Tumor Microenvironment

Edited by

Dietmar W. SiemannUniversity of Florida, Shands Cancer Center

A John Wiley & Sons, Ltd., Publication

support9780470669808.jpg

Tumor Microenvironment

Tumor Microenvironment

Edited by

Dietmar W. SiemannUniversity of Florida, Shands Cancer Center

A John Wiley & Sons, Ltd., Publication

This edition first published 2011 2011 John Wiley & Sons, Ltd.

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Library of Congress Cataloging-in-Publication Data

Tumor microenvironment / editor, Dietmar Siemann.p. cm.

Includes index.ISBN 978-0-470-74996-8 (cloth)

1. Cancer cells. 2. Carcinogenesis. I. Siemann, Dietmar W.RC269.T86 2011616.99′4071 – dc22

2010023380

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First Impression 2011

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Every day is a journey,

and the journey itself is home.

– Basho

Contents

Preface xiii

List of Contributors xv

1 The Microenvironment in Cancer 1Nicole N. Parker and Dietmar W. Siemann

1.1 Introduction 11.2 A highly selective process is required to obtain the cancer phenotype 11.3 The cancer phenotype 21.4 The extracellular matrix 31.5 Motility, invasion, and metastatic ability 41.6 Impact of the tumor microenvironment on the control of cancer 41.7 Targeting the tumor microenvironment 51.8 Summary 5References 6

2 Establishing the Tumor Microenvironment 7Allison S. Betof and Mark W. Dewhirst

2.1 Introduction 72.2 From cancerous cells to a tumor 82.3 A tumor is more than cancer cells and fibroblasts 92.4 Communication between the tumor cells and stroma 112.5 Hypoxia and angiogenesis 122.6 Conclusion 24Acknowledgements 24References 24Further reading 33

3 Contributions of the Extracellular Matrix to Tumorigenesis 35Marie Schluterman Burdine and Rolf A. Brekken

3.1 The extracellular matrix 35

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viii CONTENTS

3.2 Manipulation of the ECM during tumor development 383.3 Matricellular proteins and their complex effects on tumor development 393.4 Conclusion 47References 48

4 Matrix Metalloproteinases and Their Inhibitors – Friend or Foe 53Mumtaz V. Rojiani, Marzenna Wiranowska and Amyn M. Rojiani

4.1 Introduction 534.2 Matrix metalloproteinases 544.3 Tissue inhibitors of matrix metalloproteinases 634.4 Concluding comments 69References 69

5 Role of Tumor-Associated Macrophages (TAM) in Cancer RelatedInflammation 77Antonio Sica and Chiara Porta

5.1 Introduction 775.2 Functional plasticity of macrophages 775.3 Macrophages as key orchestrators of cancer-related inflammation 795.4 Recruitment and differentiation of TAM 815.5 Protumoral functions of TAM 835.6 Molecular determinants of TAM functions 875.7 Therapeutic targeting of TAM 895.8 Conclusions 91References 92

6 Bone Marrow Stroma and the Leukemic Microenvironment 99William B. Slayton and Zhongbo Hu

6.1 Introduction 996.2 Components and function of the normal bone marrow microenvironment 996.3 Leukemia and its microenvironment 1196.4 Summary 123References 124

7 Microenvironment Factors Influencing Skeletal Metastases 135Alessandro Fatatis, Julia A. D’Ambrosio, Whitney L. Jamieson,Danielle L. Jernigan and Mike R. Russell

7.1 Introduction 1357.2 The bone microenvironment as a target for cancer cell dissemination 1367.3 Roles of the bone microenvironment in promoting the arrest of circulating

cancer cells at the skeleton 1377.4 Concluding remarks 153References 153

CONTENTS ix

8 Premetastatic Niches 161Kevin L. Bennewith, Janine T. Erler and Amato J. Giaccia

8.1 Introduction 1618.2 ‘Seeds’ influencing the ‘Soil’ 1628.3 Cellular components of premetastatic niches 1648.4 ECM components of premetastatic niches 1668.5 Premetastatic niche formation precedes metastatic growth 1708.6 Therapeutic targeting of the premetastatic niche 1728.7 Evidence for premetastatic niches in the clinic 1748.8 Concluding remarks 174References 175

9 Hypoxia, Anerobic Metabolism, and Interstitial Hypertension 183Michael F. Milosevic

9.1 Introduction 1839.2 Pathophysiology of the tumor microenvironment 1849.3 Evaluating the tumor microenvironment 1899.4 Biologic and therapeutic implications 1959.5 Clinical implications 1999.6 Summary 201References 201

10 Hypoxia and the DNA Damage Response 207Isabel M. Pires, Rachel Poole and Ester M. Hammond

10.1 Introduction 20710.2 The DNA damage response 20810.3 Hypoxia regulation of DNA repair 21510.4 Context synthetic lethality: exploiting hypoxic deregulation

of DNA repair 22010.5 Conclusions 221References 221

11 Non-Invasive Imaging of the Tumor Microenvironment 229Bénédicte F. Jordan and Bernard Gallez

11.1 Introduction 22911.2 Imaging tumor vasculature, perfusion, and angiogenesis 22911.3 Imaging tumor hypoxia: chronic and acute 23411.4 Imaging tumor oxygen consumption 24011.5 EPR oximetry 24011.6 Imaging tumor interstitial fluid pressure (IFP) 24411.7 Imaging tumor pH 24511.8 Imaging tumor redox status 24811.9 Imaging tumor response 25011.10 Optimizing therapeutic intervention using molecular imaging 256

x CONTENTS

11.11 Conclusions 261References 261Further reading 270

12 Hypoxia-Inducible Factor 1 (HIF1) Mediated Adaptive Responsesin the Solid Tumor 271Tereza Goliasova and Nicholas C. Denko

