Cancer Stem Cells: Targeting the Roots of Cancer, Seeds of Metastasis, and Sources of Therapy Resistance

Cancer Stem Cells

Targeting the Roots of Cancer, Seeds of Metastasis, and Sources of Therapy Resistance

Huiping Liu

Case Western Reserve University, Cleveland, OH, United States

Northwestern University Feinberg School of Medicine, Chicago, IL, United States

National Center for Regenerative Medicine, Cleveland, OH, United States

Justin D. Lathia

Cleveland Clinic, Cleveland, OH, United States

Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, United States

Case Comprehensive Cancer Center, Cleveland, OH, United States

Table of Contents

Cover image

Title page

Copyright

Dedication

List of Contributors

Foreword by Jane Visvader

Foreword by Stanton L. Gerson

Preface

Acknowledgments

Section 1. CSC Overview and Methodology

Chapter 1. Introduction: Cancer Stem Cells

Introduction

Cancer Stem Cell Origins

Identification of the Cancer Stem Cell Population

Mouse Models of Cancer Stem Cell Function

Strategies for Targeting Cancer Stem Cell

Future of Cancer Stem Cell-Directed Therapies

Conclusion

List of Acronyms and Abbreviations

Chapter 2. Overview: Cancer Stem Cell Self-Renewal

Definition of Self-renewal in Cancer Stem Cells

Assays to Measure Self-Renewal Potential of Cancer Stem Cells

Modulators of Self-Renewal in Cancer Stem Cells

Conclusion

Glossary

List of Acronyms and Abbreviations

Chapter 3. Enrichment and Interrogation of Cancer Stem Cells

Introduction

Enrichment for Cancer Stem Cells

Functional Analysis

Conclusion and Future Directions

List of Acronyms and Abbreviations

Section 2. CSCs in Representative Organ Systems

Chapter 4. Critical Updates to the Leukemia Stem Cell Model

Introduction

The Original Leukemia Stem Cell Model

With Better Flow-Sorting Protocols, Leukemia Stem Cells Are Exclusively Progenitors, Not Hematopoietic Stem Cells, by Surface Phenotype

Evidence for Leukemia Stem Cells and Resistance

Other Mechanistic Explanations for Resistance

Master Transcription Factor Expression in Leukemia Stem Cells and Hematopoietic Stem Cells

The Most Frequent Mutations in Acute Myeloid Leukemia, in NPM1 and FLT3, Are in Committed Progenitors, Not Hematopoietic Stem Cells

Do Founder Mutations Expand Hematopoietic Stem Cells and/or Expand Committed Progenitors?

The Revised Model

Practical, Immediate Implications of the Revised Model

Conclusion

List of Acronyms and Abbreviations

Chapter 5. Breast Cancer Stem Cells and the Move Toward High-Resolution Stem Cell Systems

Introduction

Origins in Development

The Mammary Gland as a Classic Stem Cell–Driven System

Identification of Breast Cancer Stem Cells

Caveats of the Cancer Stem Cell Assay

Associated Cancer Stem Cell Markers and Regulators

Genome Wide Molecular Profiling in Breast Cancer

Stem Cell Programs in Breast Cancer

An Inducible Stemness

Tumor-Associated Reprogramming

Tackling Heterogeneity at Single-Cell Resolution

Conclusion

List of Acronyms and Abbreviations

Chapter 6. Lung Cancer Stem Cells: Drivers of a Genetically and Histologically Complex Disease

Normal Lung Stem Cells

The Cancer Stem Cell Model in Lung Cancer

Targeting Lung Cancer Stem Cells Therapeutically

Glossary

List of Acronyms and Abbreviations

Chapter 7. Colorectal Cancer Stem Cells

Introduction

Molecular Mechanisms and Pathways Regulating Colorectal Cancer Stem Cells

Colorectal Cancer Stem Cells and Microenvironment

Colorectal Cancer Stem Cells and Metastases

Cancer Stem Cell and Chemoresistance

Therapeutically Targeting Colorectal Cancer Stem Cells

Conclusion

Glossary

List of Acronyms and Abbreviations

Chapter 8. Targeting Bladder Cancer Stem Cells: One Step Closer to the Clinic?

Introduction

Bladder Cancer Stem Cells

Bladder Cancer Stem Cell Signaling

Prognostic Role of Cancer Stem Cells

Conclusions

List of Acronyms and Abbreviations

Chapter 9. Cancer Stem Cells as New Therapeutic Targets for Ovarian Cancer

Introduction

Ovarian Cancer Biology and Pathology

Isolation and Characterization of Ovarian Cancer Stem Cells

Ovarian Cancer Stem Cells and Drug Resistance

Therapeutic Approaches for Eradicating Ovarian Cancer Stem Cells

Conclusions

List of Acronyms and Abbreviations

Section 3. CSC Features and Mechanisms

Chapter 10. Understanding Cancer Cells of Origin in Cutaneous Tumors

Introduction

Cancer Cells of Origin Versus Cancer Stem Cells

Identifying Cancer Cells of Origin

Stem Cell Niches of the Epidermis

Cancer Cells of Origin in Cutaneous Tumors

Melanocytic Tumor Initiation

Concluding Remarks and Future Directions

List of Acronyms and Abbreviations

Chapter 11. Asymmetric Division of Cancer Stem Cells

Introduction

Asymmetric Cell Division in Unicellular Organisms

Asymmetric Cell Division in Multicellular Organisms

Drosophila Melanogaster Neuroblasts—A Model System for Classic, Intrinsic Asymmetric Cell Divisions

Asymmetric Cell Division and Cancer

Concluding Remarks and Future Directions

List of Acronyms and Abbreviations

Chapter 12. Metastasis and Metastatic Cells: A Historical Perspective and Current Analysis

Introduction

The Metastatic Cascade

Cancer Stem Cells and Metastasis: Prostate Cancer as an Example

Circulating Tumor Cells and Metastasis

The Role of Epithelial-to-Mesenchymal Transition in Metastasis

Concluding Remarks

List of Acronyms and Abbreviations

Chapter 13. Cancer Stem Cells: Metastasis and Evasion From the Host Immune Responses

Cancer Stem Cells, Metastasis, and Host Immunity: An Overview

Cancer Stem Cells and Metastasis

The Interplay Between Immune Responses and Cancer

Conclusions and Future Direction

Glossary

List of Acronyms and Abbreviations

Chapter 14. Cancer Stem Cells and Tumor-Associated Macrophages

Introduction

The Origin of Tumor-Associated Macrophages

General Properties of Tumor-Associated Macrophages

Cancer Stem Cells and Tumor-Associated Macrophages

Protumor Activities of Tumor-Associated Macrophages

Recruitment of Tumor-Associated Macrophages by Cancer Cells

Education of Tumor-Associated Macrophages by Cancer Cells

Tumor-Associated Macrophages and Cancer Therapies

Concluding Remarks

List of Acronyms and Abbreviations

Chapter 15. The Mechanisms of Therapy Resistance in Cancer Stem Cells

Evidence of Cancer Stem Cells

Evidence of Therapy Resistance

Resistance Mechanisms and Strategies to Sensitize Cancer Stem Cells

Resistance and Tumor Evolution

Conclusion

List of Abbreviations

Chapter 16. Adipose Tissue-Derived Stem Cells in Regenerative Medicine and Impact on Cancer

