Tumor-Associated Fibroblasts and their Matrix (The Tumor Microenvironment 4)

Tumor-Associated Fibroblasts and their Matrix

The Tumor Microenvironment Series Editor: Isaac P. Witz

For further volumes: http://www.springer.com/series/7529

Margareta M. Mueller • Norbert E. Fusenig Editors

Tumor-Associated Fibroblasts and their Matrix

1  3

Editors Prof. Dr. Margareta M. Mueller Research Group Tumor and Microenvironment German Cancer Research Center, Heidelberg and HFU (Hoschschule Furtwangen University) campus Villingen-Schwenningen Villingen-Schwenningen Germany [email protected]

Prof. Dr. Norbert E. Fusenig Former Division Head at the German Research Cancer Center, Heidelberg and Emeritus of the University of Heidelberg Heidelberg Germany [email protected]; [email protected]

ISBN 978-94-007-0658-3     e-ISBN 978-94-007-0659-0 DOI 10.1007/978-94-007-0659-0 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2011925928 © Springer Science+Business Media B.V. 2011 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Cover design: deblik, Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

During the last century cancer research was mainly focussed on the tumor cells alone which could be easily propagated in cell culture. During this time many important findings were obtained clearly demonstrating that cancer is a genetic disease, controlled by the activation and/or inactivation of critical control genes. However during the last two decades it has become increasingly clear that genetic alterations alone are not the sole driving force behind tumor development but that tumor growth and progression are rather intimately controlled by the microenvironment. One could almost speak of are “rediscovery” of the tumor as a highly complex tissue composed of carcinoma cells and surrounding stroma. Studies in different areas of biology including tumour biology have demonstrated that tissue structure, function and dysfunction are highly intertwined with the microenvironment and that during the development of cancer tissue biology and host physiology are subverted to drive malignant progression. It is now clear that the context is crucial and that the status of the cellular microenvironment plays a significant role in determining whether cells within a tissue retain their normal architecture or undergo tumor progression. The tumor stroma or microenvironment is made up of multiple non-malignant cell populations, including fibroblasts, adipocytes, endothelial and inflammatory cells that are embedded in a tumour specific extracellular matrix (ECM). Nowadays, there is a huge interest in tumor stroma research, and in understanding the contributions of the different stromal cell types to tumor growth and progression. One of the key components of the tumor microenvironment in carcinomas are activated fibroblasts termed cancer associated fibroblasts (CAFs). In the meantime our knowledge of CAFs has changed from being viewed as a passive bystander to becoming an important co-mediator of cancer progression. In response to cancer growth, host stromal fibroblasts undergo a dramatic morphologic and biochemical transition to form “reactive stroma” in a desmoplastic reaction much like the granulation tissues found at the site of wound healing. While the malignant cells activate fibroblasts in the tumor stroma by various stimuli, including growth factors and cytokines, cancer associated fibroblasts secrete growth factors and build a permissive soil in which the cancer cells thrive. CAFs are responsible for the elaboration of most of the connective tissue and ECM components v

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as well as, proteolytic enzymes and their inhibitors. The composition and structure of the ECM in the tumor microenvironment is essential for promoting tumor development and metastasis. The constituents of the ECM include collagens, laminins, fibronectin and several proteoglycans. They provide mechanical support for cells, facilitate cell communication and serve as substrates for cell migration. Changes in the composition or architecture of the extracellular matrix within tumors can alter integrin expression and function and promote metastatic progression, angiogenesis and lymphangiogenesis. In this unique textbook world leading experts of the area of tumor microenvironment review the most recent knowledge of the still growing complexity of the tumor microenvironment focussing on tumor associated stromal cells and the most important extracellular matrix components and summarize the role of these players in tumor progression. Moreover, novel therapeutic targets are discussed that have been discovered in the tumor microenvironment and are increasingly used in experimental and clinical tumor therapy. The message from their contributions is clear: the tumor microenvironment and its components are important and essential players in tumor progression and interesting targets for novel therapeutic strategies. However there are still many white areas on the map and we are just beginning to understand the complex interplay between tumor and stromal cells. We express our deepest gratitude to all our colleagues who have made this book the first comprehensive antology covering all major aspects of the role of the tumor microenvironment and its extracellular matrix components. Heidelberg

Margareta M. Mueller and Norbert E. Fusenig

Contents

Part I  The Tumor Microenvironment 1 Critical Roles of Stromal Fibroblasts in the Cancer Microenvironments ����������������������������������������������������������������������    3 Leland W. K. Chung Part II  Stromal Cell Diversity 2 Functional Diversity of Fibroblasts ���������������������������������������������������������   23 H. Peter Rodemann and Hans-Oliver Rennekampff 3 The Role of the Myofibroblast in Fibrosis and Cancer Progression ����   37 Boris Hinz, Ian A. Darby, Giulio Gabbiani and Alexis Desmoulière 4 The Role of Myofibroblasts in Communicating Tumor Ecosystems ����   75 Olivier De Wever, Astrid De Boeck, Pieter Demetter, Marc Mareel and Marc Bracke 5 Tumor Vessel Associated-Pericytes ����������������������������������������������������������   91 Arne Bartol, Anna M. Laib and Hellmut G. Augustin 6 The Role of Cancer-Associated Adipocytes (CAA) in the Dynamic Interaction Between the Tumor and the Host ������������������������   111 Marie-Christine Rio Part III  The Tumor ECM 7 Hyaluronan: A Key Microenvironmental Mediator of Tumor-Stromal Cell Interactions ��������������������������������������������������������   127 Naoki Itano 8  Function of Tenascins in the Tumor Stroma �������������������������������������������   145 Florence Brellier and Ruth Chiquet-Ehrismann vii

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9   Fibulins and Their Role in the ECM ���������������������������������������������������   159 Helen C. M. Cooney and William M. Gallagher 10 Tumor Fibroblast-Associated Metalloproteases ���������������������������������   175 Julie Lecomte, Anne Masset, Dylan R. Edwards and Agnès Noël Part IV  Tumor Modulating-Fibroblast Interactions 11 Multiple Fibroblast Phenotypes in Cancer Patients: Heterogeneity in Expression of Migration Stimulating Factor ���������   197 Ana M. Schor and Seth L. Schor 12 TGF-β Signaling in Fibroblasts Regulates Tumor Initiation and Progression in Adjacent Epithelia ������������������������������������������������   223 Brian R. Bierie and Harold L. Moses 13 The SDF-1-Rich Tumour Microenvironment Provides a Niche for Carcinoma Cells ����������������������������������������������������������������   245 Masayuki Shimoda, Kieran Mellody and Akira Orimo 14 Role of PDGF in Tumor-Stroma Interactions ������������������������������������   257 Carina Hellberg and Carl-Henrik Heldin 15 Radiation-Induced Microenvironments and Their Role in Carcinogenesis ����������������������������������������������������������������������������������   267 Mary Helen Barcellos-Hoff and David H. Nguyen Part V  Tumor-Modulating ECM Interactions 16 The Extracellular Matrix as a Multivalent Signaling Scaffold that Orchestrates Tissue Organization and Function ���������   285 Jamie L. Inman, Joni D. Mott and Mina J. Bissell 17 SPARC and the Tumor Microenvironment ����������������������������������������   301 Stacey L. Thomas and Sandra A. Rempel 18 Integrin-Extracellular Matrix Interactions ����������������������������������������   347 Christie J. Avraamides and Judith A. Varner 19 The Multifaceted Role of Cancer Associated Fibroblasts in Tumor Progression ����������������������������������������������������������������������������   361 Hans Petter Eikesdal and Raghu Kalluri

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Part VI  Therapeutic Application/Targeting 20 Cancer Associated Fibroblasts as Therapeutic Targets ���������������������   383 Christian Rupp, Helmut Dolznig, Christian Haslinger, Norbert Schweifer and Pilar Garin-Chesa 21 Targeting Tumor Associated Fibroblasts and Chemotherapy ����������   403 Debbie Liao and Ralph A. Reisfeld 22 Antibody-Based Targeting of Tumor Vasculature and Stroma ���������   419 Katharina Frey and Dario Neri Index ���������������������������������������������������������������������������������������������������������������   451

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Contributors

Hellmut G. Augustin  Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), 69120 Heidelberg, Germany Medical Faculty Mannheim,Vascular Biology and Tumor Angiogenesis, Heidelberg University, 68167 Mannheim, Germany Christie J. Avraamides  Moores UCSD Cancer Center, University of California, San Diego, 3855 Health Sciences Drive #0819, La Jolla, CA 92093-0819, USA e-mail: [email protected] Mary Helen Barcellos-Hoff  Departments of Radiation Oncology and Cell Biology, NYU Langone Medical Center, 566 First Avenue, 10016 New York, USA e-mail: [email protected] Arne Bartol  Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), 69120 Heidelberg, Germany Medical Faculty Mannheim, Vascular Biology and Tumor Angiogenesis, Heidelberg University, 68167 Mannheim, Germany e-mail: [email protected] Brian R. Bierie  Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, 691 Preston Research Building 2220 Pierce Ave., Nashville, TN 37232-6838, USA e-mail: [email protected] Mina J. Bissell  Life Science Division, Lawrence Berkeley National Laboratory, One Cyclotron Rd, Berkeley, CA 94720, USA e-mail: [email protected] Astrid De Boeck  Laboratory of Experimental Cancer Research, Department of Radiotherapy and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium Marc Bracke  Laboratory of Experimental Cancer Research, Department of Radiotherapy and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium

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Florence Brellier Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland Ruth Chiquet-Ehrismann Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland e-mail: [email protected] Leland W. K. Chung Uro-Oncology Research Program, Departments of Medicine and Surgery, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center and the University of California, Los Angeles, CA 90048, USA e-mail: [email protected] Helen C. M. Cooney 73 Nutley Lane, Donnybrook, Dublin 4, Ireland e-mail: [email protected] Ian A. Darby Cancer and Tissue Repair Laboratory, School of Medical Sciences, RMIT University, Bundoora, VIC 3083, Australia Pieter Demetter Department of Pathology, Erasme University Hospital, Université Libre de Bruxelles (ULB), Brussels, Belgium Alexis Desmoulière Faculty of Pharmacy, Cellular Homeostasy and Pathologies (EA 3842) and Department of Physiology, IFR 145, University of Limoges, 87025 Limoges, France Helmut Dolznig Institute of Pathology, Medical University of Vienna, Waehringer Guertel 18–20, 1090 Vienna, Austria Institute of Medical Genetics, Centre of Pathobiology and Genetics, Medical University of Vienna, Waehringer Strasse 10, 1090 Vienna, Austria e-mail: [email protected] Dylan R. Edwards School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK Hans Petter Eikesdal Department of Oncology, Haukeland University Hospital, 5021 Bergen, Norway Katharina Frey Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland e-mail: [email protected] Giulio Gabbiani Department of Pathology and Immunology, CMU, University of Geneva, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland e-mail: [email protected] William M. Gallagher UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin 4, Ireland e-mail: [email protected] Pilar Garin-Chesa Institute of Pathology, Medical University of Vienna, Waehringer Guertel 18–20, 1090 Vienna, Austria

Contributors

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Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5–11, 1130 Vienna, Austria Christian Haslinger  Boehringer Ingelheim RCV GmbH & Co KG, Dr. BoehringerGasse 5–11, 1130 Vienna, Austria Carl-Henrik Heldin  Ludwig Institute for Cancer Research, Uppsala University, Box 595, SE 751 24 Uppsala, Sweden e-mail: [email protected] Carina Hellberg  Ludwig Institute for Cancer Research, Uppsala University, Box 595, SE 751 24 Uppsala, Sweden e-mail: [email protected] Boris Hinz  Faculty of Dentistry, Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, University of Toronto, Toronto, ON M5S 3E2, Canada Jamie Inman  Life Science Division, Lawrence Berkeley National Laboratory, One Cyclotron Rd, Berkeley, CA 94720, USA Naoki Itano  Department of Molecular Oncology, Division of Molecular and Cellular Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, Nagano 390-8621, Japan e-mail: [email protected] Raghu Kalluri  Division of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center & Harvard Medical School, 330 Brookline Ave, E/CLS Room #11-090, Center for Life Sciences, 02115 Boston, MA, USA Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA e-mail: [email protected] Anna M. Laib  Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), 69120 Heidelberg, Germany Julie Lecomte  Laboratory of Tumor and Development Biology, Groupe Interdisciplinaire de Génoprotéomique Appliqué-Cancer (GIGA-Cancer), University of Liège, 4000 Liège, Belgium Debbie Liao  Department of Immunology and Microbial Sciences, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA Marc Mareel  Laboratory of Experimental Cancer Research, Department of Radiotherapy and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium Anne Masset  Laboratory of Tumor and Development Biology, Groupe Interdisciplinaire de Génoprotéomique Appliqué-Cancer (GIGA-Cancer), University of Liège, 4000 Liège, Belgium

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Kieran Mellody  CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK Harold L. Moses  Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, 691 Preston Research Building 2220 Pierce Ave., Nashville, TN 372326838, USA e-mail: [email protected] Joni D. Mott  Life Science Division, Lawrence Berkeley National Laboratory, One Cyclotron Rd, Berkeley, CA 94720, USA Dario Neri  Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland e-mail: [email protected] Agnès Noël  Laboratory of Tumor and Development Biology, Groupe Interdisciplinaire de Génoprotéomique Appliqué-Cancer (GIGA-Cancer), University of Liège, 4000 Liège, Belgium e-mail: [email protected] Akira Orimo  CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK e-mail: [email protected] Ralph A. Reisfeld  Department of Immunology and Microbial Sciences, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA e-mail: [email protected] Sandra A. Rempel  Department of Neurosurgery, Hermelin Brain Tumor Center, Henry Ford Hospital, Detroit, MI 48202, USA e-mail: [email protected] Marie-Christine Rio  Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U964, UDS, BP, 67404 Illkirch Cedex, 10142 C.U. de Strasbourg, France e-mail: [email protected] H. Peter Rodemann  Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, Eberhard Karls University Tübingen, Röntgenweg 11, 72076 Tübingen, Germany e-mail: [email protected] Hans-Oliver Rennekampff  Hospital for Plastic, Hand and Reconstructive Surgery, Medical School Hannover, Hannover, Germany Christian Rupp  Institute of Pathology, Medical University of Vienna, Waehringer Guertel 18–20, 1090 Vienna, Austria

Contributors

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Ana M. Schor  Unit of Cell and Molecular Biology, The Dental School, University of Dundee, Dundee DD1 4HR, UK e-mail: [email protected] Seth L. Schor  Unit of Cell and Molecular Biology, The Dental School, University of Dundee, Dundee DD1 4HR, UK e-mail: [email protected] Norbert Schweifer  Boehringer Ingelheim RCV GmbH & Co KG, Dr. BoehringerGasse 5–11, 1130 Vienna, Austria Masayuki Shimoda  Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan CR-UK Stromal-Tumour Interaction Group, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK Stacey L. Thomas  Department of Neurosurgery, Hermelin Brain Tumor Center, Henry Ford Hospital, Detroit, MI 48202, USA e-mail: [email protected] Judith A. Varner  Moores UCSD Cancer Center, University of California, San Diego, 3855 Health Sciences Drive #0819, La Jolla, CA 92093-0819, USA Department of Medicine, University of California, San Diego, La Jolla, CA, 920930819, USA e-mail: [email protected] Olivier De Wever  Laboratory of Experimental Cancer Research, Department of Radiotherapy and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium e-mail: [email protected]