12.1 Introduction 27112.2 Molecular consequences of tumor hypoxia 27212.3 Hypoxia inducible factor 1 27312.4 HIF-1 subunits and domain structure 27312.5 Regulation of HIF-1α protein stability and activity by post-translational

modifications 27412.6 HIF isoforms 27512.7 Oxygen-independent HIF signaling 27612.8 HIF target genes 27712.9 Hypoxia and oxygen delivery 27912.10 Hypoxia and glucose metabolism 28012.11 Hypoxia and acidosis 28112.12 Hypoxia and metastasis 28212.13 Therapeutic implications 283References 285

13 Regulation of the Unfolded Protein Response in Cancer 291Jing Zhang and Albert C. Koong

13.1 Introduction 29113.2 The UPR signaling cascade 29213.3 Hypoxia activates UPR 29513.4 UPR and expression of UPR-targeted genes in cancer 29813.5 Concluding remarks 304References 304

14 Influence of Hypoxia on Metastatic Spread 311Richard P. Hill and Naz Chaudary

14.1 Introduction 31114.2 The metastatic process 31314.3 The tumor microenvironment and metastasis 31614.4 Summary 326References 326

15 Drug Penetration and Therapeutic Resistance 329Andrew I. Minchinton and Alastair H. Kyle

15.1 Introduction 329

CONTENTS xi

15.2 Tumor microenvironment 33015.3 Drug penetration 33415.4 In vitro tumor models 33815.5 Conclusions 346References 347

16 Impact on Radiotherapy 353Michael R. Horsman, Jens Overgaard and Dietmar W. Siemann

16.1 Introduction 35316.2 The tumour vasculature and microenvironment 35316.3 Influence of tumor hypoxia on radiation therapy 35616.4 Reducing hypoxia by increasing oxygen delivery 35816.5 Radiosensitizing hypoxic cells 36316.6 Killing the resistant cell population 36516.7 Vascular targeting approaches 36616.8 Conclusions and future perspectives 367References 368

17 HIF-1 Inhibitors for Cancer Therapy 377Annamaria Rapisarda and Giovanni Melillo

17.1 Introduction 37717.2 Small molecule inhibitors of HIF-1 37817.3 Exploiting HIF-1 inhibitors in combination strategies 39117.4 Conclusions 392Acknowledgements 392References 393

18 Vascular-Targeted Molecular Therapy 401Graeme J. Dougherty and Shona T. Dougherty

18.1 Introduction 40118.2 Approaches to targeting tumor vasculature in vivo 40318.3 Alternative targeting strategies 41218.4 Concluding remarks 413Acknowledgements 413References 413

Index 421

Preface

The tumor microenvironment has long been identified as a major factor influencingtreatment resistance of cancers to radiotherapy and chemotherapy. In addition, itis now well recognized to play a critical role in neoplastic cell initiation, malignantprogression, and the metastatic spread of tumor cells.

The tumor microenvironment, or stroma, consists of cells, extracellular matrixand extracellular molecules. Important cell types that have been identified includefibroblasts, epithelial cells, infiammatory cells, immunocytes, and vascular cells.Research indicates that interactions between the neoplastic cells, the extracellularmatrix (ECM), and stromal cells are bidirectional and dynamic. The microenvi-ronment can exert both stimulatory and growth inhibitory effects on tumor cells.Indeed, cancer cells are often critically dependent on the stroma, as is the casebetween neoplastic and endothelial cells. It is well established that most tumorscannot grow larger than a few millimeters in diameter without inducing their ownvascular network, primarily through the process of angiogenesis.

Because stromal elements and their manifestations are essential for all stagesof tumor development, and lead to significant negative consequences affectingthe management of cancer by conventional anticancer therapies, a number oftreatment strategies directed against tumor microenvironmental infiuences are beingpursued. These fall into two general categories. The first strategy seeks to targetunique conditions of the tumor microenvironment which may be exploited fortherapeutic gain. A primary target in this approach has been tumor hypoxia – aconsequence of the abnormal and inadequate development of blood vessel networksin tumors. Examples of therapeutic strategies being pursued include hypoxic cellradiosensitizers, bioreductive drugs, HIF-1 targeting agents, and gene therapies. Thesecond approach targets stroma itself. Among the approaches are drugs that induceapoptosis or inhibit the function of the stromal cells, or inhibit the factors secretedby stroma that are required for tumor progression and metastasis. The developmentof novel targeting therapies which attenuate VEGF and EGF signaling illustrateprogress in the latter area.

The goal of this book is to review the importance of the tumor microenvironmentin cancer management. Particular emphasis is placed on: (i) the characterization ofthe unique features of the tumor microenvironment, (ii) the determination of factorsthat are critical in the progression and metastatic spread of tumor cells, (iii) the

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xiv PREFACE

identification of tumor cell stem cells and their interactions with stroma at sites ofdissemination, (iv) the impact of the tumor microenvironment on the response toconventional anticancer therapies, and (v) the development and assessment of noveltherapeutic strategies that target the stromal or neoplastic components of the tumormicroenvironment or its functions. Most of all, this book is about researchers’ effortsto achieve better treatment for cancer patients.