Adult Somatic Stem Cells

Adipose Tissue

Obesity and Cancer

Broad Utility of Adipose Tissue-Derived Stem Cells in Regenerative Medicine

Conclusions and Perspectives

Glossary

List of Acronyms and Abbreviations

Section 4. Clinical Applications

Chapter 17. From Research to the Clinic: Targeting Stem Cell Pathways in Cancer

Introduction

Wnt Pathway Antagonists

Notch Pathway Antagonists

Conclusions

List of Abbreviations

Chapter 18. Targeted Therapies for Glioma Stem Cells

Introduction

Tumor Niches/Microenvironments

Glioma Stem Cell as a Therapeutic Target

Conclusion

List of Acronyms and Abbreviations

Chapter 19. Circulating Tumor Cells, Cancer Stem Cells, and Emerging Microfluidic Detection Technologies With Clinical Applications

Introduction

Clinical and Biological Significance of Circulating Tumor Cells

Evolution of Circulating Tumor Cell Isolation Technologies

Microfluidic Technologies Enabling Further Circulating Tumor Cell Characterization and Downstream Analysis

Summary of Circulating Tumor Cell Characterization Approaches

Conclusion

List of Acronyms and Abbreviations

Index

Copyright

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Dedication

We would like to dedicate this book to a beloved middle school teacher Ms. Aiying Peng and others who lost their lives to cancer, the ones inspired by stem cells, and all committed to save lives and fight against cancer.

List of Contributors

Lauren Agro

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Shideng Bao,     Lerner Research Institute, Cleveland, OH, United States

Chi-Hsuan Chang,     Baylor College of Medicine, Houston, TX, United States

Keith Syson Chan,     Baylor College of Medicine, Houston, TX, United States

Samuel H. Cheshier,     Stanford University, Stanford, CA, United States

Avijeet S. Chopra,     University of Connecticut, Storrs, CT, United States

Anastasia Chumakova,     Cleveland Clinic, Cleveland, OH, United States

Michael F. Clarke,     Stanford University, Stanford, CA, United States

Salvatore Condello,     Indiana University School of Medicine, Indianapolis, IN, United States

Leanne R. Donahue,     Cornell University, Ithaca, NY, United States

Rogelio Esparza,     Stanford University, Stanford, CA, United States

Fang Fang,     Indiana University, Bloomington, IN, United States

Christine Fillmore Brainson

Stem Cell Program, Children’s Hospital Boston, Boston, MA, United States

Harvard Stem Cell Institute, Cambridge, MA, United States

Harvard Medical School, Boston, MA, United States

Austin Gurney,     OncoMed Pharmaceuticals, Inc., Redwood City, CA, United States

Andrew Haller

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Jennifer Haynes

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Diane Heiser,     Stanford University, Stanford, CA, United States

Joy Q. He,     Stanford University, Stanford, CA, United States

Masahiro Hitomi

Cleveland Clinic, Cleveland, OH, United States

Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, United States

Timothy Hoey,     OncoMed Pharmaceuticals, Inc., Redwood City, CA, United States

Jason K. Hseih,     University Hospitals-Case Medical Center & Case Comprehensive Cancer Center, Cleveland, OH, United States

Gregor Hutter,     Stanford University, Stanford, CA, United States

Awad Jarrar,     Cleveland Clinic, Cleveland, OH, United States

Carla F. Kim

Stem Cell Program, Children’s Hospital Boston, Boston, MA, United States

Harvard Stem Cell Institute, Cambridge, MA, United States

Harvard Medical School, Boston, MA, United States

Molly Kozminsky,     University of Michigan, Ann Arbor, MI, United States

Antonina V. Kurtova,     Baylor College of Medicine, Houston, TX, United States

Justin D. Lathia

Cleveland Clinic, Cleveland, OH, United States

Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, United States

Case Comprehensive Cancer Center, Cleveland, OH, United States

Cherry Leung

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Evelyne Lima-Fernandes,     Structural Genomics Consortium, Toronto, ON, Canada

Huiping Liu

Case Western Reserve University, Cleveland, OH, United States

Northwestern University, Chicago, IL, United States

Xia Liu,     Case Western Reserve University, Cleveland, OH, United States

Neethan A. Lobo

Stanford University, Stanford, CA, United States

Celgene Quanticel Research, San Francisco, CA, United States

Daniela Matei

Indiana University School of Medicine, Indianapolis, IN, United States

Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, United States

VA Roudebush Hospital, Indianapolis, IN, United States

Siddhartha S. Mitra,     Stanford University, Stanford, CA, United States

Hyeongsun Moon,     Cornell University, Ithaca, NY, United States

Sunitha Nagrath,     University of Michigan, Ann Arbor, MI, United States

Kenneth P. Nephew

Indiana University, Bloomington, IN, United States

Indiana University School of Medicine, Indianapolis, IN, United States

Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, United States

Catherine A. O’Brien

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Toronto General Hospital, Toronto, ON, Canada

Claudia Petritsch,     University of California San Francisco, San Francisco, CA, United States

Akshita Puri

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Dalong Qian,     Stanford University, Stanford, CA, United States

Sumaiyah Rehman

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Ofer Reizes

Cleveland Clinic, Cleveland, OH, United States

Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH, United States

Case Western Reserve University, Cleveland, OH, United States

Jeffrey M. Rosen,     Baylor College of Medicine, Houston, TX, United States

Kiera Rycaj,     University of Texas MD Anderson Cancer Center, Smithville, TX, United States

Yogen Saunthararajah,     Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, United States

Xiling Shen,     Duke University, Durham, NC, United States

Andrew E. Sloan,     University Hospitals-Case Medical Center & Case Comprehensive Cancer Center, Cleveland, OH, United States

Benjamin T. Spike,     University of Utah, Salt Lake City, UT, United States

Swetha J. Sundar,     University Hospitals-Case Medical Center & Case Comprehensive Cancer Center, Cleveland, OH, United States

Dean G. Tang

University of Texas MD Anderson Cancer Center, Smithville, TX, United States

Tongji University School of Medicine, Shanghai, China

University of Texas MD Anderson Cancer Center, Houston, TX, United States

Praveena S. Thiagarajan,     Cleveland Clinic, Cleveland, OH, United States

Linda J. van Weele,     Stanford University, Stanford, CA, United States

Yadong Wang

University Health Network, Toronto, ON, Canada

University of Toronto, Toronto, ON, Canada

Yinu Wang,     Indiana University, Bloomington, IN, United States

Irving Weissman,     Stanford University, Stanford, CA, United States

Andrew C. White,     Cornell University, Ithaca, NY, United States

James Wright,     University Hospitals-Case Medical Center & Case Comprehensive Cancer Center, Cleveland, OH, United States