Part I

The Tumor Microenvironment

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Chapter 1

Critical Roles of Stromal Fibroblasts in the Cancer Microenvironments Leland W. K. Chung

1.1 Introduction A mounting body of evidence suggests that the ability of cancer cells to interact reciprocally with the host microenvironment contributes to cancer growth, progression and resistance to therapeutic interventions. Interactions with stromal fibroblasts at the primary site and marrow stromal cells at metastatic sites could create a favorable microenvironment supporting cancer growth, survival, evasion of immune surveillance and resistance to therapy (Mueller and Fusenig 2004; Chantrain et al. 2008; Karnoub et al. 2007; Ronnov-Jessen and Bissell 2009; Chung et al. 2006). Changes in cancer microenvironments could also add selection pressures favorable to cancer cell evolution, increasing cancer cell heterogeneity and reciprocally causing the co-evolution of adjacent stromal fibroblasts, resulting in the development of organ- and stage-specific stromal fibroblasts capable of programming and reprogramming the fate of cancer cells (Hill et al. 2005; Sung et al. 2008; Franco et al. 2010). Over the past several years, active research has broadened our awareness of the plasticity of cancer-associated stroma, which undergo both morphologic and functional transitions supporting the pathogenesis of cancer cells (Sung et al. 2008). The dynamic presence of bone marrow-derived mesenchymal stem cells in localized and metastatic cancers contributes further to the diversity and heterogeneity of cancer-associated stroma and ultimately determines the site of cancer metastasis, while stromal fibroblasts have multiple functional roles modulating cancer growth either positively or negatively (Martin et al. 2010; Molloy et al. 2009; Rhodes et al. 2009; Zhao et al. 2009a). Figure 1.1 depicts how soluble factors, insoluble extracellular matrix proteins, and reactive oxygen species secreted by cancer cells and cells in cancer microenvironments can guide and maintain the growth and differentiation of local and distant cancers and their interactions with host microenvironments L. W. K. Chung () Uro-Oncology Research Program, Departments of Medicine and Surgery, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center and the University of California, Los Angeles, CA 90048, USA e-mail: [email protected] M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_1, © Springer Science+Business Media B.V. 2011

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Bone MET

Bone stroma

MSC Secondary metastasis

Hematogenous metastasis Blood vessel Cancer cells

BF Brain

MSC Activation

Macrophages Platelets Activated stroma T and B cells Dendritic cells

ROS GFs

Normal

Lymphatic metastasis

Transformation EMT

ECM NAF

Secondary metastasis

NAF

Benign

ROS GFs

CAF

Transformed

LF

ROS GFs CAF

Malignant

LNF Lymph node

Lung

Prostate

Fig. 1.1   Tumor-microenvironments interactions contribute to prostate cancer skeletal and soft tissue metastases. At the primary site, reciprocal cellular interactions between prostate cancer cells and local/migrating stromal fibroblasts, mediated by soluble and insoluble factors and ROS, promote the transition of normal/benign stromal fibroblasts to cancer-associated stromal fibroblasts which activate and transform prostate epithelial cells to gain increased growth and survival potential. Subsequently, through epithelial-to-mesenchymal transition (EMT), prostate cancer cells gain futher growth, survival, migratory, invasive and metastatic potential allowing them to metastasize to the skeleton via hematogeneous routes. Prostate cancer cells also frequently metastasize to lymph node and then reach the skeleton through lymphatic metastatic routes. Metastatic prostate cancer cells, after reaching the bone, adhere to and exit from marrow endothelium via transendothelial migration, undergo mesenchymal-to-epithelial transition (MET) and colonize in the bone by increased expression of cell–cell adhesive/junction proteins such as E-cadherin. The metastatic cascade is aided by the active participation of both resident fibroblasts, such as lymph node fibroblast (LNF), lung fibroblast (LF), and brain fibroblast (BF), and migrating stromal fibroblasts such as cells derived from mesenchymal stem cells (MHC) at primary and metastatic sites. The multipotent MHC can differentiate into inductive cell populations in the tumor microenvironment, including reactive stroma, macrophage, platelet, dendritic cell and T- and B-cells, in the primary and at metastatic sites, to ‘mark’ the site prior to the occurrence of secondary metastases

(Karnoub et al. 2007; Chung et al. 2006; Desgrosellier and Cheresh 2010; Ishikawa et al. 2008; Jung et al. 2009; Kaplan et al. 2005; Svineng et al. 2008). The therapeutic targeting of cancer cells, though necessary, is insufficient by itself to control the growth of localized and disseminated cancers. This has led to the acceptance of co-targeting both the cancer and its microenvironments, including stromal fibroblasts, the vascular endothelial network, immune cells and host humoral substances (Chung et al. 2005; Cress and Mohla 2004; Tu and Lin 2008; Vessella and Corey

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2006; Pollard 2009). The unique cell signaling networks linking the behavior of cancer cells (i.e. cell proliferation, resistance to apoptosis, cell migration, invasion and metastasis) with the activation of key downstream signaling pathways can be exploited as new and promising druggable targets (Chung et al. 2006; Anastasiadis et al. 2003; Kang and Altieri 2009; Tasseff et al. 2010). In this review, we use prostate cancer as a model to dissect the critical determinants that regulate growth, differentiation and progression of prostate cancer to the skeleton, the lethal human prostate cancer phenotype. Looking forward, the study of cancer-associated stromal fibroblasts is rapidly evolving and could take center stage in revealing the secrets of cancer cell evolution. Recent exciting discoveries include the reprogramming adult normal stromal fibroblasts to form induced pluripotent stem (iPS) cells capable of orchestrating the development and differentiation of an entire embryo (Takahashi and Yamanaka 2006; Yu et al. 2009; Okita et al. 2007; Park et al. 2008; Zhao et al. 2009b). These findings raise new questions about the pathways leading to stromal fibroblast heterogeneity and the potential of in situ reprogramming of adult stromal fibroblasts to undergo iPS transition in an organ-specific environment. iPS cells could serve as progenitor cells for a subsequent generation of derivative cells comprising the entire stromal microenvironments, raising the possibility of developing stroma-based cancer therapy in the future.

1.2 The Roles of Stromal Fibroblasts in the Context of Tumor Microenvironment 1.2.1  Local Microenvironment Cancer growth and evolution is intimately controlled by its microenvironment, and cancer cells also contribute reciprocally to the active process of remodeling their microenvironment (Ingber 2008; Rhee et al. 2001; Pathak et al. 1997). Cancer cells secrete soluble factors such as EGF, IGFs, PDGF, VEGF, HGF, FGFs, and TGFβs which collectively stimulate pleiotropic signaling in converging multi-signaling pathways that induce activated stromal fibroblasts or myofibroblasts, with potent growth-promoting effect on cancer cells. Local microenvironments are heterogeneous and composed of resident vascular endothelial cells, smooth muscle cells, basal/stem cells and, to a lesser extent, cells from neural and neuroendocrine lineages that could interact with cancer cells in a reciprocal manner through secreted soluble factors and insoluble extracellular matrices. In addition to resident stromal fibroblasts, migrating mesenchymal stem cells from bone marrow, with either hematopoietic or mesenchymal lineage, also contribute to stromal heterogeneity. These cells, macrophages, platelets, dendritic cells, T- and B-cells, and activated stroma, are likely to support local cancer growth, progression and distant metastasis through complex cancer–stroma, cancer–immune and cancer–stem cell interactions

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(Josson et  al. 2010; Singh et  al. 2009; Jaganathan et  al. 2007; McKeithen et  al. 2010). The local heterogeneity of stromal fibroblasts could be determined by their anatomical location. Our laboratory recently showed that human prostate stromal fibroblasts derived from the peripheral zone of the prostate gland are more inductive than stromal fibroblasts derived from the transitional or central zones, and these results are consistent with the observation that progressive prostate cancer is derived predominantly from the peripheral rather than transitional or central zones (Thalmann et al. 2009).

1.2.2  Distant Microenvironments Because of the propensity of prostate cancer to metastasize to bone, which is considered lethal, much effort has focused on defining the bone microenvironment and the mechanisms of bone turnover, including enhanced bone resorption, that contribute to the ability of cancer cells to colonize bone. Several key cell types in the bone microenvironment are of particular importance. Among these are the osteoblasts (OBs), bone-forming cells derived from bone marrow mesenchymal stem cells (MSC). Upon interaction with soluble factors such as bone morphogenic proteins, TGFβs, EGF, FGFs, or PDGF, MSC can potentially differentiate into OBs, chondrocytes, or adipocytes. In addition, there are osteoclasts (OCs), bone resorbing cells derived from monocytes of hematopoietic mesenchymal cell lineage. OCs express receptor activator of NF-kappaB (RANK), a receptor responding to RANK ligand (RANKL), secreted by osteoblasts or prostate cancer cells, promoting maturation of OCs, inducing the fusion of monocytes to form activated multinucleated OCs. These matured multinucleated OCs contribute to bone resorption or bone turnover resulting in the release of soluble growth factors, nutrients, calcium ions, and extracellular matrices (Araujo and Logothetis 2009; Buckle et al. 2010; Mizutani et al. 2009). The actions of these soluble and matrix factors alter cancer cell adhesion, proliferation and survival and also the responsiveness of the host microenvironments toward factors secreted by both cancer cells and cells in cancer microenvironments. Collectively, the factors present in the cancer milieu could determine how cancer cellinduced osteoblastic or osteolytic lesions ultimately support cancer cell colonization in bone. In addition to the local action of cancer microenvironments, the factors secreted by cancer microenvironments could conceivably govern the propensity of secondary cancer metastases to organs such as the lung, liver, brain, and kidney.

1.3 The Plasticity of the Stromal Microenvironment 1.3.1  Reactive Stroma In response to cancer growth, host stromal fibroblasts undergo a dramatic morphologic and/or biochemical transition to form “reactive stroma” in a desmoplas-

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tic reaction much like the granulation tissues found at the site of wound healing (Tuxhorn et al. 2001, 2002; Malins et al. 2006). Although the desmoplastic reaction associated with human prostate cancer is less apparent than in human breast cancer and melanoma, the transition of stromal fibroblasts to myofibroblasts at the gene expression level is quite apparent. Upon transition to reactive stromal cells, they express more abundant and diverse classes of extracellular matrix proteins with altered expression of genes associated with myofibroblasts, such as α- and γ-smooth muscle actin, fibronectin, actin bundle, paladin, Thy1, and TGF-β1 (Sung et  al. 2008; Untergasser et al. 2005; Dakhova et al. 2009). A number of soluble growth factors, when added directly to cultured stromal fibroblasts, have been shown to induce myofibroblast transition (Olaso et al. 2003; Cushing et al. 2008; Kennard et al. 2008; Kikuta et al. 2006). Direct interaction between reactive stroma and cancer cells has been observed to promote cancer growth. This is consistent with clinical observations where the detection of reactive stroma in the prostate cancer microenvironments, for example, predicts PSA recurrence and the clinical outcome in prostate cancer patients (Dakhova et al. 2009; Tothill et al. 2008; Yanagisawa et al. 2007). The engagement between cancer cells and reactive stroma could promote tissue reorganization involving the participation of the vascular network and migrating MSC and host stromal fibroblasts to lead to increased cancer growth (Zhao et al. 2009a; Santamaria-Martinez et al. 2009). The prevalence of myofibroblasts in the cancer environment has been shown in many different forms of cancer including colon, liver, lung, prostate, ovary, pancreas, and breast (Tuxhorn et al. 2001; Friedman et al. 1984; Garin-Chesa et al. 1990; Radisky and Przybylo 2008; Yao et al. 2009). The myofibroblastic appearance often precedes the onset of cancer invasion.

1.3.2  Plasticity of EMT and MET Dynamic bi-directional epithelial-to-mesenchyme (EMT) and mesenchymal-toepithelial (MET) transitions have been observed in embryonic development and in cancer progression (Chung et al. 2006; Prindull 2005; Birchmeier et al. 1996; Wells et  al. 2008; Hugo et  al. 2007). These transitions are commonly associated with a predictive switch of cancer behaviors by the affected cancer epithelium, which assumes increased migratory, invasive and metastatic potential, as assessed by changes in cell morphology and gene expression profiles. This is the rationale for designing novel EMT/MET-based targeting strategies (Sabbah et al. 2008; Moen et al. 2009; Ponzo et al. 2009). In the context of cancer microenvironments, these transitions offer a possible new explanation of the origins of the inductive stromal fibroblasts and the responding cancer epithelial cells, since both cancer epithelial cells and stromal fibroblasts can be derived from either resident or migrating pluripotent stem cells or from a selective population of transforming cancer epithelial or reactive stromal cells through interactions with specific cell types or factors in the cancer microenvironment (Santamaria-Martinez et al. 2009; Leber and Efferth 2009). A small side population of pancreatic cancer stem cells was recently reported to be particularly sensitive to EMT induction by TGF-β (Kabashima et al. 2009).

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Likewise, circulating cancer cells were shown to be sensitive to growth factor induction to undergo EMT and MET (Vessella et al. personal communication).

1.3.3  Mesenchymal Stem Cells (MSC) MSC are a class of multipotent stem cells capable of differentiating into osteoblasts, chondrocytes, and adipocytes. They can be derived from bone marrow stromal cells and also from adult linage stem cells with self-renewal capability. These cells were found to be present in cancer microenvironments, with the potential of promoting cancer growth and progression (Roorda et al. 2009), exerting immunosuppression by interfering with dendritic and T-cell functions (Spaeth et al. 2008) and ‘marking’ the sites where cancer cells subsequently metastasize (Jung et al. 2009; Kaplan et al. 2005). These functions are generally accomplished by the ability of MSC to secrete specific factors which, via circulatory network and paracrine interaction, confer migratory, invasive and metastatic potential to cancer cells at the primary site of cancer growth. They also interact at the site of metastasis, for instance by increasing bone turnover to create a favorable microenvironment supporting the dissemination of cancer cells (Kaplan et al. 2007).

1.4 The Mediators and Cell Signaling Network Governing the Plasticity of Stromal Fibroblasts Soluble and insoluble mediators secreted by cancer cells and cells in cancer microenvironments are responsible for supporting the growth and progression of cancer by interacting with selective receptors that transmit signals orchestrating a switch in cancer cell morphology and function compatible with the survival of cancer cells. In this section, specific examples defining prostate cancer interactions with soluble and matrix proteins will be used to illustrate the importance of understanding the cell signaling network to identify relevant therapeutic targets for clinical translation and develop new drugs.