This book is the culminating effort of a team of international investigators; someof whom are long-time friends, and others who are new collaborators. For manythe importance of the tumor microenvironment as it relates to cancer therapy hasbeen a long-standing passion. I feel privileged to have worked on this text with suchan outstanding group of co-authors. To them I owe a debt of gratitude, for withouttheir efforts and dedication this book would not have materialized. I thank them fortheir willingness to participate in this project, their timely contributions and theirintegrity. It has been a pleasure working with these colleagues. The quality of thebook reflects their efforts and is to their credit.

I am also grateful to all the staff at Wiley-Blackwell, especially Rachel Moore(Concept Contact), Fiona Woods (Project Editor), Izzy Canning (Publishing Assis-tant) and Sarah Abdul Karim (Production Editor), who have worked diligently tobring this book to fruition. Their assistance in all matters has been invaluable andmuch appreciated.

Too many lives are lost each year to cancer. This book reflects the dedicationof researchers throughout the world who, through their tireless pursuit to gain abetter understanding of the molecular and biologic underpinnings of cancer, seek toidentify the causes of cancer and develop strategies for treatments and cure.

Dietmar W. SiemannGainesville, FL

List of Contributors

Kevin L. Bennewith Stanford University School of Medicine, Department of Radi-ation Oncology, CCSR-South, Room 1255, 269 Campus Drive, Stanford, CA94305-5152, USA

Allison S. Betof Duke University Medical Center, Durham, NC 27710, USA

Rolf Brekken Hamon Center for Therapeutic Oncology, Simmons ComprehensiveCancer Center, Division of Surgical Oncology, UT Southwestern Medical Center,5323 Harry Hines Blvd, Dallas, TX 75390-8593, USA

Marie Schluterman Burdine Hamon Center for Therapeutic Oncology, SimmonsComprehensive Cancer Center, Division of Surgical Oncology, UT SouthwesternMedical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8593, USA

Naz Chaudary Ontario Cancer Institute, Princess Margaret Hospital and theCampbell Family Institute for Cancer Research, 610 University Avenue, TorontoON, Canada, MG52M9

Julia A. D’Ambrosio Department of Pharmacology and Physiology, Drexel Uni-versity College of Medicine, Philadelphia, PA, USA

Nicholas C. Denko Division of Radiation and Cancer Biology, Dept of RadiationOncology, Stanford University School of Medicine, 269 Campus Dr. West, CCSR-1245C, Stanford, CA 94305-5152, USA

Mark W. Dewhirst Gustavo S. Montana Professor of Radiation Oncology, Profes-sor of Pathology and Biomedical Engineering, Duke University Medical Center,Durham, NC 27710, USA

Graeme J. Dougherty Department of Radiation Oncology, Arizona Cancer Center,1501 North Campbell Avenue, Tucson, AZ 85724-5081, USA

xv

xvi LIST OF CONTRIBUTORS

Shona T. Dougherty Department of Radiation Oncology, Arizona Cancer Center,1501 North Campbell Avenue, Tucson, AZ 85724-5081, USA

Janine T. Erler Stanford University School of Medicine, Department of RadiationOncology, CCSR-South, Room 1255, 269 Campus Drive, Stanford, CA 94305-5152, USA

Alessandro Fatatis Associate Professor, Department of Pharmacology and Phys-iology, Department of Pathology and Laboratory Medicine, Drexel UniversityCollege of Medicine, 245 N. 15th Street-MS488, New College Building, Philadel-phia, PA 19102, USA

Bernard Gallez Louvain Drug Research Institute, Biomedical Magnetic ResonanceUnit, Director, Université catholique de Louvain, REMA, Avenue Mounier 73.40,B-1200 Brussels, Belgium

Amato J. Giaccia Jack, Lulu, and Sam Willson Professor of Cancer Biology,Director Division of Cancer and Radiation Biology, Stanford University Schoolof Medicine, Department of Radiation Oncology, CCSR-South, Room 1255, 269Campus Drive, Stanford, CA 94305-5152, USA

Tereza Goliasova Division of Radiation and Cancer Biology, Dept of RadiationOncology, Stanford University School of Medicine, 269 Campus Dr. West, CCSR-1245C, Stanford, CA 94305-5152, USA

Ester M. Hammond Radiation Oncology & Biology, University of Oxford, OldRoad Campus Research Building, Off Roosevelt Drive, Churchill Hospital,Oxford, OX3 7DQ, UK

Richard P. Hill Senior Scientist OCI/PMH, Professor Dept of Medical Biophysics,University of Toronto, 610 University Ave, Rm 10-113, Toronto, ON, Canada,M5G2M9

Michael R. Horsman Danish Cancer Society, Department of Experimental ClinicalOncology, Norrebrogade 44, DK-8000 Aarhus C, Denmark

Zhongbo Hu Department of Pediatrics, Division of Hematology/Oncology, Uni-versity of Florida School of Medicine, Box 100296, Gainesville, FL 32610-0296,USA

Whitney L. Jamieson Department of Pharmacology and Physiology, Drexel Uni-versity College of Medicine, Philadelphia, PA, USA

Danielle L. Jernigan Department of Pharmacology and Physiology, Drexel Univer-sity College of Medicine, Philadelphia, PA, USA

LIST OF CONTRIBUTORS xvii

Bénédicte F. Jordan Louvain Drug Research Institute, Biomedical Magnetic Reso-nance Unit, Director, Université catholique de Louvain, REMA, Avenue Mounier73.40, B-1200 Brussels, Belgium

Albert C. Koong Radiation Oncology, Stanford University School of Medicine,269 Campus Dr. West, CCSR-1245C, Stanford, CA 94305-5152, USA