Maider Zabala,     Stanford University, Stanford, CA, United States

Wenchao Zhou,     Lerner Research Institute, Cleveland, OH, United States

Foreword by Jane Visvader

Since the declaration of war on cancer in 1971, we have witnessed an exponential increase in our understanding of cancer at the molecular and cellular level. Yet, mortality from cancer remains a leading cause of death, and the incidence of most cancers continues to rise, in part due to an aging global population. Despite the development of revolutionary anticancer drugs over the past decades, the range of therapeutic armaments remains limited. The immense heterogeneity between cancer subtypes and within individual tumors poses a great challenge for biologists and clinicians. The cancer stem cell (CSC) hypothesis has stimulated great interest as a mechanism to explain intratumoral heterogeneity and therapeutic resistance. The lessons learned from stem cell regeneration and the emergence of the CSC field offer an opportunity to unravel the complexities of cancer and to develop more effective therapies. This book provides a comprehensive summary of advances in the CSC field over the past 20  years, encompassing concepts, definitions, methods and cutting-edge technologies to study CSCs, the emerging features of these cells in tumors of diverse organs, and their therapeutic potential. Moreover, it demonstrates how stem cell biology and traditional cancer models can be integrated.

Tumors show dramatic heterogeneity in their cellular morphology, signaling pathways, genetic/epigenetic lesions, and therapeutic response. How does intratumoral heterogeneity arise? On the snowy mountains of Breckenridge, Colorado, I was approached by Huiping to read this book, which addresses and seeks to answer some of the most poignant questions in the cancer field. The book boasts a list of prominent authors and provides an extraordinarily comprehensive account of CSCs, spanning both basic and translational aspects. The field has been lacking such a broad overview.

The clinical relevance of CSCs has been a crucial question. This book not only helps us to appreciate the features of CSCs in many representative systems, but also illustrates the importance of CSCs to tumor relapse and predicting patient outcome. By identifying molecular commonalities (such as self-renewal pathways) between CSC populations in diverse tumors, it has become clear that it is possible to target CSCs. Indeed, the targeting of developmental (stem cell) pathways usurped by CSCs is showing considerable promise in the clinic. The equally important issue of how CSCs evade radiation and chemotherapy is also discussed in this book. The dynamic nature of CSCs and the presence of metastatic cells with properties similar to CSCs continue to underscore the importance of studying these cells. Cancer patients usually succumb to death through metastasis. An improved understanding of the relationship between CSCs and metastatic cells will enhance the development of metastasis-targeting therapies. Despite the emerging evidence for tumors being evolving entities, the chapters herein reveal the considerable potential for targeting the unique features of CSCs in tumor development and metastasis.

This book also reviews recent developments in the field on the interplay between CSCs, their niches, and the immune microenvironment. Insights into how CSCs evade the innate and adaptive immune responses are discussed, as well as the potential of CSC-directed immunotherapy. Such therapies in synergy with conventional therapies hold the promise of affording new therapeutic strategies against cancer. The book provides a valuable source of information for trainees and scientific investigators at all levels, as well as physicians and industrial partners. Finally, this book is timely, presenting a summary of the state of the field and highlighting key priorities for future investigation. I very much enjoyed reading this book and highly commend it as a resource for the cancer biology field.

Sincerely,

Jane Visvader, PhD, Professor,     Fellow of the Australian Academy of Science, Joint Division Head, Stem Cells and Cancer, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

Foreword by Stanton L. Gerson

To provide a foreword for the definitive treatise on the state of the art of cancer stem cells by all of the authors I would like to hear from is rather overwhelming.

This text will become a reference of the field because it has assembled a timely review of critical issues in the field and will catapult the efforts forward to take what is learned about cancer stem cells into diagnostic, prognostic, therapeutic, and ultimately preventive fields of clinical oncology. In this regard, this effort is very special.

The field of stem cell characterization starts and persists to this day, in all fields, with confusion and contention about functional characteristics, isolation techniques, identification strategies, and purification to homogeneity. Every stem cell field struggles with the efforts to outwit the cells through characterization, whether they be mesenchymal, hematopoietic, neural, or cancer stem cells. To me, it is part of the endearing scientific quest for perfection, that sometimes sidelines itself in the bigger, faster, and better method of today only to be outgunned tomorrow.

Looking through these chapters, all attempt a definition of the cancer stem cell: no two definitions are the same, but the quest remains one clear focus—to identify the most problematic cell in the cancer lineage, characterized by its pleiotropic lineage potential, responsiveness to signals similar but never identical to normal stem cells, and persistence through tumor evolution and treatment. No one wants these cells in a tumor yet every tumor seems to have them. And, eliminating them is, for the moment, elusive.

One thing is clear, the outstanding compilation herein will teach about the complexity of the subject, the links between approaches, the consensus on surface markers and gene expression characterization, and the compelling approaches to isolation and targeted therapeutics. I would love five pages to critique every chapter with a provocative question and a suggestion for experimentation. But alas, my duty is to engage and encourage you to review, compare, and reflect for yourself why the field has yet to come to closure on critical issues about cancer stem cells: When do they originate in a tumor—is a single cell sufficient to set the clone and is that cell the cancer stem cell? Is the treatment resistance intrinsic to cancer and instability of gene expression and mutation profile, or a static property selected for among cancer stem cells? Will a common Achilles’ heel emerge given that there are so many similarities why should not a treatment be effective across tumor types? Do all cancer stem cells circulate and if so, as single cells covered in accessory cells and exosomes, or do they break away as clusters? What is the best clinical test of the cancer stem cell hypothesis for diagnosis, treatment, or prevention, and what is the best clinical trial design to address the role and clinical functionality of cancer stem cells? The good news is that the authors of these pages know better than anyone.

Have a good read.

Stanton L. Gerson, MD,     Case Western Reserve University School of Medicine, Case Comprehensive Cancer Center and Seidman Cancer Center, National Center for Regenerative Medicine, Cleveland, OH, United States

Preface

Cancer Stem Cells—Targeting the Roots of Cancer, Seeds of Metastasis, and Sources of Therapy Resistance is intended for scientific researchers from students to investigators as well as clinicians and industry partners who can impact daily and future cancer medicine.

Cancer has been a pathology that has continued to vex physicians and scientists, despite our ever evolving understanding of this class of diseases. In our attempts to understand its complexity and generate paradigms to guide investigation, we have turned to the elegance of development processes. Tumors have long been considered aberrant organs, and many key developmental signals reemerge during tumor initiation and progression. Another key feature shared by tumors and developing organs is cellular heterogeneity, which in normal organs, is organized in a cellular hierarchy with a self-renewing stem cell at apex. Stem cells are essential for normal development and homeostasis, and their dysfunction is linked to many degenerative diseases. Stem cells are tightly controlled by the integration of intrinsic and extrinsic regulatory mechanisms that ensure that at each cell division, at least one daughter cell retains stem cell function.

