCML Chronic myeloid leukemiaENL Eleven nineteen leukemiaFISH Fluorescent in situ hybridizationMDR Multi-drug resistanceMLL Mixed lineage leukemiaMOZ Monocytic leukemia zinc finger proteinNOD/SCID Non-obese diabetic/severe combined immunodeficiencyNOG Non-obese diabetic/severe combined immunodeficiency/IL2 recep-

tor g-nullPCR Polymerase chain reactionTIF2 Transcriptional intermediary factor 2

1.1 Historical Postulates for the Stem Cell Basis of Cancer

Today, cancer stem cells are defined as “a small subset of cancer cells within a cancer that constitute a reservoir of self-sustaining cells with the exclusive ability to self-renew and to cause the heterogeneous lineages of cancer cells that com-prise the tumor” [1]. However, this idea that primitive cells can lead to cancer is not new.

The earliest reports of a cancer stem cell hypothesis appeared in the 1800s. Similarities between teratocarcinomas and the developing embryo led biologists to postulate that cancers arise from embryonic remnants in adults [2]. Certainly, the existence of teratocarcinomas which contain cells of all three germ layers and afflict young adults along midline migration pathways between gonads to brain endorses this embryonal rest theory. Subsequent investigators further developed this theory and suggested that adult tissues may contain embryonic remnants that are normally dormant but can become cancerous if provoked [3–5].

Whereas the embryonal rest hypothesis may explain teratocarcinomas, which primarily arise in children, the hypothesis requires more elaboration to understand the genesis of acquired cancers, which arise in adulthood and not necessarily along the midline. Given evidence for tissue-resident stem and progenitor cells in the adult, it is possible that these normally self-renewing and multi-lineage differentiat-ing stem cells may be provoked by carcinogens to acquire hallmark properties of cancer, including evasion of apoptosis, growth factor independence, self-renewal, tissue invasion, and sustained angiogenesis. Hematologic malignancies, which usu-ally arise in the seventh and eighth decades of life and which coincide with normal hematopoietic stem and progenitor cells, provide a clear opportunity to define adult cancer stem cells [6].

51 Cancer Stem Cells: Historical Perspectives and Lessons from Leukemia

1.2 History of Leukemia Stem Cells

The first reports of leukemia stem cells were in the 1930s when Furth and Kahn transplanted leukemia from one mouse to another via a single undifferentiated leu-kemia cell [7]. These experiments demonstrated that a self-renewing malignant hematopoietic stem cell was present; however, without the ability to characterize source cells or define progeny, no definite comment could be made about a hierarchy of malignant stem cells which exhibit the two cardinal features of stem cells: self-renewal and multi-lineage differentiation. Defining leukemia stem cells would come decades later, after advancements in immunology and cell sorting techniques.

The first detailed investigation for leukemia stem cells came in the 1990s out of John Dick’s laboratory [8, 9]. Taking cues from normal hematopoietic stem cell biology, these investigators identified a subpopulation of CD34+CD38− human acute myeloid leukemia (AML) cells that propagated colonies in culture and reca-pitulated human leukemia in immunocompromised mice. Using limiting dilution xenotransplant experiments, AML stem cells were estimated to exist at a frequency of 1 in 250,000 CD34+CD38− AML cells. In contrast, when these investigators xenografted more committed leukemia cells expressing a CD38+ phenotype, they were unable to recapitulate AML. Together, these experiments showed that AML stem cells were present, prospectively identifiable, and rare. Moreover, an AML hierarchy was apparent, with AML stem cells giving rise to terminally differenti-ated yet malignant progeny.

Studies subsequent to these seminal discoveries have shed new light on leukemia stem cells and serve as important lessons for the field of cancer stem cell biology.

1.3 Lesson: Normal Stem Cells Aren’t Always the Origin

The fact that AML stem cells can be enriched using the same selection strategy as normal hematopoietic stem cells (e.g., immunosorting for CD34+CD38−) suggests that leukemia stem cells may be a malignant transformation of normal stem cells. However, follow-up experiments of AML stem cells found that they do not express CD90 (Thy1), in contrast to normal hematopoietic stem cells, which do express Thy1 [10]. This finding begged the question of whether malignant transformation of normal hematopoietic stem cells results in loss of Thy1 expression, or whether hematopoietic progenitors lacking Thy1 are the target of malignant transformation into leukemia stem cells. The answer depends on the type of leukemia.