Alastair H. Kyle BC Cancer Research Centre, University of British Columbia, 675West 10th Avenue, Vancouver, BC, Canada, V5Z1L3

Giovanni Melillo Head, Tumor Hypoxia Laboratory, Developmental TherapeuticsProgram, SAIC Frederick, Inc. Bldg 432, Rm 218, National Cancer Institute atFrederick, Frederick, MD 21702-1201, USA

Michael F Milosevic Department of Radiation Oncology, University of Toronto,Princess Margaret Hospital, 5th Floor Rm 634, 610 University Ave, Toronto,ON, Canada, M5G2M9

Andrew I. Minchinton BC Cancer Research Centre, University of British Columbia,675 West 10th Avenue, Vancouver, BC, Canada, V5Z1L3

Jens Overgaard Danish Cancer Society, Department of Experimental ClinicalOncology, Norrebrogade 44, DK-8000 Aarhus C, Denmark

Nicole N. Parker Department of Radiation Oncology, University of Florida ShandsCancer Center, 2000 SW Archer Road, Gainesville, FL 32610, USA

Isabel M. Pires Radiation Oncology & Biology, University of Oxford, Old RoadCampus Research Building, Off Roosevelt Drive, Churchill Hospital, Oxford,OX3 7DQ, UK

Rachel Poole Radiation Oncology & Biology, University of Oxford, Old RoadCampus Research Building, Off Roosevelt Drive, Churchill Hospital, Oxford,OX3 7DQ, UK

Chiara Porta Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy

Annamaria Rapisarda Tumor Hypoxia Laboratory, Developmental TherapeuticsProgram, SAIC Frederick, Inc. Bldg 432, Rm 218, National Cancer Institute atFrederick, Frederick, MD 21702-1201, USA

Amyn M. Rojiani Department of Pathology-BF-104, Medical College of Georgia,1120 Fifteenth Street, Augusta, GA 30912-3600

Mumtaz V. Rojiani Department of Pathology-BF-104, University of South Florida,Tampa, FL, USA

xviii LIST OF CONTRIBUTORS

Mike R. Russell Department of Pharmacology and Physiology, Drexel UniversityCollege of Medicine, Philadelphia, PA, USA

Antonio Sica Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy

Dietmar W. Siemann John P. Cofrin Professor for Cancer Research, AssociateChair, Department of Radiation Oncology, University of Florida Shands CancerCenter, 2000 SW Archer Road, Gainesville, FL 32610, USA

William B. Slayton Department of Pediatrics, Division of Hematology/Oncology,University of Florida School of Medicine, Box 100296, Gainesville, FL 32610-0296, USA

Marzenna Wiranowska Department of Pathology-BF-104, University of SouthFlorida, Tampa, FL, USA

Jing Zhang Radiation Oncology, Stanford University School of Medicine, 269Campus Dr. West, CCSR-1245C, Stanford, CA 94305-5152, USA

1The Microenvironment in Cancer

Nicole N. Parker and Dietmar W. SiemannDepartment of Radiation Oncology and University of Florida Shands Cancer Center,University of Florida, Gainesville, Florida, USA

1.1 Introduction

In the development of a cancer, the transformation of epithelial cells into a neoplasticand progressively invasive tumor occurs though the acquisition of several procancercharacteristics that can take years or decades to develop. The particular stagesof transformation have been established and a general consensus exists about theproperties of a successful malignancy. While many therapeutics have been developedto combat these properties, these therapies are not universally successful, andtheir efficacy depends on the type and site of the primary tumor, its degree ofvascularization, the proliferative compartment of the tumor, and in particular, thetumor microenvironment. The latter is the key support system of a cancer, and is animportant source of critical protumorigenic factors that facilitate growth, invasion,angiogenesis, and metastatic ability. The focus of this book is to examine how thereliance of tumors on their microenvironments for development and preservationof key cellular functions is now recognized not only as a major contributor tocancer aggression and treatment resistance but also as a potential target for noveltherapeutic intervention strategies.

1.2 A highly selective process is required to obtainthe cancer phenotype

Many studies have focused on the predetermining factors that cause hyperplasia,or the hyperproliferation of cells within their normal environment. On the pathto a cancer, regions of hyperplasticity must subsequently become dysplastic, ordisplay a highly disordered pattern of proliferation with little or no growth reg-ulation. Predisposing genomic lesions in various genes of these dysplastic cells

Tumor Microenvironment Edited by Dietmar W. Siemann 2011 John Wiley & Sons, Ltd

1

2 THE MICROENVIRONMENT IN CANCER

confer a proliferative advantage over normal cellular counterparts. Therefore, theout-proliferation exhibited by dysplastic populations requires additional geneticinstability or genomic modifications, and these cells are considered neoplastic: theyhave an advantageous rate of proliferation with a lack of regulation, and pos-sess other procancerous features at the time of transformation. Oncogenic lesions,coupled with inhibition of tumor suppressors, together contribute to cellular trans-formation. Genomic, proteomic, post-translational, and epigenetic mutations areresponsible for activating oncogenes and inhibiting tumor suppressor genes.

1.3 The cancer phenotype

Although a multitude of potential cancer etiologies may occur for cancer devel-opment, several essential characteristics are present in malignancies (Hanahan andWeinberg, 2000). A key element of malignant transformation is the loss of regula-tory control mechanisms present in normal somatic non-stem cells that are growtharrested and do not divide. Cancer cells not only possess heightened rates of cellproliferation and aberrant cell cycle checkpoints, but also lose contact-inhibitedgrowth regulation. As a result, unlike detached normal cells which die by anoikis orcell detachment-induced apoptosis, cancer cells continue to grow unabated, break-ing through basement membranes and invading extracellular spaces around tissuesand organs.