To fully illustrate the cellular heterogeneity present within tumors, investigators have been inspired by the hierarchal organization present in development to complement the stochastic model. Using transplantation assays, it was recognized that a fraction of tumor cells in a given cancer could give rise to a tumor that could serially be passaged. These tumor-initiating cells first identified in leukemias gave rise to heterogeneous tumors and had enhanced self-renewal capabilities, similar to that of normal stem cells. Out of these observations, the cancer stem cell hypothesis was born, and now cancer stem cells have been identified in a variety of advanced cancers including leukemias, breast cancer, colorectal cancer, brain tumors, pancreatic cancer, cutaneous tumors, lung cancer, bladder cancer, and other cancers. The cancer stem cell hypothesis has provided an additional model to help appreciate the complexity of cancer and complements traditional models based on genetic aberrations. By integrating the cancer stem cell hypothesis with genetic models and regulatory immune microenvironment, additional regulatory mechanisms have been identified that drive tumor growth, therapy resistance, and metastasis that claim patient lives. The first generation cancer stem cell pathway inhibitors are in clinical trials, highlighting the future potential of cancer stem cell targeting.

In this book, we have provided an overview of the current state of understanding within the field and highlight key areas that require additional investigation. In the first section, cancer stem cells are introduced along with self-renewal, a fundamental property of these cells. A synopsis on cancer stem cell enrichment methods is also included. In the second section, cancer stem cells in representative organs systems are discussed and include leukemia, breast cancer, lung cancer, colorectal cancer, bladder cancer, and ovarian cancer. In the third section, key cancer stem cell features are highlighted and include understanding the cell of origin, the role of asymmetric cell division and cell fate choice, the role of cancer stem cells in metastasis, the interaction between cancer stem cells and the immune system, and the mechanisms of therapeutic resistance. In the final section, a clinical perspective is provided for cancer stem cell–based therapies and detection, including circulating tumor cells.

We have assembled this book to engage readers of varying levels within the field and hope that you find the concepts within this book engaging and a source of inspiration. We look forward to receiving feedback and suggestions to improve the book in future editions.

Huiping Liu, MD, PhD,     Case Western Reserve University

Justin D. Lathia, PhD,     Cleveland Clinic Foundation

Acknowledgments

This book represents a true team effort, and we are extremely grateful to numerous individuals who were integral to the production of this book. First off, we would like to acknowledge our partners at Elsevier for reaching out to us and assisting with putting this book together, an exercise neither of us had previously participated in. We would especially like to thank Catherine (Cassie) Van Der Laan who assisted at the initial stages of the book conception as well as Lisa Eppich who worked with us through the entire process and was invaluable in getting the book published. We are deeply indebted to Drs. Jane Visvader and Stanton Gerson for the foreword as well as each and every one of the authors who spent time putting together high-quality chapters. Along with writing the chapters, the authors also served as peer reviewers (Dr. Jeffrey Rosen served twice). Without them, the book would not be available. We would also like to credit the following additional peer reviewers for their valuable and insightful comments: Dr. Anita Hjelmeland (University of Alabama, Birmingham), Dr. Masahiro Hitomi (Cleveland Clinic), Dr. Monica Venere (Ohio State University), Dr. Jialiang Wang (Vanderbilt University), Dr. Stefani Spranger (The University of Chicago), and Dr. Golam Kibria and Valery Adorno-Cruz (Case Western Reserve University). We are thankful to our respective laboratory members who informally contributed to the book by providing ideas and interesting points of discussion. Finally, we would like to acknowledge the many investigators in the cancer stem cell field for providing a strong foundation upon which this book was generated and sincerely apologize to the investigators whose work we were unable to include or cite due to space limitations.

With love, we really appreciate our respective family members who provided tremendous support and inspiration for the book.

Huiping Liu, MD, PhD

Justin D. Lathia, PhD

Section 1

CSC Overview and Methodology

Outline

Chapter 1. Introduction: Cancer Stem Cells

Chapter 2. Overview: Cancer Stem Cell Self-Renewal

Chapter 3. Enrichment and Interrogation of Cancer Stem Cells

Chapter 1

Introduction

Cancer Stem Cells

S.S. Mitra, J.Q. He, R. Esparza, G. Hutter, S.H. Cheshier,  and I. WeissmanStanford University, Stanford, CA, United States

Abstract

Cancer stem cells (CSCs) are tumorigenic cells that have the capacity to self-renew and differentiate into various types of cancer cells. It is hypothesized that the stemness of CSCs along with their ability to remain quiescent during periods of intense stress and to be shielded and nourished by their respective niches, allow these cells to evade chemotherapy and radiotherapy, thus making them the prime culprits for cancer recurrence even after aggressive treatment. In recent years, a multitude of models have been developed to study CSC development and propagation. These studies have suggested that successful cancer remission is contingent on the elimination of the CSC population, a task which currently is thought to require multiple synchronized therapies against the various tools these cells have developed to evade eradication. In this chapter, we will begin by characterizing CSCs and conclude with an overview of current therapeutic strategies to target this elusive and lethal cell population.

Keywords

Cancer recurrence; Cancer stem cells (CSC); CD133+; CD44+; Genetically engineered mouse models; Immunotherapy; Minimal residual disease; Stem cell niche

Contents

Introduction

Cancer Stem Cell Origins

Cancer Stem Cell Generation From a Single Cell of Origin

Two Models for Tumor Growth and Expansion

Identification of the Cancer Stem Cell Population

Features of Cancer Stem Cell

Biomarkers for Cancer Stem Cell Identification

Key Signaling Pathways Involved in Cancer Stem Cell Maintenance

Mouse Models of Cancer Stem Cell Function

Patient-Derived Xenograft Models

Germline Genetically Engineered Mouse Models

Somatic Genetically Engineered Mouse Models

Germline-Derived Allograft Models

Humanized Mouse Models

Strategies for Targeting Cancer Stem Cell

Protective Signaling Pathways

Immunotherapies Against Cell Surface Markers

Tumor Microenvironment—The Complicit Cells of the Cancer Stem Cell Niche

ATP-Binding Cassette Transporters: Multidrug and Redox Stress Resistance

Future of Cancer Stem Cell-Directed Therapies

Conclusion

List of Acronyms and Abbreviations

References

Introduction

Somatic stem cells have been known to exist since at least the 19th century through the study of lower organisms such as planarians and salamanders those were capable of remarkable tissue regeneration [1]. The subsequent study of human teratomas, germ cell tumors that could grow tissues such as bone and teeth in ectopic locations, brought the concept of stem cells—a population of cells which could remain poised and uncommitted to specific lineages even after adulthood—to human biology [2]. By the late 1950s, Conrad Waddington [3] had postulated that by regulating gene expression, cells become more specialized the more they divide—eventually becoming committed to a specific cell fate [3]. The earliest cells in the division hierarchy, termed stem cells, have the capacity not only to divide and proliferate into committed cells, but also to self-renew and create more cells with the same uncommitted identity. These asymmetric divisions thus result in the maintenance of a self-renewing pool of stem cells with a life-long responsibility to provide differentiated cell types. Of note, at each subsequent differentiation step in the cell division hierarchy, self-renewal potential is lost. Committed cells lose their ability to regenerate and therefore generally have limited cellular life spans.