1.4.1  Soluble Growth Factors The general model depicted in Fig. 1.1 shows cancer cell-secreted soluble factors promoting the activation of both cancer-associated resident stromal fibroblasts and migrating MSC and/or their derivative stromal fibroblasts. This triggers additional remodeling of tumor microenvironments, reciprocally affecting the genotype and phenotype of both cancer cells and stromal fibroblasts in the cancer microenvironment via soluble factors including growth factors, cytokines, chemokines, and

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reactive oxygen species (ROS) released in the tumor microenvironment by resident and migrating host mesenchymal or stromal cells. These factors often work in concert to induce a primarily stromal reaction manifested by activated stromal fibroblasts or myofibroblasts producing either higher levels and/or more effective combinations of soluble growth factors capable of inducing cancer growth, invasion and metastasis directly, and also of promoting the reorganization of the vascular network favoring the dissemination of cancer cells to distant organs. In other words, soluble factors produced by cancer and host stroma can be disseminated to distant sites where they become responsible for remodeling the premetastatic niche and facilitating subsequent cancer cell dissemination (Kaplan et al. 2007). In some cases, soluble factors can act at long distance at metastatic sites to modulate tumor microenvironments by providing higher concentrations or more complementary growth factors via increased bone turnover, creating a less hostile environment for the growth of cancer cells via immune suppression, or promoting osteomimicry within cancer cells and creating a metastatic bone ‘niche’ favoring overall cancer cell growth and survival at metastatic sites (Chung et al. 2006; Cooper et al. 2003). Soluble factors secreted by cancer and activated stromal cell components within the tumor microenvironment can also modulate other cell signaling networks mediated by integrin-extracellular matrix interactions (Sangaletti et al. 2008; Chiarugi and Giannoni 2005) and androgen receptor signaling pathways (Huang et al. 2006; Olapade-Olaopa et al. 1999), activating the cell signaling network to upregulate integrins and/or androgen receptor expression (Liegibel et al. 2002; Bonaccorsi et al. 2006).

1.4.2  Extracellular Matrices (ECMs) Cancer cell proliferation and survival within the tumor microenvironment depends on the ability to adhere and attach to ECMs (Desgrosellier and Cheresh 2010). Through ECM-integrin interactions, cancer cells can also gain increased invasive, migratory and metastatic potential, mediated by the activation of converging cell signaling networks downstream that confer growth and survival advantages to cancer cells (Pontier and Muller 2009). ECM-integrin interactions, known to affect embryonic development (Armant 2005), also play a directive role in determining the gene expression profiles of cancer cells, the ability of cancer cells to degrade their surrounding ECMs by matrix metalloproteinases (MMPs), to extravasate into metastatic microenvironments by adhesion to organ-specific endothelial cells (Kargozaran et al. 2007), and the status of differentiation of cancer cells.

1.4.3  ROS and Oxidative Stress High levels of ROS and oxidative stress can be created in cancer microenvironments by the continued growth and expansion of solid tumors which deplete oxygen

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supplies and build up metabolic waste at the site of tumor growth. Cancer cells with downregulated manganese superoxide dismutase (MnSOD) show increased levels of superoxide, which induce hypoxia inducing factor 1 (HIF-1α) and VEGF, causing increased angiogenesis and tumor growth (Kaewpila et al. 2008; Wang et al. 2005). An accumulation of hydrogen peroxide can also be induced by placing cancer cells under hypoxic conditions and in cells with defective catalase (Azad et al. 2009). Hydrogen peroxide is a potential mediator contributing to the co-evolution of the genotype and phenotype of both prostate cancer and bone stroma when they are in contact under 3-D co-culture conditions (Sung et al. 2006, 2008). Hypoxic conditions can also affect the transdifferentiation of cancer cells such as EMT and MET, where hypoxia induces EMT (Klymkowsky and Savagner 2009; Jiang et al. 2007) and hyperbaric oxygen treatment causes MET (Moen et al. 2009) with respective corresponding changes of either increased or decreased cancer growth and invasion in animals.

1.5 Overall Significance and Clinical Translation of Integrated Approaches to Cancer–Stromal Fibroblast Interaction Our laboratory has established a 3-dimensional (3-D) co-culture system to investigate how the information derived from cancer–stromal fibroblast interaction can be applied in the clinic and to understand the molecular pathways that determine the behaviors of cancer cells. This approach is based on our prior work showing that cancer cell phenotype and genotype can be irreversibly “programmed” when cancer cells are grown together with prostate or bone stromal cells under 3-D conditions as prostate organoids (Sung et al. 2008; Rhee et al. 2001) or in mice as tumor xenografts (Sung et al. 2008). We found several important features of these types of cellular interactions. (1) The irreversible “programming” of the phenotype and genotype of cancer cells by stromal fibroblasts is bi-directional. We observed that human stromal fibroblasts co-cultured with human prostate cancer cells under 3-D conditions can program the genotype (assessed by cytogenetics and genome-wide scan (Sung et al. 2008) and phenotype (measured by gene expression and ability to grow tumors with metastatic potential in mice (Sung et al. 2008). Remarkably, normal stromal fibroblasts from mouse, benign/normal human prostate stromal fibroblasts, and the MG-63 osteosarcoma cell line have also been observed to undergo irreversible and non-random genotypic and phenotypic changes when co-cultured with prostate cancer cells under 3-D conditions (Sung et al. 2008; Rhee et al. 2001). (2) Gene expression analyses revealed that stromal fibroblasts, after physical contact with prostate cancer cells, had increased levels of brain-derived neurotropic factor (BDNF), chemokines, CCL5 and CXCL5, versican, tenascin, connective tissue growth factor, stromal cell derived factor-1 (SDF-1/CXCL12), and HIF-1α (Sung et al. 2008). We have validated the overexpression of these biomarkers identified by our cell culture model in clinical tissue and serum samples collected from

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prostate cancer patients with confirmed bone metastasis (Sung et al. 2008). These studies highlight the bidirectional interactions and the co-evolution of tumor-stroma in prostate cancer progression.

1.6 Co-targeting Tumor and Stroma as an Effective Therapeutic Strategy for the Treatment of Cancer and Cancer Metastasis Cancer-host microenvironment communication in the primary and at distant metastatic sites, mediated by soluble factors and insoluble matrices, supports the growth and survival of cancer cells. This provides a sound rationale for co-targeting both the tumor and the host microenvironment to achieve better tumor growth control and improve overall patient survival. Cancer development is complex, involving multiple interactions between different cell types and pleiotropic signaling mechanisms leading to progression. Reciprocal interaction between prostate cancer cells and resident and migrating cells in the tumor microenvironments mediated by cell signaling networks should be considered viable targets. Prostate cancer frequently metastasizes to bone and <50% of the patients with hormonal refractory bone metastases survive more than 5 years. Our laboratory has addressed the critical issues of prostate cancer bone metastasis from both the biological and therapeutic perspectives (Chung et al. 2006; Josson et al. 2010; Chung 1993, 1995). We investigated human prostate cancer cell interaction with human osteoclasts, osteoblasts and marrow stromal cells under 3-D co-culture conditions to mimic tumor growth in vivo (Sung et al. 2008). These studies allowed us to conclude that prostate cancer survives in a tumor microenvironment by the activation of specific cell signaling networks with neighboring host cells. During this process, the cancer cells and cells in the cancer microenvironment “co-evolve” in part through their response to growth factors, extra-cellular matrices and ROS (Sung et al. 2008; Rhee et al. 2001; Thalmann et al. 1994). Cancer cells acquire several mimetic abilities, such as osteomimicry, vasculomimicry, neuromimicry and stem cell mimicry, and undergo a transition from epithelium to mesenchyme with definitive behavioral modifications (Huang et al. 2006; Zhau et al. 2008). To develop an effective targeting strategy for prostate cancer bone metastases, it is critical to consider these interactions and devise the most effective way of targeting not only tumor cells, but also cells in the tumor microenvironment (Hsieh et al. 2004; Kubo et al. 2003). Table 1.1 summarizes a number of ongoing and completed clinical trials proving the concept that the tumor-associated stroma compartment is a new and exciting target awaiting the development of novel therapeutics. Our laboratory first conducted a clinical trial co-targeting prostate cancer and bone with an adenoviral-based therapy using a therapeutic toxic gene driven by an osteocalcin promoter shared in common by cancer and bone cells (Hsieh et al. 2004; Kubo et al. 2003). A number of successful avenues have been opened, including co-targeting the interaction of prostate cancer and endothelium via VEGF-mediated signaling with an antibody (e.g.

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Table 1.1   Soluble growth factors and cells in the bone become attractive targets for the development of novel biological-based therapeutics for the management of prostate cancer local growth and distant metastases to bone and soft tissues Therapeutic targets Drugs References (growth factors, cells) Human epidermal growth factor (hEGF) Vascular endothelial growth factor (VEGF)

Transforming growth factor-beta (TGF-β) Insulin growth factor-1 receptor (IGF1R)

Trastuzumab (Herceptin®), Lapatinib (Tykerb®), Gefitinib (Iressa®), Erlotinib (Tarceva®), Cetuximab (Erbitux®), Panitumumab (Vectibix®) Bevacizumab (Avastin®), Ranibizumab (Lucentis), Lapatinib (Tykerb), Sunitinib (Sutent), Sorafenib (Nexavar), Axitinib, Pazopanib AP12009 (Trabedersen), antisense oligonucleotide against TGF-β2 (Phase III), GC1008 anti-TGF-β monoclonal antibody (Phase I) CP-751,871 (monoclonal antibody) Phase I, AP-12 (monoclonal antibody) Preclinical, 19D12 (monoclonal antibody) Preclinical, EM164 (monoclonal antibody) Preclinical, hC7C10 (monoclonal antibody) Preclinical SU-101 kinase inhibitor, Phase III, Gleevec

Platelet derived growth factor (PDGFR) Endothelin receptor Atrasentan 177 Radiolabelled anti­ Lu-labelled J591, 90 bodies—Prostate Y-labelled J591 specific membrane antigen (PSMA) AGS-PSCA (Phase I) Radiolabelled anti­ bodies—Prostate stem cell antigen (PSCA) Integrins Monoclonal antibodies targeting the extracellular domain of the heterodimer: Vitaxin Synthetic peptides containing an RGD sequence: Cilengitide; KGaA Peptidomimetics of RGD sequence: S247 Integrins-αv family CNTO 095 Osteoclasts Bisphosphonates: Non-N-containing bisphosphonates:Etidronate (Didronel), Clodronate (Bonefos, Loron) Tiludronate (Skelid)N-containing bisphosphonates: Pamidronate (APD, Aredia), Neridronate, Olpadronate, Alendronate (Fosamax), Ibandronate (Boniva), Risedronate (Actonel), Zoledronate (Zometa, Aclasta)

http://www.cancer.gov/ cancertopics/factsheet/ Therapy/targeted http://www.cancer.gov/ cancertopics/factsheet/ Therapy/targeted, http://en.wikipedia.org/ wiki/Vascular_ endothelial_growth_ factor Garber (2009)

Garber (2005)

Gibbs (2000) Lalich et al. (2007) Bander et al. (2005); Milowsky et al. (2004) David et al. (2006)

Stupp et al. (2007)

Trikha et al. (2004) http://en.wikipedia.org/ wiki/Bisphosphonate

1  Critical Roles of Stromal Fibroblasts in the Cancer Microenvironments Table 1.1  (continued) Therapeutic targets Drugs (growth factors, cells) Osteoclasts

Bone-seeking radiopharmaceuticals: Radium-223 (Alpharadin®), strontium-89, samarium-153 Osteoclasts (RANKL) Denosumab (Prolia)

13

References Nilsson et al. (2007); Tu et al. (2005) http://en.wikipedia.org/ wiki/Denosumab

bevacizumab) or small molecules to inhibit receptor tyrosine kinases (e.g. sunitinib); co-targeting interactions between prostate cancer and a number of soluble growth factors secreted by cancer, stromal fibroblasts and/or inflammatory cells using either therapeutic antibodies (Chung et al. 2005; Wu et al. 2005) or small molecule tyrosine kinase inhibitors; and co-targeting the prostate cancer/osteoblast interface with the endothelin receptor antagonists Zibotentan or Atrasentan (Kopetz et al. 2002). Cotargeting extracellular matrix-prostate cancer interactions with integrin antagonists against αvβ3 (e.g. Vitaxin) or αv (e.g. CNTO95) has been tested. Bone-directed cotargeting with a RANKL antibody, denosumab, or osteoclast antagonists, bisphosphonates, has helped to reduce bone pain and skeletal-related events (SRE) (Keller 2002; Saad et al. 2009). Co-targeting patients with confirmed bone metastases with radiopharmaceutics, 89Sr, 153Sm (β-emitters), or 188Re (γ-emitter) radionuclides, plus chemotherapy (e.g. docetaxel, mitoxantrone, estramustine and etoposide), long-term androgen suppression and/or external beam radiation has also improved quality of life and prostate cancer patient survival (Sartor 2009; Tu et al. 2005). A common drawback in these trials is the severe marrow toxicity encountered by patients treated with the co-targeting agents. So far, this has compromised the prospect of repeated administration of these highly effective agents to prostate cancer patients for potential “cure”. Recently, an exciting therapeutic development using bone-seeking 223Ra, an α-emitter, for the treatment of prostate cancer bone metastases has been explored (Nilsson et al. 2005, 2007). 223Ra has the advantage of emitting high linear energy transfer (LET) radiation with a short track length in tissues for up to just a few mm. In a randomized, multicentre placebo-controlled phase II trial (Nilsson et al. 2005, 2007), 223Ra was found to be well-tolerated, sparing myelotoxicity while reducing serum bone-alkaline phosphatase concentration, a marker indicative of prostate cancer growth in bone in patients with bone metastases. 223Ra prevented SRE and improved overall survival in patients with hormonerefractory prostate cancer. 223Ra is currently in Phase III trials in the US and Europe in patients with metastatic prostate cancer bone metastases.

1.7 The Frontier of Future Stromal Fibroblast Research The plasticity of stromal fibroblasts and their ability to induce cancer cell growth, migration, invasion and metastasis, to promote cancer cell survival, and to alter cancer cell sensitivity toward chemotherapy and radiation therapy raises the fol-

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lowing questions, which can be considered as some of the future frontiers of stromal fibroblastic research. (1) What are the regulatory mechanisms determining the plasticity and differentiation status of stromal fibroblasts? The intriguing biology of iPS cells taught us the lesson that a normal adult stromal fibroblast can be reprogrammed by the introduction of a cassette of transcription factors, Oct4, Sox2. Klf4 and c-Myc, to become pluripotent stem cells capable of forming cells and organs of diverse lineages including inductive stromal fibroblasts and MSCs. The critical question that needs to be addressed is whether the engineered transcription factor protein(s) produced within tumor microenvironments can play the reprogramming roles of an adult stromal fibroblast and explain the heterogeneity of stromal fibroblasts. This speculation has now been supported by a stunning laboratory demonstration where recombinant transcription factor proteins, when added to cultured adult stromal fibroblasts, reprogrammed these cells to express markers indicative of a stem cells phenotype (Cho et al. 2010; Tang et al. 2010; Rhee et al. 2011). This suggests the possibility that proteins secreted by cancer cells and cells in the tumor microenvironments could reprogram adult cells and that this could be the molecular basis of the reactivation of embryonic growth potential of the stroma, proposed more than three decades ago by McNeil as a contributing factor to benign hyperplastic growth of the prostate gland (BPH) commonly found in the aging male (McNeal 1978). (2) What are the critical soluble factors and ECMs produced by cells in tumor microenvironments that can dictate the growth, survival and metastasis of malignant prostate cancer cells? Published data suggest that a host of factors, including classical soluble growth and survival factors, ROS, chemokines and cytokines, ECMs and their fragments, can modulate cancer–cancer and cancer–stroma interactions. It is becoming increasingly important to reclassify these factors and their combinations based on their molecular actions with special emphasis on factors that confer lethal phenotypes to cancer cells. Developing better tools to predict the clinical outcome of prostate cancer will support the concept and its implementation in personalized and predictive oncology for improved diagnosis, prognosis and treatment of patients with prostate cancer. (3) What are the most effective means of co-targeting tumor and stroma to prevent cancer cells from developing therapeutic resistance? There are an increasing number of clinical trials based on the concept of co-targeting cancer and cancer-associated microenvironments. An improved fundamental understanding of how tumor–stroma interacts, and how the genotype and phenotype of cancer cells may be “co-evoluted” will help us developing better and more effective co-targeting strategies for the management of lethal prostate cancer. Acknowledgements  The author wishes to thank Dr. Sajni Josson for her help in the construction of the figure and the table. The editorial assistance from Gary Mawyer is also appreciated. This work was supported in part by the following grants: R01 CA122602 and P01 CA098912 from National Caner Institute and W81 XWH-01–1-0172 from Department of Defense, United States Army.