Extracellular matrix remodeling and cellular changes in adhesion molecules areboth required for a cancer cell to become more motile. Rearrangement of theactin cytoskeleton facilitates cell motility and plasticity, as does downregulation ofadhesion proteins that bind tightly to the extracellular matrix (Chapter 3). A cancercell modifies its adhesive properties and implements a program of non-adhesionthrough multiple modifications.

The progressive growth of a tumor ultimately results in an inability of normal tissueblood vessels to oxygenate and provide nutrients to tumor cells most distal to theblood supply. As a consequence oxygen-deficient (hypoxic) regions develop withinthe tumor (Chapters 2 and 9). The ability of transformed cells to survive hypoxicconditions requires a switch from aerobic to anaerobic glycolysis, a major approachby which cancer cells circumvent the cytotoxic effects of oxygen deprivation (Gatenbyand Gawlinski, 2003). As a result of glycolysis, lactic acid byproducts accumulatein cells undergoing this process. Although this acidification is generally toxic tocells, cancer cells upregulate acid transporter proteins and efficiently secrete acidproducts into the surrounding environment. A side effect of acid secretion is anincrease in local extracellular acidity, fluid retention, and subsequently, an increasein interstitial pressure (Chapter 9). Still, the ability of cancer cells to metabolicallyadapt by preferentially undergoing glycolysis even in the presence of oxygen notonly provides a survival advantage over non-transformed cells but also ensures thepersistence of only the most successful cancer cells (Gatenby and Gillies, 2004).

The outgrowth of a tumor that is beyond the diffusion limits of nearby bloodvessels, which supply nutrients and oxygen, leads to another critical phenotypicadvantage of cancer cells: the ability to induce angiogenesis, or the process

THE EXTRACELLULAR MATRIX 3

of developing new blood vessels from existing vascular structures. Cancer cellsaccomplish this through the upregulation and release of proangiogenic factors thatcan destabilize endothelial cells and induce vascular outgrowth from normal bloodvessels. Endothelial cells proliferate toward the source of the chemoattractant angio-genic factors to form a new capillary network for the tumor mass. However, unlikenormal vasculature, which is extremely ordered, this newly developed tumor neo-vasculature is highly aberrant in structure, lacking organization and vessel integrity.Although many proangiogenic factors have now been identified, vascular endothelialgrowth factor (VEGF) is believed to be the major inducer of tumor angiogenesis.It has not only been implicated in many cancer types (Fukumura et al., 1998) butimportantly, VEGF expression has been shown to correlate with tumor angiogenesisand aggression, poor patient outcome, and is a predictor for metastasis and hightumor grade in multiple cancer types (Brychtova et al., 2008). Interestingly, somestudies have demonstrated that acidic microenvironments can induce vesicle lysing,thereby secreting VEGF into the tumor microenvironment and contributing to afeed-forward mechanism in which tumor-induced hypoxia and cellular acidificationlead to the formation of neovasculature.

It is generally believed that tumors cannot grow to a size larger than a fewcubic millimeters without inducing a neovasculature. Once cancer cells inducerevascularization, thereby ensuring a more constant nutrient supply, this growthrestriction is effectively removed. The requirement for additional tumor spacenecessitates the ability of cancer cells to invade into surrounding tissue. It is mostadvantageous for cancer cells to digest adjacent extracellular matrix and force thelocal reorganization of normal epithelia and surrounding stromal elements.

1.4 The extracellular matrix

The extracellular matrix is comprised of various cell types and secreted proteinsthat help maintain the organization of higher-order cellular structures. In additionto containing various cell types, the matrix is deposited as a mix of such proteins ascollagens, fibronectin, laminins, hyoluronan, plasminogens, proteases, and numerousothers, which collectively form an inflexible scaffold to which cells attach. Inaddition, other secreted cellular proteins such as cytokines and extracellular matrixremodeling proteins normally reside in the extracellular matrix (Chapters 3 and 4).These proteins are released when the matrix is degraded, and upon their releasebecome activated due to proteases and other activating enzymes present in theextracellular environment, further contributing to the regulation of extracellularmatrix turnover.

Many cell types are present in the extracellular matrix and the tumor milieu.Examples include fibroblasts, which are an integral inducer of matrix remodeling,as well as endothelial cells, hematopoietic-derived cells, and immune cells, whichnormally monitor this environment for foreign (i.e., non-host) bodies (Chapter 5).In cancer, particularly at the later stages of transformation and invasion, normalimmune functions are subverted, leading to recognition of the tumor as part of thehost, rather than as an invading foreign entity.

4 THE MICROENVIRONMENT IN CANCER

1.5 Motility, invasion, and metastatic ability

Successful and evolutionarily adapted cancer cells are motile, have no majorattachments to extracellular substrata, and can more easily move through theextracellular and intracellular space due to decreased cell adhesion and an increasein factors which facilitate extracellular matrix degradation and remodeling. A nat-ural effect of motility is that cancer cells invade into surrounding tissue, colonizeand populate the area given a favorable microenvironment. Further, cancer cellmotility facilitates the movement of the cancer cell through layers of endothelial cellssurrounding blood vessels, enabled in part through cancer cell secretion of vasculardestabilizing factors. As a result the vasculature is perturbated and cancer cells gainaccess to the circulation, which is the major mode of transport for cancer cells toreach distant organs.