Cancer stem cells (CSCs) are a subset of tumor cells which have escaped cell cycle regulatory mechanisms, cell death, and yet have retained the immense self-renewing and proliferative potential of stem cells [4]. In this manner, they have the capacity to regenerate entire tumors from a limited number of cells. Their existence was first documented by Bonnet and Dick [5] in transplantation studies of human acute myeloid leukemia (AML) in mice with severe combined immunodeficiency disease (SCID). Of the transplanted leukemic cells, only an estimated population of between 0.01 and 1% of the total cell population was capable of initiating AML in the immunocompromised mice. Termed SCID leukemia–initiating cells, they were found to undergo rapid clonal expansion and appeared to be at the top of a cancer cell hierarchy. Later, it was shown that most AML tumor-initiating cells were at the stage of the multipotent progenitors (MPPs), and not the hematopoietic stem cells (HSCs) [6], opening the question as to how does a normally non-self-renewing cells gain both self-renewal and unlimited expansion. The introduction of the CSC hypothesis has led to new developments and insights into the nature of tumor propagation and possible therapeutic targets. Yet, much is still unknown about this important tumor cell subtype in each of the human and experimental cancers. In this chapter, we will discuss the biological properties of CSCs, current research on their identification and function, and future goals for CSC-directed therapies.

Cancer Stem Cell Origins

Cancer Stem Cell Generation From a Single Cell of Origin

The cells within a tumor are derived from tissues and organs which contain normal stem cells, progenitors, and lineage committed cells, eg, the lineage hierarchy of the blood system. Identifying the complete roadmap of transitions from HSC through MPPs lacking self-renewal (short term-HSC, MPPs [7,8]) to common myeloid progenitor in mice [9] and humans [10], and Common lymphoid progenitor (CLP) [11], and downstream from them ever more committed progenitors (eg, granulocyte–macrophage progenitor (GMP) and megakaryocyte/erythroid progenitor) [9,12] allowed Weissman and colleagues to phenotypically isolate the cell types from which gave rise to leukemias. The critical development of a strain of mice where two pathways important in programmed cell death was blocked in hematopoetic cells [13]. Serial transplantation of leukemias from mice that developed AML could only be achieved with GMP cells. They further inferred that the progression to leukemia required at least five to seven rare events, either genetic or epigenetic; however, most of these events could not confer self-renewal, and so must have occurred in self-renewing cells to persist sufficiently to form a clone that was leukemic [14]. The pathways from the cell type that acquired the first oncogenic mutation, also known as the cell of origin to CSC, are varied and complex. However, it has been shown that in in vitro models of hematopoietic malignancy, certain oncoproteins may activate genetic programs involved in self-renewal, thereby conferring stemness to committed malignant cells and leading to the creation of CSCs [15–17]. In vivo studies using an MLL1–AF9 mouse model of AML confirmed this finding but with an additional caveat: the amount of translocation product expressed determined the efficiency of CSC generation [18]. Below a certain threshold, oncogenic capacity was limited, demonstrating the importance not only of oncoprotein presence, but also of gene dosage, in tumor formation. It is important to note, however, that the CSC population of a tumor is almost always genetically distinct from the cell of origin. Two models have been proposed to explain why this may be the case.

Two Models for Tumor Growth and Expansion

Two models exist to explain the heterogeneity of tumor growth. The first, which emphasizes a population hierarchy in which all stemness derives from rare CSCs at the top and the tumor bulk is comprised of differentiated CSCs, is referred to as the hierarchical model. By contrast, the second, which emphasizes the functional heterogeneity within the tumor population and posits that all tumor cells contribute to tumor maintenance, is referred to as the clonal evolution (CE) model. It is important to note that these two pathways are not mutually exclusive, and that both pathways may very well occur in the same tumor. In both cases, it is postulated that an acquired mutation confers a growth and proliferative advantage on a certain malignant cell type, allowing it to outcompete surrounding cell types and thus forms a tumor [19]. Yet the CE model postulates that the heterogeneity of tumor cells—specifically, the sheer number of different mutations often found in a single tumor—make it likely that tumors form from a multitude of genetic and epigenetic errors [20]. A dominant CSC may later give way to a cell downstream that has acquired an even more explosive growth advantage [21]. While the hierarchical model works well to characterize somatic stem cells, in the CE model, such structure is often obliterated by the rapid accumulation of mutation process that accelerates as essential DNA repair and cell cycle checkpoint processes are increasingly disrupted [20].

Identification of the Cancer Stem Cell Population

Features of Cancer Stem Cell

CSCs are robust cells which may have acquired characteristics similar to their normal tissue stem cell. The expression of ABC transporters and telomerase and glutathione synthetase are properties of normal tissue stem cells that are extended into their CSC progeny [22–27] that allow for cell survival and proliferation even after exposure to anticancer therapeutics. For example, certain gastrointestinal cancer cell lines show increased resistance to oxidative stress via interactions between CD44 and cell surface cystine–glutamate exchange transporters which result in increased synthesis of reduced glutathione, a key molecule involved in the neutralization of reactive oxygen species [15]. In this manner, they are able to gain a survival advantage in inflammatory environments. Other studies have shown that CSCs are also capable of extensive metabolic reprogramming [16,17], rapid DNA damage repair [18,28], as well as enhanced drug excretion through ATP-binding cassette (ABC) transporters [29] of particular concern in the context of chemotherapeutic agents and other anticancer drugs.

Given the aggressively proliferative nature of CSCs, it is no wonder that they have been found to contribute significantly to the formation of minimal residual disease (MRD) [30,31]. These cells, which can escape targeting by anticancer therapy and remain undetected even as the tumor bulk regresses, are capable of regenerating entire tumors from an astonishingly small starting population. Moreover, because of the selection pressures introduced by chemotherapy, cancer recurrence after drug administration often grows more aggressively than the original tumor. Kurtova et al. found that the administration of chemotherapeutic agents triggered apoptosis in healthy cells which subsequently released prostaglandin E2, inducing previously dormant keratin14+ bladder CSCs to proliferate again [32]. Overcoming these hyperaggressive characteristics of CSC, especially the paradoxical enrichment often found after incomplete prior therapy, is of particular interest in the current development of novel therapeutics.