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Pontier SM, Muller WJ (2009) Integrins in mammary-stem-cell biology and breast-cancer progression—a role in cancer stem cells? J Cell Sci 122(Pt 2):207–214 Ponzo MG, Lesurf R, Petkiewicz S et al (2009) Met induces mammary tumors with diverse histologies and is associated with poor outcome and human basal breast cancer. Proc Natl Acad Sci U S A 106(31):12903–12908 Prindull G (2005) Hypothesis: cell plasticity, linking embryonal stem cells to adult stem cell reservoirs and metastatic cancer cells? Exp Hematol 33(7):738–746 Radisky DC, Przybylo JA (2008) Matrix metalloproteinase-induced fibrosis and malignancy in breast and lung. Proc Am Thorac Soc 5(3):316–322 Rhee HW, Zhau HE, Pathak S et al (2001) Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel. In Vitro Cell Dev Biol Anim 37(3):127–140 Rhee YH, Ko JY, Chang MY et  al (2011) Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease. J Clin Invest 121(6):2326–2335 Rhodes LV, Muir SE, Elliott S et al (2009) Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence. Breast Cancer Res Treat 113:293–299 Ronnov-Jessen L, Bissell MJ (2009) Breast cancer by proxy: can the microenvironment be both the cause and consequence? Trends Mol Med 15(1):5–13 Roorda BD, ter Elst A, Kamps WA, de Bont ES (2009) Bone marrow-derived cells and tumor growth: contribution of bone marrow-derived cells to tumor micro-environments with special focus on mesenchymal stem cells. Crit Rev Oncol Hematol 69(3):187–198 Saad F, Abrahamsson PA, Miller K (2009) Preserving bone health in patients with hormone-sensitive prostate cancer: the role of bisphosphonates. BJU Int 104(11):1573–1579 Sabbah M, Emami S, Redeuilh G et  al (2008) Molecular signature and therapeutic perspective of the epithelial-to-mesenchymal transitions in epithelial cancers. Drug Resist Updat 11(4– 5):123–151 Sangaletti S, Di Carlo E, Gariboldi S et al (2008) Macrophage-derived SPARC bridges tumor cellextracellular matrix interactions toward metastasis. Cancer Res 68(21):9050–9059 Santamaria-Martinez A, Barquinero J, Barbosa-Desongles A et al (2009) Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis. Exp Cell Res 315(17):3004–3013 Sartor O (2009) Radiopharmaceutical and chemotherapy combinations in metastatic castrate-resistant prostate cancer: a new beginning? J Clin Oncol 27(15):2417–2418 Singh S, Singh R, Singh UP et al (2009) Clinical and biological significance of CXCR5 expressed by prostate cancer specimens and cell lines. Int J Cancer 125(10):2288–2295 Spaeth E, Klopp A, Dembinski J, Andreeff M, Marini F (2008) Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene Ther 15(10):730–738 Stupp R et al (2007) Integrin inhibitors reaching the clinic. J Clin Oncol 25:1637–1638 Sung SY, Kubo H, Shigemura K et al (2006) Oxidative stress induces ADAM9 protein expression in human prostate cancer cells. Cancer Res 66(19):9519–9526 Sung SY, Hsieh CL, Law A et  al (2008) Coevolution of prostate cancer and bone stroma in three-dimensional coculture: implications for cancer growth and metastasis. Cancer Res 68(23):9996–10003 Svineng G, Ravuri C, Rikardsen O, Huseby NE, Winberg JO (2008) The role of reactive oxygen species in integrin and matrix metalloproteinase expression and function. Connect Tissue Res 49(3):197–202 Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676 Tang Y, Lin CJ, Tian XC (2010) Functionality and transduction condition evaluation of recombinant klf4 for reprogramming iPS cells. Cell Reprogram 13:99–112

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Tasseff R, Nayak S, Salim S, Kaushik P, Rizvi N, Varner JD (2010) Analysis of the molecular networks in androgen dependent and independent prostate cancer revealed fragile and robust subsystems. PLoS One 5(1):e8864 Thalmann GN, Anezinis PE, Chang SM et al (1994) Androgen-independent cancer progression and bone metastasis in the LNCaP model of human prostate cancer. Cancer Res 54(10):2577–2581 Thalmann GN, Rhee H, Sikes RA et  al (2009) Human prostate fibroblasts induce growth and confer castration resistance and metastatic potential in LNCaP cells. Eur Urol 58:162–172. Tothill RW, Tinker AV, George J et al (2008) Novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome. Clin Cancer Res 14(16):5198–5208 Trikha M et al (2004) CNTO 95, a fully human monoclonal antibody that inhibits alphav integrins, has antitumor and antiangiogenic activity in vivo. Int J Cancer 110(3):326–335 Tu SM, Lin SH (2008) Current trials using bone-targeting agents in prostate cancer. Cancer J 14(1):35–39 Tu SM, Kim J, Pagliaro LC et al (2005) Therapy tolerance in selected patients with androgen-independent prostate cancer following strontium-89 combined with chemotherapy. J Clin Oncol 23(31):7904–7910 Tuxhorn JA, Ayala GE, Rowley DR (2001) Reactive stroma in prostate cancer progression. J Urol 166(6):2472–2483 Tuxhorn JA, Ayala GE, Smith MJ, Smith VC, Dang TD, Rowley DR (2002) Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling. Clin Cancer Res 8(9):2912–2923 Untergasser G, Gander R, Lilg C, Lepperdinger G, Plas E, Berger P (2005) Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation. Mech Ageing Dev 126(1):59–69 Vessella RL, Corey E (2006) Targeting factors involved in bone remodeling as treatment strategies in prostate cancer bone metastasis. Clin Cancer Res 12(20 Pt 2):6285s–6290s Wang M, Kirk JS, Venkataraman S et al (2005) Manganese superoxide dismutase suppresses hypoxic induction of hypoxia-inducible factor-1alpha and vascular endothelial growth factor. Oncogene 24(55):8154–8166 Wells A, Yates C, Shepard CR (2008) E-cadherin as an indicator of mesenchymal to epithelial reverting transitions during the metastatic seeding of disseminated carcinomas. Clin Exp Metastasis 25(6):621–628 Wu JD, Odman A, Higgins LM et al (2005) In vivo effects of the human type I insulin-like growth factor receptor antibody A12 on androgen-dependent and androgen-independent xenograft human prostate tumors. Clin Cancer Res 11(8):3065–3074 Yanagisawa N, Li R, Rowley D et al (2007) Stromogenic prostatic carcinoma pattern (carcinomas with reactive stromal grade 3) in needle biopsies predicts biochemical recurrence-free survival in patients after radical prostatectomy. Hum Pathol 38(11):1611–1620 Yao Q, Qu X, Yang Q, Wei M, Kong B (2009) CLIC4 mediates TGF-beta1-induced fibroblast-tomyofibroblast transdifferentiation in ovarian cancer. Oncol Rep 22(3):541–548 Yu J, Hu K, Smuga-Otto K et al (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324(5928):797–801 Zhao HF, Chen J, Xu ZS, Zhang KQ (2009a) Distribution and differentiation of mesenchymal stem cells in tumor tissue. Chin Med J (Engl) 122(6):712–715 Zhao XY, Li W, Lv Z et al (2009b) iPS cells produce viable mice through tetraploid complementation. Nature 461(7260):86–90 Zhau HE, Odero-Marah V, Lue HW et al (2008) Epithelial to mesenchymal transition (EMT) in human prostate cancer: lessons learned from ARCaP model. Clin Exp Metastasis 25(6):601–610

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Part II

Stromal Cell Diversity

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Chapter 2

Functional Diversity of Fibroblasts H. Peter Rodemann and Hans-Oliver Rennekampff

2.1 Introduction Solid tumours are multi-cellular tissues comprised of tumour cells and stromal cells, including fibroblasts, endothelial cells and inflammatory cells. When a cancer cell metastasizes, it first will be exposed to cancer associated fibroblasts in the immediate tumour microenvironment and subsequently to normal fibroblasts as it traverses the underlying connective tissue on its way to the bloodstream. So far, the interactions of tumour cells with stromal fibroblasts influence tumour biology by mechanisms that are not yet fully understood. It is known that cells of the tumour parenchyma and stroma are in extensive crosstalk, and the composition of the stroma and the nature of tumour stromal interactions change over time with tumour progression (Beacham and Cukierman 2005; Proia et al. 2005). The tumour-stroma crosstalk markedly influences not only tumour growth by modifying and controlling angiogenesis, suppressing or subverting immune responses of the host, but also by modulating extracellular matrix composition, and secreting factors which in turn stimulate cells to further alter cell physiology as well as the cellular and acellular composition of the tumour microenvironment (Stuelten et  al. 2010; Olumi et  al. 1999; Liotta and Kohn 2001). Fibroblasts as ubiquitous stromal cells are accessory cells which influence other neighbouring cell types through secretion of cytokines as well as growth and differentiation factor factors (Baglole et al. 2006; Micke and Ostman 2005). While in the early phase fibroblasts can have tumour suppressing activity the phenotype of fibroblasts can alter to a tumour promoting state as carcinogenesis progresses (Proia et al. 2005). This phenotypic switch does occur in two stages in which a reversible “primed” fibroblasts type is generated first followed the manifestation of an irreversible phenotype of fibroblasts which present tumour-promoting properties H. P. Rodemann () Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, Eberhard Karls University Tübingen, Röntgenweg 11, 72076 Tübingen, Germany e-mail: [email protected] M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_2, © Springer Science+Business Media B.V. 2011

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(Beacham and Cukierman 2005). These so-called cancer associated fibroblasts (CAF) have properties distinct from normal fibroblasts and actively promote tumorigenesis as it has been described for example for prostate and breast cancer (Olumo et al. 1999; Orimo et al. 2005). The topic of cancer associated fibroblasts, however, is discussed in separate chapters of this book. In the following various aspects of the functional diversity of normal not cancer associated fibroblast will be discussed as the capacity of fibroblasts to produce and organize the extracellular matrix and to communicate with other cell systems makes them a central component of tissue biology.

2.2 Fibroblast Phenotypes Fibroblasts from different anatomical regions display characteristic phenotypes. Early work on the heterogeneity of fibroblasts performed by Castor and co-workers (Castor et al. 1962) demonstrated metabolic differences between mesothelial fibroblasts, fibroblasts of the skin, articular tissues and periosteum. Studies by Chang et al. (2002) and Rinn et al. (2006, 2008) characterized the gene expression profile of fibroblasts derived from different anatomical regions of the body with cDNA microarray technology. These authors were able to show a site specific gene expression pattern with a striking division into: anterior-posterior, proximal-distal and dermal-non dermal. In addition they demonstrated diversity in topographic expression for genes involved in extracellular matrix synthesis, growth and differentiation, cell migration as well as genes involved in genetic syndromes. Work by Chipev and Simon (2002) indicated that fibroblasts from different body sites differ in size, with palmar fibroblasts being smaller than non-glabrous derived fibroblasts. In addition growth kinetics and TGF (Transforming Growth Factor) -β1 Receptor II expression as well as the ability to contract collagen lattices were found to differ with palmoplantar skin derived fibroblasts having lower receptor levels and an increased mitotic rate. The authors speculated that this regional diversity may in part account for localized susceptibility to disease manifestation like scarring and keloid formation. Recent work on oral mucosal fibroblasts has demonstrated the differences in the capacity of these fibroblasts to reorganize collagen lattices by an increased matrix metalloproteinase (MMP)-2 expression as compared to skin derived fibroblasts (Stephens et al. 2001). Furthermore, it was shown that oral mucosa derived fibroblasts proliferated more rapidly and had a higher capacity for cell doublings. A functional correlation was linked to the fact that cultured oral fibroblasts secret more hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) than skin fibroblasts (Gron et al. 2002). It was suggested that both effects may contribute to a ‘fetal’ wound healing phenotype of oral fibroblasts. Beside the differences in fibroblasts from different anatomical sites fibroblasts separated from a single tissue are similarly not composed of a homogeneous population but rather consist of subsets of different fibroblasts. Sorrell and Caplan (2004) have reviewed differences between papillary fibroblasts, which reside in the

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superficial dermis and reticular fibroblasts which are located in the deeper dermis, highlighting the difference in matrix molecule production from these two distinct cell populations. Versican is produced at low levels by papillary fibroblasts while reticular fibroblasts produce high levels of this molecule. In contrast, decorin is produced in high levels by papillary fibroblast but only in small amounts by reticular fibroblasts. Collagen type I and collagen type III production and the subsequent ratio of these two dermal collagens were not found to differ between these populations of fibroblasts. Studies from the laboratory of the author also point toward the existence of different morphological and biochemical subsets of fibroblasts within the same tissue. Two major populations of fibroblasts have been detected in the dermis, lung and kidney termed mitotically active, i.e. replicative progenitor fibroblasts (MF) and irreversible postmitotic fibrocytes (PMF). These two subsets have subsequently been classified by cytomorphological and biochemical characteristics (Bayreuther et al. 1988; Rodemann 1989; Rodemann and Mueller 1990; Rodemann and Bamberg 1995; Lara et al. 1996; Rodemann et al. 1996; Burger et al. 1998; Herskind et al. 1998; Hakenjos et al. 2000). Based on in vitro and ex vivo-in vitro experiments it was shown that fibroblasts of human, rat and mouse skin but also lung as well as renal fibroblasts differentiate in vivo and in vitro along a lineage of replicative progenitor fibroblast cell types and non replicative, postmitotic functional fibrocytes of man, rat and mouse (Mollenhauer and Bayreuther 1986; Bayreuther et al. 1988; Rodemann et al. 1991). According to their cytomorphology (Fig. 2.1), replicative potential and ability to synthesize specific cytokines and growth factors, like TGF-β and Keratinocyte Growth Factor (KGF), MF-progenitor fibroblasts can be further classified into the cell types MF I, MF II, and MF III (Rodemann 1989; Herskind et al. 1998; Burger et al. 1998; Hakenjos et al. 2000). Subcloning experiments revealed that these MF-cell types differentiate along the lineage MF I, MF II,

Parameter

MFl

MFll

MFlll

PMF

Cell division potential

+++

++

+

–

SA-ß-Gal expression

–

(+)