In the metastatic cascade, the tumor microenvironments of both the primarytumor and the target sites colonized by cells shed from the primary tumor are ofcritical importance to the successful spread of neoplastic cells (Joyce and Pollard,2009) (Chapters 6–8 and 12). The classic ‘seed and soil’ mechanism describes asituation in which only permissive target microenvironments enable the attachmentand subsequent proliferation by a metastatic cell. In addition, cells in the primarysite of a cancer shed factors and progrowth signals that contribute to the tumormicroenvironment at the secondary sites of tumor formation (Chapter 8). In thisway, metastatic tumor cells establishing new colonies continue to receive progrowthsupport signals while they are colonizing the secondary site and during subsequentphases of secondary tumor growth.

In addition, the evolution of the microenvironment can impact premetastatic cellsin a manner that leads to an invasive, advanced, and evolutionarily favored metastaticphenotype that can survive extravasation, intravasation, and can establish newtumors at distant sites. Accumulating evidence suggests that the hypoxic conditionsthat select for successful tumor types also contribute to the metastatic potential ofthat tumor (Chapter 14). Therefore, eradication of the hypoxic regions of a cancerhas short-term and long-term benefits (Chapters 16–18), in that both tumor bulkand metastatic capability are reduced.

1.6 Impact of the tumor microenvironmenton the control of cancer

The tumor microenvironment is a growing target for consideration of cancertherapeutics due to its varied influence on the cells and on the physical aspectsof chemotherapeutic delivery (Chapters 2 and 15). Several drawbacks to tradi-tional chemotherapies that do not account for the microenvironment are: the tumorvasculature, which is highly disordered and leaky; tumor core hypoxia, whichconfers radiation resistance on tumor cells in this state; cells furthest from bloodvessels become growth-arrested, preventing efficacious chemotherapeutic inhibitionof proliferating cells; and the upregulation of acid transporter and other trans-porter proteins, which efficiently excrete chemotherapeutics from cancer cells and

SUMMARY 5

hinder successful cancer treatment (Chapters 2, 9, and 15). Importantly, offspringof chemotherapeutic survivors can pass this genetic property to daughter cells, mak-ing subsequent populations of tumor cells highly resistant to subsequent therapy.One further consideration regarding the tumor microenvironment is the stem cellpopulation, a slow-growing subset of cancer cells, which is inherently resistant totherapies targeting cells that are actively cycling. Stem- or stem-like cancer cellsare pluripotent, highly plastic, and dedifferentiated entities that easily and steadilyrepopulate tumors following therapy. Considering these scenarios, targeting thetumor microenvironment becomes an increasingly logical and attractive therapeuticoption in cancer management.

1.7 Targeting the tumor microenvironment

Classical anticancer therapies including radiotherapy and chemotherapy are toxicto cancer cells but such treatments are typically also associated with inadvertentdamage to critical normal tissues. Newer and more specific therapies have becomemore prevalent in the treatment of specific cancers as the molecular mechanisms ofcarcinogenesis become better characterized. The approach of uncovering molecularetiologies of cancer coupled with the development of targeted therapies that exploitessential signaling pathways (Chapters 10, 13 and 17) will undoubtedly contributeto the future arsenal of anticancer therapeutics.

Because the microenvironment of tumors not only severely impairs the treatmentefficacy of conventional anticancer therapies but also differs significantly from thosefound in normal tissues, research is beginning to focus on the tumor microenviron-ment as a separate cancer-associated entity that may be targeted (Chapters 10, 13 and17). Indeed, several strategies have already been identified that exert an anticancereffect through the specific targeting of the tumor microenvironment. Oxygen-poorcells display greater resistance to radiotherapy, and methods for reversing theradioprotective effects of hypoxia in order to enhance the treatment efficacy ofradiotherapy have received considerable attention (Chapters 9 and 16). The othercompartment of the tumor microenvironment that has been extensively targeted isthe tumor vasculature (Chapter 18). As an essential part of tumor survival, such astrategy seeks to deprive the tumor of critical nutrients and means to spread. Theuse of antiangiogenic and vascular disruptive therapies provide powerful adjuncts toconventional anticancer treatments. All such potential therapeutic interventions willbe critically dependent upon the establishment of novel approaches to non-invasiveimaging of the tumor microenvironment (Chapter 11).

1.8 Summary

The microenvironment of tumors creates a significant hindrance to the controlof cancers by conventional anticancer therapies. The physical conditions presentare imposing and manifold, and include elevated interstitial pressure, localizedextracellular acidity, regions of oxygen and nutrient deprivation, and contraction

6 THE MICROENVIRONMENT IN CANCER

of the extracellular matrix. No less important are the functional consequencesexperienced by the tumor cells residing in such environments: adaptation to hypoxia,cell quiescence, modulation of transporters, and enhanced metastatic potential.Together these factors lead to therapeutic barriers that may render the chance oftumor elimination as minimal.

However, the aberrant nature of the tumor microenvironments also offers uniquetherapeutic opportunities. Reducing tumor hypoxia can improve drug delivery andenhance radiotherapy. The inhibition of fibroblasts and other cell types exploitsthe tumor’s reliance on the microenvironment for various factors and propertiesnecessary for its survival. Targeting the tumor vasculature would destroy thenutritional support network of the tumor. These approaches and many othersdirected against the tumor microenvironment are under active investigation in thelaboratory and the clinic.