In leukemias that harbor the fusion oncogene BCR-ABL (which can be found in patients with chronic myeloid leukemia [CML], acute lymphoblastic leukemia [ALL] and AML with translocation of chromosomes 9 and 22), the cancer-initiating cell is believed to be at the level of the hematopoietic stem cell or higher. Forced expression of BCR-ABL in hematopoietic progenitor cells resulted in a proliferation of leukemia cells; however, the transformed hematopoietic progenitors could not

6 C.R. Cogle

self-renew and recapitulate disease [11]. In other types of leukemia, hematopoietic progenitors may serve as the origin for transformation. For example, forced expres-sion of oncogene fusions such as MLL-ENL or MOZ-TIF2, which can be found in patients with AML, endow hematopoietic progenitor cells with the ability to self-renew and differentiate [11, 12]. Together, these results show the heterogeneity of leukemia origin and may explain the heterogeneity in clinical behavior.

In context to cancer stem cells in solid tumors, the hunt for the source should not be restricted to the organ-resident stem cell. Candidates for oncogenic transformation should also include more committed tissue progenitor and differentiated cells, espe-cially in epithelial situations where field cancerization and dysplasia can be found.

1.4 Lesson: Don’t Underestimate the Microenvironment

In early leukemia stem cell experiments, when investigators replaced the severe combined immunodeficiency (SCID) mouse with the more immunocompromised non-obese diabetic (NOD)/scid strain, xenotransplanted human AML CD34+CD38− cells more readily repopulated secondary mice, thus demonstrating in vivo self-renewal typical of stem cells. Use of even more immunodeficient mice, such as NOD/scid/IL2R-g−/− (NOG) mice [13], resulted in even higher engraftment levels of human AML cells [14]. Moreover, in these NOG mice, consistent AML engraft-ment can be found in secondary and tertiary xenograft recipients. Interestingly, female NOG mice are more tolerant of AML stem cell engraftment than male mice [15]. Taken together, these data implicate the host microenvironment as a key factor in determining the presence and frequency of cancer stem cells. Careful consideration and scrutiny should be applied to the model system used to detect, quantify, and characterize putative cancer stem cells. Discoveries from one lab may not replicate in another lab simply due to differences in host model and/or manipulations of the host model. For example, conditioning transplant recipients with ionizing irradia-tion or antibodies to immune cells may enhance the gain when reading out putative cancer stem cell engraftment.

Although differences in the host microenvironment may complicate consensus on the definition of cancer stem cells, these differences may also be explored as opportunities to discover which situations support cancer survival. Once defined, these host microenvironmental factors may then be targeted as novel therapeutic strategies. For example, blood vessels in the bone marrow microenvironment are important for leukemia stem cell survival and proliferation [16–18]. Targeting these blood vessels in the microenvironment causes regression of leukemia and may be a promising therapeutic for patients with this cancer [19, 20]. As another example, given evidence of robust AML engraftment in severely deficient animals, host immune response to leukemia stem cells is likely important. In fact, leukemia stem cells were shown to over-express CD47, a surface protein that inhibits macrophage recognition [21]. Clinically, patients whose leukemia cells expressed high levels of CD47 had inferior outcomes after chemotherapy, which suggests the importance of

71 Cancer Stem Cells: Historical Perspectives and Lessons from Leukemia

macrophage immunosurveillance in leukemia [22]. Modulating host immune response to overcome leukemia’s evasion may therefore represent a novel potential therapeutic strategy.

1.5 Lesson: Surface Molecules Aren’t Just Markers

Immunophenotyping is a common method for identifying and selecting cancer stem cells after advancements in immunology and cell sorting technology (e.g., flow cytometry, magnetic separation). Increasingly, investigators have used the term “marker” to describe a unique surface molecule or constellation of surface mole-cules on putative cancer stem cells. However, the term “marker” is a restrictive term that disregards the molecule’s biological function.

As an example, the normal hematopoietic stem cell expresses CD44 receptors, which tether it to stromal adhesion molecules like hyaluronic acid, osteopontin, col-lagens, and matrix metalloproteinases. Leukemia stem cells also express CD44 iso-forms [23]. Recognizing that CD44 is more than a “marker” of leukemia stem cells, investigators have blocked CD44 stroma binding and found impairments in leuke-mogenesis. When BCR-ABL leukemia CD44 receptors were mutated, leukemia pro-liferation was inhibited. Furthermore, the application of blocking antibodies to CD44 inhibits leukemia stem cell engraftment [24].