+

+++

Total collagen synthesis

(+)

+

+

+++

KGF-production

(+)

+

++

+++

+

+

+++

+++

TGFß1-production

Fig. 2.1   Differentiation lineage, marker and collagen expression, as well as growth factor production profile of progenitor fibroblasts and postmitotic fibrocytes. The differentiation lineage of progenitor fibroblasts (MF) and postmitotic fibrocytes (PMF) along the sequence MFI-MFIIMFIII-PMF cell types was analysed on the basis of cell biological and biochemical parameters. Quantitative and qualitative differences could be demonstrated for the parameter cell division potential, SA-β-galactosidase (SA-β-Gal) expression, total collagen synthesis, KGF and TGFβ1 production. A relative indicator system (− negative; (+) very low; + low; ++ moderate; +++ high) is chosen for cell type specific comparison of these parameters

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MF III, before differentiation into irreversible postmitotic fibrocytes (PMF) occurs (Bayreuther et al. 1988, 1989; Rodemann et al. 1996). MF I-type progenitor fibroblasts have the highest replicative capacity. MFI progenitor fibroblasts of human origin can go through about cell divisions before differentiating into MF II type cells. This cell type can divide approximately 15–20 times before differentiating into cell type MF III which has the potential for only 5–8 further division cycles before differentiating spontaneously into irreversible postmitotic fibrocytes (Rodemann et al. 1989). Based on its biochemical activity the latter cell type reflects the functional cell type of the fibroblast-fibrocyte cell system. Per cell it produces about 5–8 times more total collagen as compared to progenitor fibroblasts and provides the correct ratio of interstitial collagens type I, III, and V needed for homeostasis and maintenance of interstitial tissues (Rodemann et al. 1989). The cellbiological and biochemical parameters of progenitor fibroblasts and postmitotic fibrocytes are summarized Fig. 2.1. As reported earlier for human skin (Bayreuther et  al. 1988; Rodemann 1993; Herskind et al. 1998) as well as rat lung (Hakenjos et al. 2000) a constant ratio in the in vivo proportion of progenitor fibroblasts and functional postmitotic fibrocytes of 2:1 exists and seems to be independent of the age of the donor. Thus it can be assumed that biochemical homeostasis of the dermis as well as of interstitial tissues in various organs depends on a correct ratio of progenitor fibroblasts and functional postmitotic fibrocytes. Disturbance of this ratio by exogenous or endogenous factors may lead to an altered cellular homeostasis in the affected tissue and can result in pathologic tissue remodelling, similar to that observed in renal, lung and skin fibrosis (Rodemann and Bamberg 1995; Rodemann et al. 1996; Burger et al. 1998; Herskind et al. 1998; Rodemann and Mueller 1990; Rodemann et al. 1991; Hakenjos et al. 2000; von Pfeil et al. 2002). Under in vitro conditions, progenitor fibroblasts and functional postmitotic fibrocytes can be classified according to the specific expression of the senescence activated-β-Galactosidase activity (SA-β-Gal), which is also known as pH6-activity of β-galactosidase. Postmitotic fibrocytes show a strong expression of SA-β-Gal activity whereas MF-progenitor fibroblasts do not (Hakenjos et  al. 2000). The expression of this enzyme activity has been used in attempts to identify cellular ageing processes in fibroblasts cultures (Dimri et al. 1995). The expression of pH6β-galactosidase activity could only be demonstrated in fibroblasts which have undergone replicative senescence, i.e. in irreversible postmitotic cells (Dimri et  al. 1995). These fibroblast populations are predominantly composed by the functional and irreversible postmitotic fibrocytes (Herskind et al. 1998; Hakenjos et al. 2000) (for summary see Fig. 2.1). Decreased proliferative potential of fibroblasts has also been noticed in a stress-induced senescence-like phenotype. Replicative senescence can be induced via pathways that accelerate shortening of the telomere, while premature stress-induced senescence can be caused by DNA damage via telomere-independent mechanisms e.g. oxidative stress or UVB exposure (Von Zglinicki et al. 1995; Pascal et al. 2005; Debacq-Chainiaux et al. 2005). The analysis of purified subpopulations of human MF-progenitor fibroblasts derived from skin and obtained

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through subcloning procedures indicated that MFI, MFII and MFIII progenitor fibroblasts show increased telomere-shortening along the differentiation sequence MF1-MFII-MFIII (Herskind et al. unpublished results). As demonstrated for the rabbit kidney two biochemically diverse subpopulations of fibroblasts have been characterized in the cortex and papilla (Rodemann et al. 1991; Knecht et al. 1991). Papillary fibroblasts presented an about 50% longer mitotic lifespan than cortical fibroblasts before both subtypes differentiate into postmitotic fibrocytes. In addition to differences in protein expression, these two renal fibroblast subtypes respond differentially to growth factor stimulation as well as to stimuli from co-cultured epithelial cells. While no differences in growth response of these two subtypes could be observed after mitogenic stimulation with either epidermal growth factor (EGF) or insulin-like growth factor-1 (IGF-1) a significant differential response could be demonstrated for platelet derived growth factor (PDGF). PDGF exerted a potent mitogenic stimulus for papillary fibroblast but was only weakly mitogenic for cortical fibroblasts (Knecht et al. 1991). Additional patterns of fibroblasts heterogeneity have been identified through the study of wounding and wound healing. Wound healing proceeds in three interrelated phases (reviewed in Clark et  al. 1993): inflammation phase, proliferation phase, and maturation phase. Although fibroblasts have long been recognized as key cells in those process, it is only recently that we have begun to understand the origin and phenotypic differences of fibroblasts as related to the specific phases of wound healing. The initial inflammatory phase consists of an orderly extravasation of leucocytes like PMN and monocytes/macrophages into the wound. Bucala et al. (1994) have shown that a population of fibroblasts which arise from blood-borne circulating progenitor cells migrate into the wound site and may account for as many as 10% of the cells that infiltrate sites of acute tissue injury. These cells were also termed fibrocytes and express markers for cells of both hematopoietic and mesenychmal origin. These fibrocytes have to be clearly distinguished from the fibrocytes described by author and which resemble postmitotic fibroblasts. In addition it is unclear whether Bucala’s fibrocyte of hematopoetic origin enters the fibroblast lineage described above. The collagen+/vimentin+/CD 34+/CD 13+/CD45+ positive fibrocyte cells may contribute to normal wound repair by antigen presentation but may also participate in pathologic fibrotic responses (Chesney et al. 1997). Tredget and his group (Yang et al. 2005) identified this cell type in hypertrophic scarring of the skin. During the proliferative phase of wound healing granulation tissue and fibrovascular ingrowth occurs. Rossio-Pasquier et  al. (1999) have demonstrated that in full thickness dermal loss, fibroblasts migrating into the wound originate from the subcutaneous wound bed rather than from the non injured surrounding dermis. Van den Bogaerdt et  al. (2002) have shown that mitotic and contractile activity differs significantly between these dermal and fat derived fibroblasts. Middelkoop (2005) suggested that these differences may have a great impact on the use of either acellular dermal substitutes which repopulate from the subcutaneous fat or dermalderived fibroblast-seeded matrices.

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In the final phase of wound healing, a specialized population of differentiated fibroblasts occur (Gabbiani et  al. 1971, 2003). It has been demonstrated that fibroblasts under mechanical tension and under the influence of Platelet Derived Growth Factor (PDGF) express stress fibers. This cell type is termed proto-myofibroblast. Additional tension, TGF-β and a splice variant of fibronectin promote further differentiation into the myofibroblasts. These contractile myofibroblasts express alpha smooth muscle actin and are involved in the production of extracellular matrix and tissue contraction. Germain et al. (1994) provided evidence that human wound fibroblasts represent a functionally different population than fibroblasts isolated from unwounded dermis. In their analysis they concluded that 30–40% of wound-derived fibroblasts were myofibroblasts whereas only 1% of normal fibroblasts were alpha smooth muscle actin positive. Early studies from Bell et al. (1979) demonstrated that fibroblasts of high population doubling which have left the cell cycle can carry out lattice contraction at least as effectively as cycling cells from early passages, leading the authors to conclude that the contraction of wounds is not due to myofibroblasts. In vivo data indicated that the number of myofibroblasts in the wound is dependent upon the amount of transplanted deeper dermal portion (Rudolph et  al. 1991). Unfortunately it remains unclear whether this effect is due to the matrix or the resident cell population. Regardless of whether myofibroblasts are the true motile force for wound contraction, there is general agreement that wound contraction is an active cellular phenomenon depending on the activity of viable fibroblasts. Biological control of these cells either by the matrix or by surrounding cells may minimize scar contraction (Bell 1995; Bell et al. 1981). Interestingly, and of great importance for the understanding of the biology of chronic wounds, is the fact that a different fibroblast phenotype has been identified in diabetic ulcers when compared to normal undiseased skin as well as healthy skin from non-diabetic volunteers (Loots et  al. 1999). Fibroblasts from non-insulin-dependent diabetic ulcers exhibited a lower proliferation capacity and more flattened morphologic appearance (Loots et al. 1999). It is tempting to speculate that this fibroblast population resembles postmitotic fibroblasts. Whether these phenotypic differences in fibroblasts are the underlying cause for nonhealing diabetic ulcers or a secondary phenomenon reflecting an altered growth factor profile in this type of wound was not examined. Studies by Morocutti et al. (1996, 1997) revealed that dermal fibroblasts from insulin dependent diabetic patient with progressive fibrosis exhibit a remarkably decreased potential for proliferation as well as increased collagen production correlating with an increased proportion of postmitotic fibrocytes. Interestingly, these altered fibroblasts were not derived from lesional skin but from otherwise healthy skin of these patients. Hasan et al. (1997) also presented evidence that fibroblasts from chronic venous ulcer wounds are phenotypically altered, as a slower proliferation rate of wound derived fibroblasts was noticed. In addition a decreased expression of type 2 TGFβ receptors of ulcer fibroblast was accompanied by a failure to phosphorylate Smad 2, Smad 3, and p42/44 mitogen activating protein kinase (MAPK).

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2.3 Functional Effects of Fibroblast Subpopulations Besides the well established matrix building effect of fibroblasts, there is experimental evidence for a fibroblast-derived mesenchymal–epithelial crosstalk regulating epithelial growth factors (Clark 2003). For example reepithelialisation of cutaneous wounds as a concert of proliferation and migration of keratinocytes is strongly dependent on mesenchymal cell- derived factors. Fibroblasts are able to secrete IL-6, IL-8, HGF, and KGF all of which are known to stimulate keratinocyte proliferation and migration. Fusenig and his group (Fusenig 1994; Maas-Szabowski et  al. 1996) have demonstrated that growth-arrested X-ray-irradiated fibroblasts and proliferating fibroblasts profoundly differ in their profiles of IL-1 alpha stimulated cytokines and growth factor secretion. IL-1 alpha stimulated postmitotic fibroblasts depicted lower transcriptional levels (mRNA) of KGF, IL-1 alpha, IL-8, while HGF mRNA was dramatically increased. However, on the protein level IL-1 alpha-stimulated irradiated fibroblasts presented lower HGF levels than proliferating dermal fibroblasts. In previous work by others (Limat et al. 1989; Waelti et al. 1992) the growth promoting effect of postmitotic fibroblasts on keratinocytes was clearly demonstrated in vitro. As recently published (Nolte et  al. 2008), non-stimulated subpopulations of dermal derived MF-progenitor fibroblasts or postmitotic fibrocytes constitutively secrete significantly different amounts of KGF. When compared to progenitor fibroblasts MF II the amount of KGF produced per cell increases significantly in MF III type progenitor cells by a factor of about 2.2 (see also Fig. 2.1). Postmitotic fibrocytes, however, produce more than 3-times more KGF than MFII-type progenitors and app. 1.5-times more than MFIII type progenitor fibroblasts. These observations indicate a specific role of the subtypes of progenitor fibroblasts and postmitotic fibrocytes in the mesenchymal–epithelial interaction and specifically in the homeostasis of the epithelial cell system in skin. As previously described, TGF-β, another important growth factor is secreted by fibroblasts and counteracts the mitotic effect of KGF on keratinocytes. Among the three isoforms of TGF-β, TGF-β1 is the most prominent regulator. TGF-β1 was shown to down regulate epithelial growth and induce differentiation and apoptosis in keratinocytes (Mansbridge and Hanawalt 1988). Hakenjos et al. (2000) and von Pfeil et al. (2002) have previously demonstrated that TGF-β1 is predominantly produced by MF III type progenitor fibroblasts as well as postmitotic fibrocytes (see also Fig. 2.1). This cytokine plays an important role in the autocrine regulation of the differentiation process of progenitor fibroblasts to functional fibrocytes through the induction of cell cycle inhibitor proteins like p21 and p27 mediating permanent cell cycle arrest in G0 (Lee and Bae 2002). The importance of fibroblast/fibrocyte interaction with tissue specific epithelia is also demonstrated by the use of fibroblasts to construct skin substitutes. Fibroblasts seeded into a collagen-GAG matrix were shown to promote rapid epithelial outgrowth on the collagen-GAG matrix as compared to a non seeded collagen-GAG (Boyce et al. 1988, 1995, 2000; Boyce 2001). In contrast, the feeder layer system used to culture keratinocytes utilizes postmitotic fibrocytes, originally derived from

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the irradiated transformed mouse 3T3 cell line for co-culture (Rheinwald and Green 1975). There is strong evidence from radiobiological research that irradiation does not necessarily kill the feeder cells, but rather induces terminal differentiation with irreversible growth arrest (Rodemann and Bamberg 1995), yet largely preserves physiologic function in producing growth factors and extracellular matrix proteins (Rodemann and Bamberg 1995; Lara et al. 1996; Hakenjos et al. 2000; von Pfeil et  al. 2002). So far however, the functional influence of the secreted cocktail of growth factors and cytokines by the various subpopulations of fibroblasts on keratinocyte proliferation remains unclear. Despite the possible qualitative and quantitative differences between the different subpopulations it is well described that a direct cell-cell contact is crucial to promote keratinocyte growth because fibroblast conditioned medium cannot substitute for feeder cells (Briggaman and Wheeler 1968; Krejci et al. 1991). In addition to growth promoting experiments, a novel line of research is focussing on site specific interaction of fibroblast subtypes on development and tissue homeostasis. In an elegant set of experiments (Yamaguchi et al. 2005) it was shown that fibroblasts derived from soles and palms are able to induce keratin 9 mRNA in cultured non-palmoplantar keratinocytes. Non-palmoplantar keratinocytes cultured alone or in co-culture with non-palmoplantar fibroblasts failed to express keratin 9, indicating the extrinsic regulation by signals from site specific fibroblasts. In a similar set of experiment site specific regulation of melanocytes and pigmentation was also demonstrated (Yamaguchi et al. 2005). In the context of functional epitheliazation there is evidence that fibroblasts play a major role in basement membrane formation either alone or in conjunction with overlying keratinocytes (Contard et al. 1993; Cooper et al. 1993; Sahuc et al. 1996). These studies have demonstrated that keratinocytes alone were either unable to or limited in the production of laminin 1, collagen IV and laminin 5. In contrast, human fibroblasts, alone or in combination with overlying keratinocytes showed significant production of laminin 1 and collagen IV, and laminin 5. Subsequently, fibroblasts seeded into a collagen-GAG matrix were shown to promote basement membrane formation and epidermal homeostasis as compared to a non seeded collagen-GAG. Total collagen production by fibroblasts is dependent on the subpopulation of fibroblasts with postmitotic fibroblasts producing 5 to 8 times more collagen type I, III or V as compared to progenitor fibroblasts (Rodemann et al. 1989). Moreover, cultured postmitotic fibrocytes in comparison to progenitor fibroblasts produce the in vivo-like proportion of interstitial collagens me, III and V indicating again the important role of this cell type for tissue and ECM homestasis.