Because the molecular underpinnings of cancer development are becoming increas-ingly well characterized, future studies will undoubtedly identify distinct molecularmarkings that are characteristic of the tumor microenvironment. Such advanceswill lead to the development of targeted therapies that will selectively impair theneoplastic cell populations residing in these environments. Ultimately, by combiningsuch therapies with conventional anticancer treatments it may be possible to bringcancer growth, invasion, and metastasis to a halt.

References

Brychtova, S., Bezdekova, M., Brychta, T., and Tichy, M. (2008) The role of vascularendothelial growth factors and their receptors in malignant melanomas. Neoplasma, 55,273–279.

Fukumura, D., Xavier, R., Sugiura, T. et al. (1998) Tumor induction of VEGF promoteractivity in stromal cells. Cell, 94, 715–725.

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Reviews Cancer, 9, 239–252.

2Establishing the TumorMicroenvironment

Allison S. Betof1 and Mark W. Dewhirst21Department of Pathology, Duke University Medical Center, Durham, USA2Department of Radiation Oncology, Duke University Medical Center, Durham, USA

2.1 Introduction

For years cancer researchers focused on a seemingly obvious target: tumor cells.While this research yielded invaluable knowledge, a comprehensive understanding oftumor behavior remains elusive. Just as children are influenced by their environmentas they grow, so too are developing tumors. Tumors do not grow in a vacuum.Instead, they are surrounded by the extracellular matrix, blood vessels, immunecells, and other supporting structures that make up human organs. Together thissupport system comprises a tumor’s microenvironment. In recent years, cancerresearch has experienced a paradigm shift toward trying to understand the role ofthe microenvironment in tumor development, growth, metastasis, and treatment.This chapter will focus on the establishment of that environment.

There are two principal fields of microenvironmental research. One focuseson cellular components, cell–cell interactions, and the matrix that makes upthe tumor parenchyma in addition to tumor cells. The other component is thephysiological microenvironment. This involves the exchange of oxygen, nutrients,and waste products through tumor vasculature. While they are often discussedseparately, these two components of the microenvironment do not exist in isolation.Though each component of the microenvironment will be addressed separately, thischapter will also review areas of overlap and interaction between the physical andphysiological microenvironments.

National Institutes of Health/National Cancer Institute

NIH/NCI CA40355-25

Tumor Microenvironment Edited by Dietmar W. Siemann 2011 John Wiley & Sons, Ltd

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2.2 From cancerous cells to a tumor

The complex, heterogeneous structures we know as tumors arise from cancercells that proliferate within the space dedicated to another organ. The presenceof malignant cells within normal tissue alters the environment and interruptstissue homeostasis. Reciprocal interactions with the host stroma facilitate tumorproliferation, invasion, and metastasis. However, the question of which of thesechanges occurs first has become a central issue. Do cancer cells transform andthen change the microenvironment where they are located to suit their needs? Orare there changes in the previously normal tissue that promote transformation andtumorigenesis in a particular location?

The answer, it now seems, is that both of these alterations can result in theformation of a tumor. For evidence that stromal changes can result in tumorigenesisin the absence of cancer cells, the reader is directed to one of several excellentreviews (Ariztia et al., 2006; Bhowmick, Neilson, and Moses, 2004b). However, formost tumors that are epithelial in origin, the initiating event is usually an epithelialmutation followed by key paracrine signals from stromal fibroblasts that pro-mote neoplastic progression (Lengauer, Kinzler, and Vogelstein, 1998; Bhowmick,Neilson, and Moses, 2004b). Reciprocal interactions between differentiating epithe-lia and adjacent stromal cells are essential to reaching proper tissue maturationin normal tissue (Bhowmick, Neilson, and Moses, 2004b). In contrast, malignanttransformation of epithelial cells is preceded by or occurs simultaneously with hoststromal activation, inducing angiogenesis, fibroblast proliferation, and recruitmentof inflammatory mediators (Tomakidi et al., 1999; Ronnov-Jessen, Petersen, andBissell, 1996; Tlsty and Hein, 2001). Stromal fibroblasts respond to tumor-derivedpathological signals by producing collagen and activating myofibroblast-associatedproteins such as α-smooth muscle actin, vimentin, and desmin (Tlsty and Hein,2001). In addition, activated fibroblasts proliferate and migrate more in vitro thanfibroblasts from benign tissue (Schor, Schor, and Rushton, 1988; Schor et al., 1985).

Cancer-associated fibroblasts (CAFs) are stromal fibroblasts with key roles intransformation, proliferation, and invasion, three of the hallmarks of cancer. Thesefibroblasts secrete growth factors and chemokines that signal critical changes inthe extracellular matrix (ECM) and contribute oncogenic signals that increaseproliferation and invasion (Kalluri and Zeisberg, 2006). For example, CAFs are ableto stimulate both growth and transformation of immortalized prostate epithelialcells in vitro and in vivo, whereas normal fibroblasts are not (Olumi et al., 1999). Inaddition, Parrott et al. used an ovarian cancer model to show that tumor fibroblastsmay not just assist in tumorigenesis, but may actually be necessary to form a tumorfrom cancer cells (Parrott et al., 2001). This group then used histology to show thatthe ovarian cancer cells recruited adjacent stroma from the normal mouse tissue toform the stroma of the tumor. Thus, the CAFs play a crucial role in establishing thephysical microenvironment of a growing tumor.