Biomarkers for Cancer Stem Cell Identification

No specific set of markers have yet been identified that both sensitively and specifically detect the CSC population. For example, while cell surface protein CD133 has been widely implicated in multiple cancer types and is currently used as part of the standard practice for CSC identification from a heterogeneous cell population, it has been shown that separation of CD133+ and CD133− populations and subsequent functional assays reveal two genetically distinct cell populations that have different growth dynamics but nevertheless both retain tumorigenic potential [33,34]. Moreover, in the setting of pancreatic cancer, it was noted that CD44+/CD24− cells, which expressed CSC-specific markers first isolated in breast cancer, exhibited variable overlap with CD133+ cells [35]. These two studies indicated that although these markers were apparently specific to the CSCs described in these studies, different CSCs have different markers; thus these markers were not universal, just as normal stem cell markers for one tissue stem cell do not tell us much about other tissue stem cells, were not necessarily sensitive, and were unable to identify large portions of the CSC population. Nevertheless, the combination of the various surface markers along with high ALDH1 levels did yield a functionally enriched CSC population with a remarkably low cellular threshold needed to initiate tumor formation [36]. Table 1.1 illustrates which tumors currently have a verified CSC population and includes their specific markers.

The gold standard assay for identifying a CSC population is the in vivo limiting-dilution assay, in which primary tumor cells are mechanically disassociated, cellular fractions are separated, and the cellular mixtures are injected into the homologous organ or tissue in immune-deficient mice to form xenografts in recipient mice [73]. By monitoring tumor development in these mice, it is possible to set a lower limit for the frequency of CSCs in a given cell population [74]; the in situ CSC, which do not need to have genes expressed to protect them between isolation and transplantation, neither of which are physiological. The observation of tumors in recipient mice supports the existence of a CSC population that is proliferative, capable of self-renewal, and able to make the bulk of the differentiated tumor cells that lack stemness. Moreover, because it is possible to separate tumor cells into distinct fractions through techniques such as fluorescence-activated cell sorting (FACS), one can also use this assay to probe the existence of a hierarchical cellular structure within different tumor compartments [73].

Table 1.1

Verified Cancer Stem Cell Populations and Their Corresponding Markers in Various Cancers

An alternative to the in vivo limiting-dilution assay is the in vitro limiting-dilution assay, which utilizes cell culture techniques to measure tumorigenicity rather than transplantation. In this approach, tumor cells are again mechanically dissociated and cellular fractions separated, with the difference being that culture then takes place in microtiter wells [75]. Stemness is assayed by staining for stem cell associated markers, as well as by functional assays such as the formation of cellular progenitor structures (eg, tumorspheres) and the ability to maintain self-renewal after serial passages [47,75–77]. However, any interpretation from in vitro experiments is tempered by the hyperoxic in vitro environment and the lack of a stem cell niche.

In vitro measures are often used as a convenient and cost-effective alternative to animal studies. However, whether cell culture is representative of in vivo work is an ongoing concern [78]. For example, the receptor EGFRvIII, overexpressed in up to 70% of glioblastoma (GBM) tumors, is a promising therapeutic target with multiple ongoing clinical trials selectively targeting EGFRvIII-amplifying cells [79,80]. Use of EGFRvIII+ cells in murine xenografts accurately model CSC attributes, including preferential residence in the perivascular niche and the ability to differentiate into endothelial cells [49]. However, normal in vitro culture conditions select against EGFRvIII expression, and cultured human-derived cell lineages quickly lose the marker after serial passaging [81,82]. EGFRvIII expression can be retained by culturing cells in niche-specific serum-free neurosphere media [49]. Thus, whether in vitro studies may be representative of in vivo studies may ultimately depend on how well culture conditions reflect the composition of the native CSC niche.

Key Signaling Pathways Involved in Cancer Stem Cell Maintenance

CSCs maintain their tumorigenicity and stemness through several key signaling pathways, including PI3K/AKT, NF-kB, Notch, Hedgehog, Wnt/β-catenin, and others [83–87]. Aberrant signaling in one or more of these pathways results in the formation of cells that are hyperproliferative, yet resistant to normal cellular checkpoint mechanisms. For example, Notch signaling is implicated in cell self-renewal and plasticity, and Notch4 has been found to be upregulated in breast CSCs [87,88]. Similarly, Wnt has signaling also been associated with maintenance of stemness in CSC populations, with abnormal Wnt/β-catenin signaling contributing to leukemias as well as solid organ malignancies such as colon and breast carcinoma [89,90]. In colon carcinoma specifically, abnormal Wnt expression has been found to inappropriately activate an epithelial–mesenchymal transition in gut cells, leading the cells to gain invasive properties and increasing potential for cancer metastasis [91]. PI3K/AKT affects Bcl-2 and other apoptotic inhibitory proteins, thereby conferring increased resistance to apoptosis in leukemias and other cancers such as melanoma, prostate, and endometrial [65,92]. NF-kB also affects Bcl-2 but also regulates the cellular inflammatory response to cytokines [86]. Aberrant NF-kB expression in CSCs thus promotes a hyperinflammatory state while increasing CSC resistance to inflammation, resulting in cancer progression.

Mouse Models of Cancer Stem Cell Function

Mouse models have contributed significantly to the understanding of CSC biology. In preclinical CSC research, there is now a multitude of transplantable patient-derived or animal-derived models that can be placed orthotopically (in the same anatomical location where the tumors arose from) or heterotopically (in a different anatomical location where the tumors arose from) into new syn-, allo-, or xenogeneic hosts. Additionally, organ- and cancer-specific genetically engineered mouse (GEM) models, further stratified into either germline or somatic (non-germline), serve as an important tool to study cancer initiation, progression, and metastasis. Recently, optimized humanized mouse models have also been developed. We will give a brief overview of each.

Patient-Derived Xenograft Models

The current gold standard for tumor induction is the transplantation of either low-passage CSC lines from patient-derived primary or metastatic tumors, or of directly FACS-sorted subsets of potential CSC candidates, either heterotopically or orthotopically into immunocompromised host mice. These tumors, called patient-derived xenografts (PDXs), can also be serially transplanted into subsequent hosts to confirm the tumorigenic potential of the putative CSC candidate, although this presents the tumors with selections that may not occur in humans, and within a few serial transplants tumors are commonly selected that grow more rapidly and have increased instances of new genetic and epigenetic alterations.