1.6 Lesson: There May Be More Than One Cancer Stem Cell Population

Clear evidence shows that leukemia stem cells can be found in the CD34+CD38− subpopulation of leukemic bone marrow. However, there is also evidence that leuke-mia stem cells can be found in the CD34− subpopulation [25–27]. Whether leukemia stem cells lose CD34 expression after oncogenic transformation or whether CD34-negative leukemia stem cells represent transformation of a very primitive bone marrow–derived stem cell is yet to be defined.

Leukemia stem cells have also been defined by their functional characteristics. For example, aldehyde dehydrogenase (ALDH) is important for eliminating intrac-ellular toxins. Normal hematopoietic stem cells are known to have higher levels of this enzyme and can thereby be prospectively identified based on functional ALDH activity [28]. Taking cues from normal stem cell biology, leukemia investigators have reported enrichment of leukemia stem cells by selecting leukemic bone mar-row cells with high ALDH activity [29]. Another functional assay exploits the drug efflux capacity of stem cells. In normal stem cell biology, side-population cells, defined by their ability to efflux the DNA-binding dye Hoechst 33342, have shown self-renewal and multi-lineage differentiation [30, 31]. Following suit, leukemia

8 C.R. Cogle

investigators have also identified a small subpopulation of leukemia stem cells that reside within this side-population of leukemic bone marrow [32, 33].

At face value, these multiple and overlapping reports may suggest contradic-tions. But it is more likely that there are different leukemia stem cell populations for different types of leukemias. In addition, it has yet to be determined whether there are multiple leukemia stem cells within each patient’s leukemia.

1.7 Lesson: Treatment Failure May Be Due to Cancer Stem Cell Resistance

The identification of self-renewing leukemia stem cells that reside in protective microenvironments suggests that these cells may be sources of primary refractory and relapsed disease. If so, then these leukemia stem cells must be less sensitive to conventional therapies than their differentiated progeny.

Given the important role of multiple drug resistance (MDR) transporters in stem cells (a family of at least 48 human ATP binding cassette [ABC] transporters dis-covered to date), this mechanism has been suggested as cause for leukemia stem cell resistance to conventional chemotherapies [34]. In younger patients with AML, MDR1 is less frequent, which may explain better responses to therapy [35]. Administration of MDR inhibitors as adjuvant therapy does bring about improve-ments in remission rates [35, 36]. However, it is not clear whether the more effective response rates are due to MDR inhibition in leukemia stem cells and increased sen-sitivity to chemotherapy, or increases in circulating chemotherapy levels due to altered chemotherapy metabolism related to side effects of the MDR inhibitor.

For patients with CML, the BCR-ABL fusion oncogene can be targeted with the tyrosine kinase inhibitor, imatinib. Imatinib directly targets the BCR-ABL–encoded tyrosine kinase activity in CML leading to decreased proliferation of myeloid progenitors. However, despite cytogenetic responses measured by fluorescent in situ hybridization (FISH), molecular eradication of the disease measured by more sensi-tive quantitative polymerase chain reaction (PCR) is difficult to achieve and the current standard of care is to keep patients on imatinib indefinitely or until disease relapse or progression. The persistence of CML despite tyrosine kinase inhibitor therapy within imatinib is a result of resistance by quiescent CML stem cells [37]. Several strategies are now being developed to target resistant CML-initiating cells.

1.8 Conclusions

Traced back far enough, the roots of cancer can be found in developmental biol-ogy. From the embryonal rest theory, more detailed investigations of cancer have uncovered rare cancer stem cells with the potency to self-renew and differentiate.

91 Cancer Stem Cells: Historical Perspectives and Lessons from Leukemia

Because of advances in normal hematopoietic stem cell biology and immunology, significant progress has been made in defining leukemia stem cells. Translating technology from the normal to malignant setting has illuminated mechanisms of leukemogenesis, resistance to treatment, and relapse. This enlightened understand-ing empowers physician scientists to move beyond brute force cytotoxicity and closer to strategic strikes.

Several lessons stand out from the leukemia stem cell experience that are relevant to most cancer stem cell investigations. These lessons all have in common the central idea that cancer is a heterogeneous mixture of primitive and differentiated cells that each has multidirectional relationships with each other and the host microenviron-ment. The idea that multiple subpopulations enriching for cancer stem cells are sup-ported by many microenvironmental interactions is more likely than the concept of one cancer stem cell dependent on only one pathway. Certainly, it is easier to present and think about cancer stem cell data in one dimension, but creating new therapies and optimizing old ones will require us to broaden our scientific considerations.

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Part IICancer Stem Cells in Solid Tumors