2.4 Functional Effects of Extracellular Matrix on Fibroblasts While it is well accepted that fibroblasts produce and modulate extracellular matrix there is strong evidence that extracellular matrix material as well as the specific lattice shape regulate cell shape and function of fibroblasts and may act as a form of

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switch between proliferative and differentiate states. Eckes et al. (1993) examined fibroblast behaviour in a three-dimensional contracted collagen matrix and compared them to fibroblast monolayers. They found that culture of dermal fibroblasts seeded in collagen gel leads to a time-dependent depression of pro alpha 1(I) collagen mRNA levels. It was not however investigated whether under these conditions fibroblasts changed in morphology or in their receptor expression. There is evidence that activation of the integrin alpha1 beta1 leads to down-regulation of collagen synthesis (Langholz et al. 1995). Studies by Nakagawa et al. (1989) on skin fibroblasts demonstrated that collagen embedding influences the cellular response and subsequent growth factor release of some but not all growth factors, e.g. IL-1β. Contracting lattices placed under tension lead to synthesis of collagen and regulated the expression of cytokines like TGF- beta 1 differently from relaxed collagen lattices embedding (Eckes et al. 2000; Kessler et al. 2001). Unfortunately phenotypic changes of these embedded fibroblasts were not investigated. Different matrix materials were also shown to have a significant influence on fibroblast function. Normal human cultured fibroblasts undergo programmed cell death in a three- dimensional contractile collagen gel while fibrin gels did not exert this effect (Fluck et al. 1998). In addition fibroblasts did contract the fibrin gel but did not proliferate. In anchored collagen gels fibroblasts showed a doubling time of 2 days and no apoptotic cell death. Despite all this evidence only limited in vivo studies have been undertaken to objectively evaluate scarring as a function of different dermal matrices, which is of crucial interest for dermal engineering. Mansbridge and his group (Kern et al. 2001, 2002) analysed fibroblasts behaviour in monolayer collagen cultures and scaffold based three-dimensional cultures with special attention to the expression of molecules associated with immune system activation. In monolayers of dermal fibroblasts expression of HLA class I but not of HLR-DR was found. Induction of HLA-DR and an increase in HLA -class I expression was observed after interferon-gamma stimulation. In contrast fibroblasts cultured in three-dimensional scaffolds consisted of a fraction of fibroblasts which were non-responsive to gamma interferon. After isolation and subsequent monolayer culture this effect was reversible. Interestingly, collagen-gel-based three-dimensional fibroblast cultures did not exert a similar unresponsiveness and it was concluded that the interaction of fibroblasts with naturally deposited extracellular matrix other than collagen was responsible for this effect. Kern et al. (2002) examined the expression of CD40 on fibroblasts as the interaction of fibroblasts and T-lymphocytes through the CD40 receptor was suggested to contribute to fibrogenesis. Human dermal fibroblasts express CD40 in a manner that is inversely related to proliferation and it was concluded that CD40 expression is greater in the stationary phase than in the log phase. In three-dimensional scaffold based fibroblast cultures responsiveness to interferon-gamma with subsequent CD40 expression was significantly reduced providing a possible etiology for the lack of rejection of allergenic three-dimensional scaffold based fibroblast cultures. This effect appears to be significantly dependent upon the scaffold utilized as Navsaria and his group (Griffiths et al. 2004) and Lamme et al. (2002) have shown that allergenic fibroblasts seeded in a collagen matrix do not persist but rather induce a fibrotic response.

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2.5 Concluding Remarks As reviewed in all investigated tissue and organ systems fibroblasts represent a functional diverse population of cells. Phenotypic differences are manifested in a variety of ways: extracellular matrix production and organization as well as the secretion of and response to growth factors. The functional diversity of the different fibroblast subtypes have to be considered when aspects of tissue homeostasis, especially with respect to stromal or mesenchymal-epithelial interactions and crosstalk are discussed. For example as stated by Sorrel and Caplan (2004) for dermal fibroblasts the term “fibroblast” is an oversimplification. All investigations into phenotypic characteristics of stromal fibroblasts indicated that these cells represent a dynamic and diverse population of functional cell types. A specific nomenclature should reflect this fact and greater care should be taken when defining the population of fibroblasts used in experimental studies. The terminology fibrocyte should be reserved for the postmitotic subtype of fibroblasts as the term ‘cyte’ implicates a non-proliferative cell population.

References Baglole CJ, Ray DM, Bernstein SH, Feldon SE, Smith TJ (2006) More than structural cells, fibroblasts create and orchestrate the tumor microenvironment. Immunol Invest 35:297–325 Bayreuther K, Rodemann HP, Francz PI, Maier K (1988) Differentiation of fibroblast stem cells. J Cell Sci Suppl 10:115–130 Bayreuther K, Rodemann HP, Hommel R, Dittmann K, Albiez M, Francz PI (1989) Human skin fibroblasts in vitro differentiate along a terminal cell lineage. Proc Natl Acad Sci U S A 85:5112–5116 Beacham DA, Cukierman E (2005) Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol 15:329–341 Bell E (1995) Strategy of the selection of scaffolds for tissue engineering. Tissue Eng 1:163–179 Bell E, Ivarsson B, Merrill C (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci U S A 76:1274–1278 Bell E, Ehrlich HP, Sher S, Merrill C, Sarber R, Hull B, Nakatsuji T, Church D, Buttle DJ (1981) Development and use of a living skin equivalent. Plast Reconstr Surg 67:386–392 Boyce ST (2001) Design principles for the composition and performance of cultured skin substitutes. Burns 27:523–533 Boyce ST, Christianson D, Hansbrough JF (1988) Structure of a collagen-GAG skin substitute optimized for cultured human epidermal keratinocytes. J Biomed Mater Res 22:939–957 Boyce ST, Goretsky MJ, Greenhalgh DG, Kagan RJ, Rieman MT, Warden GD (1995) Comparative assessment of cultured skin substitutes and native skin autograft for the treatment of full thickness burns. Ann Surg 222:743–752 Boyce ST, Supp AT, Wickett RR, Hoath SB, Warden GD (2000) Assessment with the dermal torquemeter of skin pliability after treatment of burns with cultured skin substitutes. J Burn Care Rehabil 21:55–63 Briggaman RA, Wheeler CE (1968) Epidermal-dermal interactions in adult human skin: role of dermis in epidermal maintenance. J Invest Dermatol 51:454–465 Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A (1994) Circulating fibrocytes define a new leukocyte subpopulations that mediates tissue repair. Mol Med 1:71–81

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Burger A, Löffler H, Bamberg M, Rodemann HP (1998) Molecular and cellular basis of radiation fibrosis. Int J Radiat Biol 73:401–408 Castor CW, Prince RK, Dorstewitz EL (1962) Characteristics of human “fibroblasts” cultivated in vitro from different anatomical sites. Lab Invest 11:703–713 Chang HY, Chi JT, Dudoit S, Bondre C, Van de Rijn M, Botstein D, Brown PO (2002) Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci 99:12877–12882 Chesney J, Bacher M, Bender A, Bucala R (1997) The peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming naive T cells in situ. Proc Natl Acad Sci U S A 94:6307–6312 Chipev CC, Simon M (2002) Phenotypic differences between dermal fibroblasts from different body sites determine their responses to tension and TGFbeta1. BMC Dermatol 2:13 Clark RAF (2003) Epithelial-mesenchymal networks in wounds: a hierarchal view. Commentary. J Invest Dermatol 120:9–11 Clark RAF, Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen KF (eds) (1993) Mechanisms of cutaneous wound repair; dermatology in general medicine. McGraw Hill, New York, pp 473–486 Contard P, Bartel RL, Jacobs L, Perlish JS, MacDonald ED, Handler L, Cone D, Fleischmajer R (1993) Culturing keratinocytes and fibroblasts in a three-dimensional mesh results in epidermal differentiation and formation of a basal lamina-anchoring zone. J Invest Dermatol 100:35–59 Cooper ML, Andree C, Hansbrough JF, Zapata-Sirvent RL, Spielvogel Rl (1993) Direct comparison of a cultured composite skin substitute containing human keratinocytes and fibroblasts to an epidermal sheet graft containing human keratinocytes on athymic mice. J Invest Dermatol 101:811–819 Debacq-Chainliaux F, Borlon C, Pascal T, Royer V, Eliaers F, Ninane N, Carrard G, Friguet B, de Longueville F, Boffe S, Remacle J, Toussaint O (2005) Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-beta1 signaling pathway. J Cell Sci 118:743–758 Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelly C, Medrano EE, Linskens M, Rubel JJ, Pereira-Smith O, Peacocke M, Campisi J (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367 Eckes B, Mauch C, Hüppe G, Krieg T (1993) Downregulation of collagen synthesis in fibroblasts within three-dimensional collagen lattices involves transcriptional and posttranscriptional mechanisms. FEBS 216:129–133 Eckes B, Kessler D, Aumailley M, Krieg T (2000) Interactions of fibroblasts with the extracellular matrix: implications for the understanding of fibrosis. Springer Semin Immunopathol 21:415–429 Fluck J, Querfeld C, Cremer A, Niland S, Krieg T, Sollberg S (1998) Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels. J Invest Dermatol 110:153–157 Fusenig NE (1994) Epithelia-mesenchymal interactions regulate keratinocyte growth and differentiation in vitro. In: Leigh I, Lane B, Watt F (eds) The keratinocyte handbook. Cambridge University Press, Cambridge, pp 71–94 Gabbiani G (2003) The myofibroblast in wound healing and fibrocontractive diseases. J Pathol 200:500–503 Gabbiani G, Ryan GB, Majno G (1971) Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction. Experientia 27:549–550 Germain L, Jean A, Auger F, Garrel DR (1994) Human wound healing fibroblasts have greater contractile properties than dermal fibroblasts. J Surg Res 57:268–273 Griffiths M, Ojeh N, Livingstone R, Price R, Navsaria H (2004). Survival of Apligraf in acute human wounds. Tissue Eng 10:1180–1195 Gron B, Stoltze K, Andersson A, Dabelsteen E (2002) Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes. APMIS 110:892– 898

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Hakenjos L, Bamberg M, Rodemann HP (2000) TGF-β1-mediated alterations of rat lung fibroblast differentiation resulting in the radiation-induced fibrotic response. Int J Radiat Biol 76:503–509 Hasan A, Murata H, Falabella A, Ochoa S, Zhou L, Badiavas E, Falanga V (1997) Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factor-beta1. J Dermatol Sci 16:59–66 Herskind C, Bentzen SM, Overgaard J, Bamberg M, Rodemann HP (1998) Differentiation state of skin fibroblast cultures versus risk of subcutaneous fibrosis after radiotherapy. Radiother Oncol 47:263–269 Kern A, Liu K, Mansbridge J (2001) Modification of fibroblast gamma-interferon responses by extracellular matrix. J Invest Dermatol 117:112–118 Kern A, Liu K, Mansbridge J (2002) Modulation of interferon-gamma response by dermal fibroblast extracellular matrix. Ann N Y Acad Sci 961:364–367 Kessler D, Dethlefsen S, Haase I, Plomann M, Hirche F, Krieg T, Eckes B (2001) Fibroblasts in mechanically stressed collagen lattices assume a “synthetic” phenotype. J Biol Chem 276:36575–36585 Knecht A, Fine LG, Kleinman KS, Rodemann HP, Müller GA, Woo DD, Norman JT (1991) Fibroblasts of rabbit kidney in culture. II. Paracrine stimulation of papillary fibroblasts by PDGF. Am J Physiol 261:F292–F299 Krejci NC, Cuono CB, Langdon RC, McGuire J (1991) In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis. J Invest Dermatol 97:843–848 Lamme EN, van Leeuwen RTJ, Brandsma K, van Marle J, Middelkoop E (2000) Higher number of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar-tissue formation. J Pathol 190:595–603 Lamme EN, van Leeuwen RT, Mekkes JR, Middelkoop E (2002) Allogeneic fibroblasts in dermal substitutes induce inflammation and scar formation. Wound Repair Regen 10:152–160 Langholz O, Rockel D, Mauch C, Kozlowska E, Bank I, Krieg T, Eckes B (1995) Collagen and collagenase expression in three-dimensional collagen lattices are differentially regulated by alpha 1 beta 1 and alpha 2 beta 1 integrins. J Cell Biol 131:1903–1915 Lara PC, Russell NS, Smolders IJ, Bartelink H, Begg AC, Coco-Martin JM (1996) Radiationinduced differentiation of human skin fibroblasts: relationship with cell survival and collagen production. Int J Radiat Biol 70:683–692 Lee KY, Bae SC (2002) TGF-beta-dependent cell growth arrest and apoptosis. J Biochem Mol Biol 35:47–53 Limat A, Hunziker T, Boillat C, Bayreuther K, Noser F (1989) Postmitotic human dermal dermal fibroblasts efficiently support the growth of human follicular keratinocytes. J Invest Dermatol 92:758–762 Liotta LA, Kohn EC (2001) The microenvironment of the tumor-host interface. Nature 411:375– 379 Loots MA, Lamme EN, Mekkes JR, Bos JD, Middelkoop E (1999) Cultured fibroblasts from chronic diabetic wounds on the lower extremity (non-insulin-dependent diabetes mellitus) show disturbed proliferation. Arch Dermatol Res 291:93–99 Maas-Szabowski N, Fusenig NE (1996) Interleukin-1-induced growth factor expression in postmitotic and resting fibroblasts. J Invest Dermatol 107:849–855 Mansbridge JN, Hanawalt PC (1988) Role of transforming growth factor beta in the maturation of human epidermal keratinocytes. J Invest Dermatol 90:336–341 Micke P, Ostman A (2005) Exploring the tumor environment: cancer associated fibroblasts as targets in cancer therapy. Expert Opin Ther Targets 9:1217–1233 Middelkoop E (2005) Fibroblast phenotypes and their relevance for wound healing. Int J Low Extrem Wounds 4:9–11 Mollenhauer J, Bayreuther K (1986) Donor-age-related changes in the morphology, growth potential, and collagen biosynthesis in rat fibroblast subpopulations in vitro. Differentiation 32:165–172