One possible mechanism for the tumor promoting effects of CAFs comes fromtheir overexpression of fibroblast secreted protein-1 (FSP1) (Grum-Schwensen et al.,

A TUMOR IS MORE THAN CANCER CELLS AND FIBROBLASTS 9

2005). When FSP1 knockout mice are injected with carcinoma cells, tumor formationis decreased compared to wild-type mice and the tumors that do form do notmetastasize. Furthermore, adding FSP1+/+ fibroblasts in addition to the carcinomacells restores the wild-type phenotype. A second possible mechanism is that stromalCAFs secrete stromal cell-derived factor 1α (SDF-1α, also known as the cytokineCXCL12), which promotes proliferation of mammary carcinoma cells when it bindsto the receptor CXCR4 on the cancer cells (Orimo et al., 2005). These signalingmolecules clearly contribute to the developing tumor microenvironment, but theirprecise effects and the contributions of other factors remain unknown.

2.3 A tumor is more than cancer cells and fibroblasts

In addition to the tumor cells and CAFs already discussed, the microenvironmentconsists of normal parenchymal and epithelial cells, ECM, normal stromal fibrob-lasts, soluble growth factors and cytokines, components of the immune system,nerves, and blood and lymphatic vessels. However, this is more than just a physicalspace. Implied within the term ‘microenvironment’ is a venue for these factorsto interact.

2.3.1 The extracellular matrix

Within a carcinoma, epithelial cells are supported by a three-dimensional structureknown as the ECM. The proteins that make up the ECM are produced by fibroblasts.The stroma is separated from the epithelium and endothelium by the basementmembrane, a specialized type of ECM composed of collagen IV, laminin, andheparan sulfate proteoglycans (Kalluri, 2003).

There are considerable differences in matrix composition between tumor stromaand the stroma associated with normal tissue. Cells from the tumor secrete avariety of proteins into the ECM that are involved in cell adhesion, motility,communication, and invasion (Mbeunkui and Johann, 2009). Notably, some ofthese molecules are involved in degrading the ECM. This degradation occurs nearthe tumor-host interface, where the tumor-derived proteases overwhelm the host’sendogenous inhibitors leading to extensive remodeling and stimulating alterna-tive signals from the cell surface (Handsley and Edwards, 2005). Remodelingis achieved by combined efforts of secreted and membrane-anchored matrixmetalloproteinases (MMPs), adamalysin-related membrane proteases, bone mor-phogenic protein-1-type metalloproteinases, endoglycosidases, and tissue serineproteases including tissue plasminogen activator, urokinase, thrombin, and plas-min (Carmeliet, 2003; Werb, 1997). Interestingly, most of these factors involved inremodeling the tumor-host interface originate from host cells, not from the grow-ing carcinoma (Bowden et al., 1999; Coussens et al., 2000; Nakahara et al., 1997;Sternlicht et al., 1999).

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Infiltrating inflammatory cells release MMP-9, MMP-12, and MMP-8 fromintracellular stores, but they also release cytokines including IL-1β and tumor necro-sis factor-α (TNF-α) that stimulate fibroblasts to produce more MMPs (Van Kempenet al., 2003). In addition to fibroblasts and inflammatory cells, paracrine signalsstimulate other stromal cells to be the predominant source of microenvironmentalMMPs during tumorigenesis (Van Kempen et al., 2003; Coussens and Werb, 2002).

Secreted MMPs degrade both ECM and other proteins in the microenvironment,including growth factors, cytokines, and receptors. Therefore, the effects of indi-vidual MMPs on the microenvironment are diverse, but they are critical playersin establishing the environment surrounding tumor cells. One potent example isMMP-7, which is expressed by malignant breast epithelial cells. MMP-7 degradesthe ECM, disrupts the basement membrane, and cleaves E-cadherin, weakening theconnection between breast epithelial cells (Fingleton et al., 2001; Noe et al., 2001).MMPs are found in the microenvironment in zymogen form, and they require acti-vation by other MMPs and related molecules. The reader is cautioned that MMPoverexpression alone, which has often been reported in tumors, does not explainMMP activity in the microenvironment, since immunohistochemistry often doesnot distinguish between the zymogen and active forms (Coussens, Fingleton, andMatrisian, 2002). Under normal conditions MMPs are also regulated by endogenoustissue inhibitors of metalloproteinases (TIMPs), so the balance between TIMPs andMMPs critically affects the microenvironment (Mbeunkui and Johann, 2009).

To balance these remodeling and degradative enzymes, synthesis of matrix com-ponents is upregulated by both the tumor and host. Activated fibroblasts synthesizecollagen type I and fibronectin in response to the binding of mast cell tryptase toprotease-activated receptor-2 (Coussens and Werb, 2002; Frungieri et al., 2002).Macrophages also contribute interleukin (IL)-1β and nitric oxide synthase, both ofwhich augment type I collagen synthesis (Van Kempen et al., 2003). The newlysynthesized collagen that forms the peritumoral stroma is loosely woven and disor-ganized, contributing to the overall disorder surrounding a developing tumor (Ruiteret al., 2002).

2.3.2 Immune cells in the microenvironment

The immune system makes an invaluable contribution to the tumor microenviron-ment. Inflammatory cells secrete growth factors, cytokines, and chemokines thatstimulate epithelial proliferation and generate reactive oxygen species (ROS) thatdamage DNA, promoting tumor initiation and progression (Coussens and Werb,2002). They also release proteolytic enzymes, leading to matrix remodeling andangiogenesis. The first cells to respond to tumor growth factors are mast cells,which are attracted by stem cell factor (SCF), vascular endothelial growth factor(VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), andplatelet-derived growth factor (PDGF) (Hiromatsu and Toda, 2003). Upon arrival,mast cells degranulate to release VEGF (Norrby, 2002), serine proteases (Yong,1997), and MMP-9 (Fang et al., 1997).