NSG, NOG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ: NSG and NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic: NOG) and Rag1—Il2Rg—mice, which are B- and T-cell-deficient and lack natural killer (NK) cell activity represent a well-established model system to study the engraftment and mobilization of human peripheral blood stem cells and are excellent hosts for growing experimental leukemias and solid tumors [9,10,93,94]. The understanding and identification of CSC in hematopoietic malignancies, especially in leukemias [95] and lymphomas [5] has largely profited from this approach, but recently, CSCs in solid cancers such as breast [36], colon [14,96], skin [72,97], melanoma [71], and brain [47] emerged using the SCID repopulation approach as proof of tumorigenicity. However, the degree of compromise of the host immune system can be permissive for the tumorigenic potential of unselected patient-derived cells as shown by Quintana et al. in melanoma-derived xenografts and indicated that the tumor-initiating potential of cells within the tumor bulk may vary significantly depending on the tumor type [78]. However, Quintana et al. transplanted small numbers of melanoma cells in high-protein matrigel, a basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins used by very few laboratories. While the use of high-protein matrigel does bring up interesting questions of using the correct niche for xenotransplant models it also brings in the added possibility of the contribution from the matrigel components in increased tumor initiation.

While heterotopic models, eg, those created via subcutaneous injection of CSCs or cancer organoids, are simpler to implement versus orthotopic models, their most prominent disadvantage is the absence of the original supporting stem cell niche or microenvironment. This problem could partially be solved by co-transplanting tumor cells and human stroma cells or basement membrane matrix proteins (eg, Matrigel) [98]. However, most stromal cell compartments such as endothelial cells and fibroblasts will rapidly be replaced by mouse tissue [99]. While heterotopic PDX models may be used in high throughput testing of anti-CSC drugs, the microenvironment, nontumor cell stroma, lymphatic drainage, and vascularization of heterotopic tumors are not the same as from where the tumor originated, thus limiting the external validity of this model [100].

In contrast, orthotopic models possess tissue-specific stromal cell types and extracellular matrix components that support the cancer type inherent CSC niche [101]. Due to the lack of certain components of the immune system, however, PDX tumors tend to progress faster than tumors otherwise would in patients, which might render the model more sensitive to targeted anti-CSC drugs than the true physiologic response [102]. Using fluorescent and luminescent labeling techniques, tumor growth and effect of anti-CSC drug candidates can be monitored noninvasively or using intravital microscopy to specifically visualize the interaction of the cancer cells within the tumor microenvironment. For example, Lee et al. showed that the homing of hematopoietic cancer cells and normal stem cells to their niche in the bone marrow could be modeled using implantable marrowlike microenvironments and monitored by intravital microscopy and fluorescence computed tomography [103].

Germline Genetically Engineered Mouse Models

Of all murine cancer models, GEM models provide the most complete representation of cancer development; cancers develop from initiation through progression, coevolve with intrinsic stroma, and possess an intact immune system [104]. Techniques to engineer the germline of mice include constitutive and conditional knock-in/knock-out strategies, targeted transgene expression, and inducible transgene expression. In the last several decades, numerous organ-specific germline deletions of tumor suppressor genes or mutations of oncogenes have been created that shed light on tumorigenesis [105]. In some instances, spatial and temporal induction or deletion of oncogenes/tumor suppressor genes, mainly by exploiting countless variations of the powerful organ-specific recombination tools, Cre/LoxP and FLP/FRT, resulted in a sequence of oncogenic events mirroring human tumorigenesis to a great extent [105]. Bacterial Cre and yeast FLP enzymes are site-specific recombinases that catalyze specific recombination between defined loxP and FRT sites, respectively [24,106,107]. Therefore, in the presence of Cre or FLP protein expression, homologous recombination is induced between loxP or FRT sites that flank the gene of interest thus recombining out the flanked genetic sequence and deleting the gene of interest. If a STOP codon is flanked by loxP or FRT site, potentially oncogenic mutations can be driven as well. The fact that Cre recombinases have been engineered to be inducible and driven by tissue-specific promoters renders this approach an invaluable tool to simulate oncogenesis [108].

Based on dissociation of germline GEM-derived tumors, it was possible to isolate tumor-initiating cells and propagate and characterize them further in allograft or xenograft models; exemplified by CD15+ cells sorted from Ptch mutant mice with medulloblastoma [109]. Yet, GEM cancer models provide the only opportunity to evaluate drug delivery, therapeutic response, and biomarker expression for cancers evolving within their natural microenvironment (autochthonous cancers) [104]. Recently, the discovery of the CRISPR/Cas9 technique has decreased the time to achieve germline genome editing significantly [110].

Somatic Genetically Engineered Mouse Models

Given the low penetrance rate and high heterogeneity of traditional germline GEM models, other approaches considering direct manipulation of somatic cells have been developed. By utilizing genetically engineered murine embryonic stem cells for chimera-formation and transplantation, these models have the additional benefit of requiring less time and fewer resources to develop, two major obstacles to preclinical trials [111].

Chimeric mouse models take advantage of genetic mutations that predispose toward tumor formation. They are created by transplanting genetically engineered embryonic stem cells into the inner cell mass of preimplantation-stage embryos, commonly at the blastocyst stage. These blastocytes are then implanted into pseudopregnant mice, eventually generating chimeric mice whose cells are a mosaic of engineered and original host cells. One such chimeric model by Zhou et al. employed ESCs engineered to contain tissue-specific, inducible oncogenes and a luciferase marker, and deletions of prominent tumor suppressors to generate chimeras which were thus predisposed to developing specific tumor types [112].

Transplantation mouse models harness the ability of stem and tumor cells to proliferate and compete with the native cells in a host mouse. The models often involve the implantation of genetically modified stem or tumor cells into adult tissue, and rely on the ability of the implanted cells to hone to their respective stem cell niches in the host animal and act as new cells of origin. One such example is a transplantation model by Alcantara Llaguno et al., that described the localization of GBM cells-of-origin to neural stem cells in the subventricular zone [113].

Germline-Derived Allograft Models

GEM-derived allograft (GDA) models combine the human cancer similarity of GEM models with the relative simplicity of transplantation approaches. Importantly, the host mouse is a genetic-background matched, immunocompetent animal that enables researchers to study the immune-related microenvironment of the CSC niche as well. GDA models represent a better alternative to assess immunomodulatory therapies against CSCs, since a fully functional immune system is in many instances necessary to validate the effect of immunomodulatory agents [114].

Humanized Mouse Models

To overcome the immune-related limitations of PDX models, humanized mouse models (eg, those created by transferring functional human immune cells or purified HSCs into immunocompromised mice) have been developed and optimized in recent years. Novel humanized mouse model with much better engraftment of human myeloid cells will prove very useful in monitoring the full potential of human immune cells against CSC xenografts [115].

Strategies for Targeting Cancer Stem Cell

As previously discussed, the stemness of CSCs confers undesirable traits that promote cancer progression and metastasis, and contributes to the formation of cancers that are refractory to chemo- and radiotherapy. Here, we will describe several strategies to target CSCs based on these acquired characteristics.

Protective Signaling Pathways

Protective signaling pathways such as antiapoptotic pathways have generated much interest among researchers attempting to halt the proliferation of CSCs. For example, inhibitors of NF-kB have been shown to improve cancer response to chemotherapy. Combined treatment with NF-kB and chemotherapeutic agents such as the DNA intercalating antibiotic doxorubicin leads to a marked reduction in multidrug resistance. Moreover, inhibitors of the Hedgehog pathway such as cyclopamine have shown the ability to inhibit metastasis in orthotopic mouse models of pancreatic cancer and in in vitro adherent glioma models of GBM [116,117].