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Morocutti A, Earle KA, Sethi M, Piras G, Pal K, Richards D, Rodemann HP, Viberti GC (1996) Premature senescence of skin fibroblasts from insulin-dependent diabetic patients with kidney disease. Kidney Int 50:250–256 Morocutti A, Earle KA, Rodemann HP, Viberti GC (1997) Premature cell ageing and evolution of diabetic nephropathy. Diabetologia 40:244–246 Nakagawa S, Pawelek P, Grinnell F (1989) Long-term culture of fibroblasts in contracted collagen gels: effects on cell growth and biosynthetic activity. J Invest Dermatol 93:792–798 Nolte S, Xu W, Rennekampff H-O, Rodemann HP (2008) Diversity of fibroblasts—a review on implications for skin tissue engineering. Cell Tiss Org 187:165–176 Olumi AF, Grossfeld GD, Hayward SW, Carroll PR, Tisty TD (1999) Carcinoma-associated fibroblasts direct tumor progression of initiated human prostate epithelium. Cancer Res 59:5002– 5011 Orimo A, Gupta PB, Segroi DC, Renzana-Seisdedos F, Delaunay T (2005) Stromal fibroblasts present in invasive human breast carcinomas promote growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121:335–348 Pascal T, Debacq-Chainliaux F, Chrétien A, Bastin C, Dabée AF, Bertholet V, Remacle J, Toussaint O (2005) Comparison of replicative senescence and stress-induced premature senescence combining differential display and low-density DNA arrays. FEBS Lett 579:3651–3659 Proia DA, Kuperwasser C (2005) Stroma: tumor agonist or antagonist. Cell Cycle 4:1022–1025 Rheinwald JG, Green H (1975) Feeder layer system: serial cultivation of strains of human epidermal keratinocytes. Cell 6:331–343 Rinn JL, Bondre C, Gladstone HB, Brown PO, Chang HY (2006) Anatomic demarcation by positional variation in fibroblast gene expression programs. PloS Genet 2:1084–1096 Rinn JL, Wang JK, Liu H, Montgomery K, van de Rijn M, Chang HY (2008) A systems biology approach to anatomic diversity of skin. J Invest Dermat 128:776–782 Rodemann HP (1989) Differential degradation of intracellular proteins in human skin fibroblasts of mitotic and mitomycin C(MMC)-induced postmitotic differentiation states. Differentiation 42:37–43 Rodemann HP (1993) Differential gene expression, protein synthesis and degradation in ageing fibroblasts. In: Bernd A, Bereiter-Hahn J, Hevert F Holzmann H (eds) Cell culture models for dermatological research. Springer, Berlin, pp 272–277 Rodemann HP, Bamberg M (1995) Cellular basis of radiation-induced fibrosis. Radiother Oncol 35:83–90 Rodemann HP, Mueller GA (1990) Abnormal growth, clonal proliferation and 35S-methionine polypeptide pattern of fibroblasts derived from kidneys with interstitial fibrosis. Proc Soc Exp Biol Med 195:57–63 Rodemann HP, Bayreuther K, Francz PI, Dittmann K, Albiez M (1989) Selective enrichment and biochemical characterisation of seven fibroblast cell types of human skin fibroblast populations in vitro. Exp Cell Res 180:84–93 Rodemann HP, Müller GA, Knecht A, Norman JT, Fine LG (1991) Fibroblasts of rabbit kidney in culture: I. characterization and identification of cell-specific markers. Am J Physiol 261:283– 291 Rodemann HP, Binder A, Burger A, Löffler H, Bamberg M (1996) The underlying cellular mechanisms of fibrosis. Kidney Int 49:32–36 Rossio-Pasquier P, Casanova D, Jomard A, Dermarchez M (1999) Wound healing of human skin transplanted onto the nude mouse after a superficial excisional injury: human dermal reconstruction is achieved in several steps by two different fibroblast subpopulations. Arch Dermatol Res 291:591–599 Rudolph R, Vande J, Berg G, Pierce F (1991) Changing concept in myofibroblast function and control. In: Janssen H, Rooman JIS (eds) Wound healing. Wrightson Biomedical Publishing Ltd., Petersfield, pp 103–115

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Sahuc F, Nakazawa K, Berthod F, Collombel C, Damour O (1996) Mesenchymal-epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermalepidermal junction formation in a skin equivalent model. Wound Rep Regen 4:93–102 Sorrell, JM, Caplan AI (2004) Fibroblast heterogeneity: more than skin deep. J Cell Sci 117:667– 675 Stephens P, Davies KJ, Occleston N, Pleass RD, Kon C, Daniels J, Khaw PT, Thomas DW (2001) Skin and oral fibroblasts exhibit phenotypic differences in extracellular matrix organization and matrix metalloproteinase activity. Br J Dermatol 144:229–237 Stuelten CH, Busch JI, Tang B, Flanders KC, Oshima A, Sutton E, Karpova TS, Roberts AB, Wakefield LM, Niederhuber JE (2010) Transient tumor-fibroblast interactions increase tumor cell malignancy by TGF-β mediated mechanism in a mouse xenograft model of brest cancer. PlosOne 5:e9832 Van Den Bogaerdt AJ, van Zuijlen PPM, van Galen M, Lamme EN, Middelkoop E (2002) The suitability of cells from different tissues to be used in tissue engineered skin substitutes. Arch Dermatol Res 294:135–142 von Pfeil A, Hakenjos L, Herskind C, Dittmann K, Weller M, Rodemann HP (2002) Irradiated homozygous TGF-1 knockout fibroblasts show enhanced clonogenic survival as compared with TGF-1 wild-type fibroblasts. Int J Radiat Biol 78:331–339 Von Zglinicki T, Saretzki G, Docke W, Lotze C (1995) Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp Cell Res 220:186–193 Waelti ER, Inaebnit SP, Rast HP, Hunziker T, Limat A, Braathen LR, Wiesmann U (1992) Coculture of human keratinocytes on post-mitotic human dermal fibroblast feeder cells: production of large amounts of interleukin-6. J Invest Dermatol 98:805–808 Yamaguchi Y, Hearing VJ, Itami S, Yoshikawa K, Katayama I (2005) Mesenchymal-epithelial interactions in the skin: aiming for site specific tissue regeneration. J Dermatol Sci 40:1–9 Yang L, Scott PG, Dodd C, Medina A, Jiao H, Shankowsky HA, Ghahary A, Tredget EE (2005) Identification of fibrocytes in postburn hypertophic scars. Wound Repair Regen 13:398–404

Chapter 3

The Role of the Myofibroblast in Fibrosis and Cancer Progression Boris Hinz, Ian A. Darby, Giulio Gabbiani and Alexis Desmoulière

3.1 Introduction The discovery of the myofibroblast (Gabbiani et al. 1971; Tomasek et al. 2002) has opened a new perspective in the understanding of phenomena such as connective tissue remodeling and epithelial-mesenchymal interactions that play crucial roles in normal and pathological processes including organ shaping during development, tension production in pulmonary alveoli, wound contraction, tissue deformation during fibrotic diseases and, more recently, epithelial tumor invasion or metastasis formation. The myofibroblast has been shown to: (1) produce mechanical force, thanks to the neo-expression of α-smooth muscle actin (α-SMA), the actin isoform typical of vascular smooth muscle cells (SMCs) (Hinz et  al. 2002) and the formation of specialized junctional complexes with the extracellular matrix (ECM) (Dugina et al. 2001; Goffin et al. 2006) (Fig. 3.1), and (2) synthesize collagen type I and III (Tomasek et al. 2002); all these changes take place under the stimulation of local mechanical forces and of transforming growth factor β1 (TGFβ1), produced by infiltrated macrophages or local cells, in conjunction with the ectodomain A (ED-A) sequence of cellular fibronectin (FN) (Tomasek et al. 2002). Thus, the myofibroblast appears as a major player in connective tissue rearrangement. More recently it has been shown that, at least in certain situations, e.g. epithelial tumor invasion, myofibroblasts can influence directly or indirectly the behavior of epithelial cells (De Wever et  al. 2008; Desmouliere et  al. 2004). This activity opens a new perspective for the understanding of tumor progression and is in line with the concept of myofibroblast heterogeneity that has been proposed on the basis of its multiple origins, according to the physiological or pathological process as well as to the organ involved (Hinz et al. 2007). While it is accepted that most myofibroblasts derive from local fibroblastic cells (although it remains open as to whether all fibroblasts can modulate into myofibroblasts), numerous works have G. Gabbiani () Department of Pathology and Immunology, CMU, University of Geneva, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland e-mail: [email protected] M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_3, © Springer Science+Business Media B.V. 2011

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Fig. 3.1   Cytoskeletal features of a myofibroblast. Cultured bone marrow-derived mesenchymal stem cells were treated for 5 days with transforming growth factor β1 and stained by means of immunofluorescence for α-smooth muscle actin ( blue), F-actin ( green) and focal adhesion protein vinculin ( red)

demonstrated alternative origins, e.g. from perisinusoidal cells in the liver, arterial SMCs in the atheromatous plaque, epithelial or endothelial cells, through epithelial (endothelial)-mesenchymal transition (EMT), in organs such the lungs and the kidney, and, finally, circulating bone marrow (BM)-derived cells that have been called fibrocytes (Table 3.1). The purpose of this article is to review the main biological features as well as the mechanisms of force production by the myofibroblast and to discuss the role of this cell in the development of fibrotic changes and in epithelial cancer progression.

3.2 The Myofibroblast—An Overview 3.2.1  Benefits and Consequences of Myofibroblast Appearance Myofibroblasts are generated from different precursors to populate and to repair injured tissues by secreting and organizing ECM in a contractile process (Desmouliere et al. 2005; Hinz et al. 2007; Tomasek et al. 2002). Although, strictly spoken, tumors are not injured tissues, their local environment closely resembles that of wounds and activates fibroblastic cells in many similar ways (Dvorak 1986). When organs and tissues are damaged the inherent repair mechanisms of our body have to fulfill two urgent tasks: (1) establishing tissue homeostasis, fighting inflammation and discarding debris all of which is carried out by immune and inflammatory cells and (2) rapidly providing mechanical tissue coherence by forming a scar

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Table 3.1   Where the myofibroblast comes from—an overview on possible myofibroblast precursors. The main myofibroblast progenitor after injury of different tissues appears to be the locally residing fibroblast. In the liver, myofibroblasts are additionally recruited from hepatic stellate cells following an activation process. In tumours and in fibrotic liver, lung, and kidney, differentiated myofibroblasts can arise from epithelial- and endothelial-to-mesenchymal transition (EMT), respectively. During atheromatous plaque formation, de-differentiating SMC (i.e. that lose late SMC markers) from the media are considered to be the major source of myofibroblastic cells. The relative contribution of bone marrow-derived circulating fibrocytes to the formation of differentiated myofibroblasts in different fibrotic lesions is unclear at present; it is conceivable that fibrocyte transdifferentiation terminates at the proto-myofibroblast stage. Finally, MSC have been shown to acquire the differentiated myofibroblast phenotype in vitro and in vivo Myofibroblast precursor

Condition

References

Astrocyte

Glial scar

Chondrocyte Endothelial cell (via EndMT)

Cartilage repair Heart fibrosis

Silver and Miller 2004; Moreels et al. 2008 Wang et al. 2000 Zeisberg et al. 2007b

Kidney fibrosis Stroma reaction to epithelial tumors

Epithelial cell (via EMT)

Fibroblast

Kidney fibrosis Liver fibrosis Lung fibrosis Tumor development Fibrotic reactions to body implants Kidney fibrosis Liver fibrosis Skin wound healing Stroma reaction to epithelial tumors

Fibrocyte

Asthma Atherosclerosis Cardiomyopathy, post-myocardial infarct Kidney fibrosis Liver fibrosis Lung fibrosis Skin wound healing Tumor development Vascular remodeling

Kizu et al. 2009; Zeisberg et al. 2008; Li et al. 2009) Zeisberg et al. 2007a; Potenta et al. 2008 Iwano et al. 2002; Zeisberg and Kalluri 2008 Zeisberg et al. 2007c Kim et al. 2006; Chilosi et al. 2003 Kalluri and Zeisberg 2006; Thiery 2002; Radisky et al. 2007) Ariyan et al. 1978; Suska et al. 2008 Qi et al. 2006; Desmouliere et al. 2003 Ramadori and Saile 2004; Li et al. 2007 Darby et al. 1990; Gabbiani et al. 1971 De Wever and Mareel 2002; Desmouliere et al. 2004) Schmidt et al. 2003 Fujita et al. 2007; Sata et al. 2002 Haudek et al. 2006; van Amerongen et al. 2008; Mollmann et al. 2006 Wada et al. 2007; Sakai et al. 2006 Forbes et al. 2004; Kisseleva et al. 2006 Phillips et al. 2004; Andersson-Sjoland et al. 2008; Ishii et al. 2005 Ishii et al. 2005; Mori et al. 2005; Direkze et al. 2003; Fathke et al. 2004 Ishii et al. 2005; Direkze et al. 2004; Ishii et al. 2003 Frid et al. 2006; Varcoe et al. 2006

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Table 3.1  (continued) Myofibroblast Condition precursor Hepatic stellate cell Pericyte Osteoblast Mesenchymal stem cell

Smooth muscle cell

Liver fibrosis Systemic sclerosis, dermal scarring Vascular repair Bone repair Chronic renal disease Gut repair Liver fibrosis Lung injury Skin wound healing Tumor development Atheromatous plaque evolution

References Guyot et al. 2006; Bataller and Brenner 2005; Friedman 2004b Sundberg et al. 1996; Rajkumar et al. 2006 Rajkumar et al. 2006 Kinner et al. 2002a Ninichuk et al. 2006 Brittan et al. 2005; Brittan et al. 2002 di Bonzo et al. 2008 Yan et al. 2007 Ye et al. 2006; Yamaguchi et al. 2005; Sasaki et al. 2008 Ishii et al. 2003 Hao et al. 2006; Bochaton-Piallat and Gabbiani 2006

which is the task of fibroblasts and myofibroblasts (Baudino et  al. 2006; Brown et al. 2005; Desmouliere et al. 2005; Gurtner et al. 2008; Werner and Grose 2003). Under physiological conditions the contractile and secretory activities of myofibroblasts are terminated when the tissue is sufficiently remodeled and repaired. Under most circumstances ‘sufficient repair’ signifies that the damaged tissue regains mechanical coherence but does not necessarily means the restoration of functionality since myofibroblasts are specialized to repair but not to regenerate. At the end of the repair process, myofibroblasts disappear by massive apoptosis, leaving the mature scar behind (Desmouliere et al. 1995). The control of myofibroblast apoptosis is still unclear though it seems likely that this could be regulated by a number of factors including local levels of growth factors such as TGFβ1 and endothelin-1 (Kulasekaran et al. 2009) as well as mechanical forces (see below). In contrast, persisting myofibroblast activity results in tissue deformation by contracture. This is the case for the formation of hypertrophic scars such as those developing after burns (Atiyeh et  al. 2005), in scleroderma (Strehlow and Korn 1998; Varga and Abraham 2007) and in the palmar fibromatosis of Dupuytren’s disease (Tomasek et al. 1999). Myofibroblast-generated contractures further characterize the fibrosis that affects vital organs including liver (Gressner and Weiskirchen 2006; Iredale 2007), heart (Baudino et al. 2006; Brown et al. 2005), lung (Chiappara et al. 2001; Phan 2002; Thannickal et al. 2004) and kidney (Lan 2003). Cells with a myofibroblastic phenotype further contribute to the development of the atheromatous plaque after blood vessel injury (Bochaton-Piallat and Gabbiani 2006). A number of different biomaterials have been shown to induce myofibroblasts which for example create tissue constrictions around solid body implants (Anderson et al. 2008; Comut et al. 2000; Li et al. 2007a; Suska et al. 2008) or contract silicone breast implants (Coleman et al. 1993; Siggelkow et al. 2003). As discussed in detail further below, myofibroblasts are instrumental in the stroma reaction to epithelial tumors and promote

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cancer progression by creating a stimulating microenvironment for the transformed cells (Bhowmick and Moses 2005; De Wever and Mareel 2003; Desmouliere et al. 2004). Before discussing the role of myofibroblasts in fibrosis and tumor biology, we will give an overview on the typical features of these intriguing cells.