Also of great research interest is the development of antagonists to the Notch and Wnt/β-catenin pathways, which have been implicated in metastasis and tumorigenicity in breast and colon cancer [118,119]. Notch signaling, mediated by γ-secretase, was found to be important in the development of brain metastases from breast cancer in mouse models. Inhibition of Notch1 in these mice reduced the CD44+ and CD24− CSC subpopulation and lowered the incidence of brain metastases [119].

CSC quiescence—the activation of a hypometabolic state of dormancy—when faced with unfavorable cellular environments, is also under consideration as a potential therapeutic target. Since dormant CSCs are largely refractory to cytotoxic agents, which often target actively replicating cells through disruption of DNA replication, strategies to lock out these cells from their resting G0 cell cycle phase have gained prominence. One such strategy involves Fbw7, a component of the E3 ubiquitin ligase which is involved in the degradation of proto-oncogenic proteins such as Notch and c-Myc [120–122]. Fbw7 inactivation promotes the transition of CSCs from dormancy into a proliferative state, allowing for their targeting by conventional chemotherapeutics. For example, in chronic myeloid leukemia CSCs which had acquired resistance to the tyrosine kinase inhibitor imatinib, Fbw7 inhibition improved drug response by stimulating CSCs to proliferate, leaving them vulnerable once again to antiproliferative therapeutics [123].

Finally, as mentioned previously, the protective role of the CD44-xCT-GSH axis against oxidative stress has been exploited extensively by CD44+ CSCs to gain a competitive advantage in hyperinflammatory environments [15]. This feature of CSCs is currently being targeted by anti-xCT therapies, including the drugs sulfasalazine and auranofin, conventionally used to treat rheumatoid arthritis and other autoimmune disorders [124]. Interestingly, while sulfasalazine treatment preferentially targets CD44+ cells, the epidermal growth factor receptor-targeted drug cetuximab selectively targets the CD44− population [125,126]. Combination therapy with sulfasalazine and cetuximab therefore might be an effective strategy to treat tumors that may otherwise develop marked drug resistance.

Immunotherapies Against Cell Surface Markers

Another key strategy to target CSCs has been the development of monoclonal antibodies to surface markers found specifically on the CSC population. As we have discussed, no unifying surface antigen has been found to represent the entire CSC population, but antibody therapy against specific markers have been associated with depletion of the CSC population in certain cancers. For example, in the case of AML, in which markers such as CD33, CD44, and IL-3R are expressed predominantly in the CSC population, drugs such as gemtuzumab ozogamicin (an anti-CD33 humanized mouse monoclonal antibody conjugated to the cytotoxic drug calicheamicin) have been developed for use in clinical treatment [127]. Anti-CD44 and anti-IL-3R therapies have shown to be promising in mice; however, it appears that the bone marrow niche may be in part shielding CSCs from the brunt of the cytotoxic damage via activation of the PI3K/Akt pathway in CSCs [128,129].

Tumor Microenvironment—The Complicit Cells of the Cancer Stem Cell Niche

The role of the stem cell microenvironment, or niche, in maintaining stemness is well known. For HSCs, the bone marrow niche comprising cellular and noncellular elements provide a fertile ground for stem cell maintenance [130]. The cellular elements include specialized compartments of the bone marrow such as osteoblastic [131] and vascular niche [132]. Similar to HSC, leukemic stem cells (LSC) require the marrow niche to sustain their perpetual, malignant self-renewal state. The niche promotes angiogenesis to nourish tumors and secretes important growth factors and chemoresistance signals, thus serving as a shield from chemotherapeutic agents and other proapoptotic factors [133]. For example, bone marrow stromal cells which serve as important niche cells for leukemia CSCs have been found to secrete stromal cell-derived factor-1 (SDF-1/CXCR12). CXCR12 could allow tumor cells, which have the associated receptor CXCR4, to home to the niche for nourishment and replenishment. While it is known that leukemic cells co-opt the bone marrow niche and create an abnormal microenviroment to sequester transplanted human CD34+ cells [134], it is possible that the receptor–ligand interaction ensures close associate of LSC with stromal cells to manipulate the stromal cells into secreting additional growth and antiapoptotic signals.

Similarly, solid tissue stem cells also reside within stem cell niches; highlighting the fundamental importance of these specialized microenvironments for normal stem cell biology [135]. It was shown as far back as in 1940s that tumor cells migrate into the normal brain around blood vessels (perivascular satellitosis), suggesting that glioblastoma cells might develop a special relationship with the surrounding vasculature [136]. However, only in the last decade that a functional relationship between the tumor vasculature and glioblastoma CSC has emerged where CD133+ GBM CSC produced high levels of vascular endothelial growth factor (VEGF) which lead to highly vascular and hemorrhagic tumors in immunocompromised mice. We now know that CSC from GBM and other brain tumors are maintained within vascular niches that mimic the neural stem cell niche [137].

Tumor angiogenesis through the secretion of VEGF is another characteristic of the niche that promotes tumor growth and survival. Studies in mice have shown that administration of bevacizumab, a recombinant antibody against VEGF-A, decreases the number of GBM CSCs by disrupting tumor vasculature, thereby compromising the CSC niche [137,138]. Moreover, combination therapy of VEGF antibodies with cyclophosphamide, a cytotoxic drug, was more effective against glioma xenografts in vivo than either therapy alone, providing more support for simultaneously targeting CSCs via multiple pathways to achieve maximal antitumor effect [139].

ATP-Binding Cassette Transporters: Multidrug and Redox Stress Resistance

In addition to tumor angiogenesis and activation of antiapoptotic pathways, CSCs also appear to express high levels of ABC transporters that can pump a variety of structurally unique drugs out of the cell, thus conferring an additional degree of resistance to CSCs and contributing to MRD [114,140,141]. The ABC superfamily includes multidrug resistance proteins, breast cancer resistance protein, and P-glycoprotein [142]. Currently, Chen et al. are attempting to synthesize inhibitors of ABC function using 20(S)-protopanoxadiol (PPD) derivatives. Of note, PPD12 was found to increase the concentration of cytotoxic drugs adriamycin and rhodamine123 in chemoresistant cells, presumably by directly binding to an inhibitor of the transporter itself [143]. Because of their extensive capacity to contribute to MRD and tumor recurrence and metastasis, ABC transporters are a highly promising target for future therapeutics.

Future of Cancer Stem Cell-Directed Therapies

CSC-directed immunotherapies are among the most promising novel agents, yet also have the greatest potential to conflict with current standards of care. Immunotherapies are dependent on a controlled activation of the immune system to selectively target and attack cells carrying a preselected antigen. An often highlighted advantage of active immunotherapies is their long-lasting