3.2.2  What Is a Myofibroblast? In the early 1970s, Gabbiani and coworkers have identified specialized fibroblasts as the active component in dermal wound contraction, which were then named myofibroblasts to account for their ultrastructural similarity to SMCs (Gabbiani et al. 1971). The original myofibroblast definition was based on the co-existence of fibroblastic morphological features including a developed endoplasmic reticulum and SMC features such as actin filament bundles and contractile activity. Specific molecular markers were only defined subsequently. Other morphological features of the myofibroblast include high ECM synthesizing activity, development of cellto-cell and cell-to-matrix adhesions (fibronexus), and secretion of growth factors (for reviews see Eyden 2008; Hinz 2007). Myofibroblasts can be of very heterogeneous origins as discussed further below; however, their development follows a well-established sequence of events. The progress of myofibroblast differentiation can be separated into two phases, each of which is characterized by specific cytoskeletal features (Fig. 3.2). First, myofibroblast precursor cells acquire de novo contractile bundles. These in vivo stress fibers generate sufficient forces to pull cells forward to populate tissue spaces in a migration process and to pre-remodel the ECM (Hinz et al. 2001b). To discriminate such activated and mildly contractile cells from quiescent fibroblastic cells which are devoid of any contractile features, we have previously proposed the term ‘proto-myofibroblast’ (Tomasek et al. 2002) (Fig. 3.2). In the presence of mechanical stress and TGFβ1, proto-myofibroblasts can further evolve into differentiated myofibroblasts, hallmarked by the neo-expression of α-SMA and its incorporation into pre-existing stress fibers (Clement et al. 2005; Goffin et al. 2006; Tomasek et al. 2002) (Fig. 3.2). It is this stress fiber incorporation of α-SMA that renders myofibroblasts highly contractile (Hinz et al. 2001a). In standard cell culture, during which fibroblasts inevitably form stress fibers, the term ‘myofibroblast’ generally describes only the α-SMA expressing cells. De novo expression of α-SMA is also the most widely used criterion to identify tissue myofibroblasts and to diagnose myofibroblast-related diseases. On the basis of α-SMA expression, myofibroblasts are classified as a predominant subpopulation of cancer-associated fibroblasts (CAF) in the tumor stroma (Orimo and Weinberg 2007; Sugimoto et al. 2006). In addition, differentiated myofibroblasts in vivo and in vitro can de novo express the cell-cell contact protein OB-cadherin that has not yet been reported on the surface of α-SMA-negative fibroblasts (Hinz et al. 2004). OB-cadherin-positive stroma myofibroblasts were shown to accumulate around epithelial tumors (Shibata et al. 1996; Tomita et al. 2000). Although α-SMA expression will discriminate differentiated myofibroblasts from normal fibroblasts,

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Fig. 3.2   The mechanical feedback loop in myofibroblast development. Fibroblasts in intact tissue are stress-shielded by a functional extracellular matrix (ECM); they do not develop contractile features and cell-matrix adhesions. After injury, inflammatory signals activate fibroblasts to spread into the provisional wound matrix. Local cell remodeling activity leads to gradual increase in global matrix stiffness that counteracts cell traction forces. The resulting formation of small focal adhesions (FAs) and stress fibers that contain only cytoplasmic actins characterize the proto-myofibroblast. Transforming growth factor β1 and the ectodomain A (ED-A) of cellular fibronectin (FN) stimulate proto-myofibroblasts to express α-smooth muscle actin (α-SMA), which at first remains cytoplasmic and is not incorporated into stress fibers. Continuing ECM fiber alignment creates larger surfaces for adhesion formation; larger adhesions permit development of stronger stress fibers and generation of higher contractile forces. When intracellular tension reaches a critical level it allows incorporation of α-SMA into pre-existing stress fibers. The force generated by α-SMA-containing stress fiber is significantly higher compared to cytoplasmic actin stress fibers leading to further FA enlagement and ECM contraction, thereby establishing a mechanical feedback loop. Myofibroblasts may exit this cycle when the original structure of the ECM is reconstituted and again takes over the mechanical load; stress-released myofibroblasts eventually undergo apoptosis. (Reprinted with permission from Hinz 2007)

this marker obviously fails to make a distinction with vascular SMCs that populate the tumor stroma. In normal adult tissue, SMCs express a number of late differentiation markers that are usually not in the repertoire of the myofibroblast, including smooth muscle myosin heavy chain (Benzonana et al. 1988), h-caldesmon (Eyden 2007), and smoothelin (van der Loop et al. 1996). The muscle intermediate filament

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protein desmin is an often reliable and widely used exclusion criterion but can be expressed in myofibroblasts in some particular conditions (Hinz et al. 2001b; Skalli et al. 1986). Moreover, SMCs in normal vessel tissue not only express desmin but also (or sometimes only) vimentin; both intermediate filament proteins are therefore not reliable markers for one or other cell type (Frank and Warren 1981; Gabbiani et al. 1981). Equally problematic is the differentiation between myofibroblasts and pericytes, which can express vimentin and desmin, are α-SMA positive and smooth muscle myosin negative (Armulik et al. 2005; Eyden 2007; Hughes 2008). However, pericytes are characterized by their close interaction with endothelial cells and lack of contractile features in normal tissues (Eyden 2007). Discriminating myofibroblasts from SMCs and pericytes is a daunting task in conditions of smooth muscle injury, angiogenesis, and in cell culture, in particular if the provenance of the cells is unclear. For example, remodeling of injured arteries is thought to be predominantly driven by SMCs from the media de-differentiating into myofibroblasts but the contribution from adventitial fibroblasts to the myofibroblast population has also been suggested. In both conditions, SMCs lose their late differentiation markers desmin, smooth muscle myosin and smoothelin and acquire a myofibroblastic and synthetic phenotype (Benzonana et al. 1988; BochatonPiallat and Gabbiani 2006; Christen et al. 2001; Hao et al. 2006; Larson et al. 1984; Sartore et al. 2001; Zalewski et al. 2002). On the other hand, some of the late SMC markers can be induced at least in cultured fibroblasts by treatment with TGFβ1 (Chambers et al. 2003; Malmstrom et al. 2004). Hence, considering the expression profile of cytoskeletal proteins, the myofibroblast appears to exist in a continuous differentiation spectrum between fibroblasts and SMCs.

3.2.3  On the Origin of Myofibroblast Species Closely related to the problem of how to identify the myofibroblast is the question of where these cells come from—often with the aim to prevent their formation and pathological accumulation. Indeed, it emerges that myofibroblasts, similar to fibroblasts, are a tremendously heterogeneous population of cells. Thus it may be more appropriate to consider the myofibroblast as a phenotype rather than a cell type. This view is supported by the fact that myofibroblasts can be recruited from a variety of precursor cells depending on the injured tissue and the microenvironment (Hinz et al. 2007) (Fig. 3.3). In many organs, locally residing fibroblasts are considered to be the major source of α-SMA-positive myofibroblasts in response to injury and during fibrosis development, such as in skin (Gabbiani 2003; Hinz 2007), in fibrotic reactions to body implants (Ariyan et al. 1978; Suska et al. 2008), in liver (Li et al. 2007b; Ramadori and Saile 2004), kidney (Desmouliere et al. 2003; Qi et al. 2006), and in the stroma reaction to epithelial tumors (De Wever and Mareel 2002; Desmouliere et al. 2004). EMT is another mechanism of myofibroblast generation from epithelial and endothelial precursors during tumor development (Kalluri and Zeisberg 2006; Thiery 2002; Zeisberg et  al. 2008), as well as in kidney fibrosis (Iwano et al. 2002; Kizu et al. 2009) and possibly lung fibrosis (Chilosi et al. 2003;

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Fig. 3.3   Myofibroblast origins. The main myofibroblast progenitor after injury of different tissues appears to be the locally residing fibroblast (see Fig. 3.1). In the liver, myofibroblasts are additionally recruited from hepatic stellate cells that follow an activation process. In tumors and in fibrotic liver, lung, and kidney, differentiated myofibroblasts can arise from epithelial- and endothelial-to-mesenchymal transition (EMT), respectively. During atheromatous plaque formation, de-differentiating smooth muscle cells (i.e. that lose late smooth muscle cell markers) from the media are considered to be the major source of myofibroblastic cells. The relative contribution of bone marrow-derived circulating fibrocytes to the formation of differentiated myofibroblasts in different fibrotic lesions is unclear at present; it is conceivable that fibrocyte transdifferentiation also terminates at the proto-myofibroblast stage. Finally, mesenchymal stem cells have been shown to acquire the differentiated myofibroblast phenotype in vitro and in vivo. See also Table 3.1. (Modified from Hinz 2009a)

Kim et al. 2006). EMT has been demonstrated to contribute to fibrosis of the heart (Zeisberg et al. 2007b) and liver (Zeisberg et al. 2007c). In fibrotic liver, hepatic stellate cell (HSCs) are another important source of myofibroblasts (Bataller and Brenner 2005; Friedman 2004b; Guyot et al. 2006), whereas de-differentiation of SMCs contributes to the generation of myofibroblasts in the atheromatous plaque (Bochaton-Piallat and Gabbiani 2006; Hao et al. 2006). In systemic sclerosis, vessel repair and dermal scarring, pericytes have been suggested to acquire contractile myofibroblast features (Rajkumar et al. 2006; Sundberg et al. 1996). Although the repair processes of injured brain exhibit many specific features compared with other organs, astrocytes seem to develop a myofibroblastic phenotype in the glial scar (Moreels et al. 2008; Silver and Miller 2004).

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Other myofibroblast sources that have attracted broad interest recently are BM derived, blood-circulating cells, which are locally recruited to inflamed and remodeled tissue. Two cell types crystallize as being prominent blood-borne precursors for the myofibroblast: fibrocytes and mesenchymal stem cells (MSCs); both are not always clearly discriminated. Fibrocytes are characterized by the co-expression of the fibroblast markers collagen types I and II and FN, of monocyte markers CD13 and CD11b, and hematopoietic progenitor markers CD34, CD45, and CD105 (Bucala et al. 1994). More exhaustive lists of fibrocyte features have recently been reviewed (Abe et al. 2001; Bellini and Mattoli 2007; Mattoli et al. 2009; Metz 2003). In culture, TGFβ1-induced and spontaneous up-regulation of α-SMA in fibrocytes correlates with down-regulation of CD34 and CD45 (Schmidt et al. 2003). Consistent with this, in an animal model of wound healing, CD34 and CD45 disappear within hours of fibrocyte recruitment/maturation in the inflamed tissue. This loss of the fibrocyte markers makes it difficult to determine in vivo whether fibrocytes actually differentiate into myofibroblasts or whether they simply localize to sites of myofibroblast accumulation (Mori et al. 2005; Quan et al. 2006). To answer the question of fibrocyte-to-myofibroblast differentiation, a number of studies used irradiated wild-type mice, engrafted with BM obtained from GFP-expressing transgenic mice or from sex-mismatched animals. BM-derived cells were associated in varying proportions with myofibroblast-containing lesions in the context of tumor development (Direkze et  al. 2004; Ishii et  al. 2005) and in animals subjected to fibrotic stimuli in different organs (Andersson-Sjoland et  al. 2008; Forbes et  al. 2004; Haudek et  al. 2006; Ishii et  al. 2005; Kisseleva et  al. 2006; Moeller et  al. 2009; Phillips et al. 2004; Sakai et al. 2006; Schmidt et al. 2003; Wada et al. 2007), during vascular remodeling (Frid et al. 2006; Fujita et al. 2007; Varcoe et al. 2006), and in dermal wound healing (Direkze et al. 2003; Fathke et al. 2004; Mori et al. 2005). Other studies seem to exclude that BM-derived fibrocytes contribute to myofibroblast formation in liver (Kisseleva et al. 2006) and lung fibrosis (Hashimoto et al. 2004); the definitive answer to the question of fibrocyte-to-myofibroblast differentiation remains open. Another population of BM-derived cells, possibly giving rise to tissue myofibroblasts are circulating MSCs (Roufosse et al. 2004). MSCs in culture are generally identified by the conjunct expression of specific cell surface proteins such as CD105, CD90, CD44, CD73, CD166, CD29, CD106 and Stro-1 (Minguell et al. 2001; Pittenger et al. 1999; Simmons and Torok-Storb 1991). MSCs are different from fibrocytes by being negative for the monocyte surface proteins CD13 and CD11b and the hematopoietic markers CD34 and CD45 (He et al. 2007; Pittenger et al. 1999). MSCs can differentiate into a variety of cell types that are of potential use for regenerative medicine and tissue engineering (Caplan 2007). Even listing only the envisaged applications of MSCs would by far exceed the scope of this chapter, and we concentrate here on their involvement in tumor biology. Systemically transplanted MSCs target to the stroma environment of epithelial tumors (Hall et al. 2007; Hung et al. 2005; Menon et al. 2007; Studeny et al. 2004). This specific homing together with the immuno-inactivity of MSCs is possibly useful for the tumor-specific delivery of anti-cancer drugs, cytokines and viruses (Komarova et al.

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2006; Studeny et al. 2002) and to suppress tumor development as such (Khakoo et al. 2006). However, the tumor environment seems to activate myofibroblast differentiation of MSCs similar to CAFs (Mishra et  al. 2009). BM-MSCs, cultured in tumor-conditioned medium differentiate into α-SMA-positive myofibroblasts (Mishra et al. 2008). Recent studies evaluating the interaction between MSCs and epithelial tumor cells in vivo and in vitro indicate that acquisition of the myofibroblast phenotype by MSCs can reduce the success of an envisaged MSC therapy and may even amplify the disease. MSCs that have become activated by mildly invasive human breast carcinoma cells in culture enhance the metastatic potential of the cancer cells when injected subcutaneously; this effect is mediated in a paracrine feedback loop (Karnoub et  al. 2007). BM-MSCs can also spontaneously express α-SMA and up-regulate their contractile activity in standard culture and in response to pro-fibrotic TGFβ1 (Bonanno et al. 1994; Cai et al. 2001; Kinner et al. 2002b; Peled et al. 1991). Proteomic profiling of BM-MSCs revealed a typical myofibroblast differentiation program after treatment with TGFβ1 (Wang et al. 2004). It has to be noted that α-SMA-positive MSCs in culture are not necessarily myofibroblasts but may represent intermediate stages of differentiation into SMCs or pericytes (Bianco et al. 2001; Charbord et al. 1990). It remains to be shown whether MSCto-myofibroblast differentiation is rather a risk or whether it can be beneficial in the context of the tumor environment and for regenerative medicine.

3.3 Formation and Control of the Myofibroblast From this amazingly heterogeneous selection of possible precursor cells it is conceivable that myofibroblasts are a family of different cell types that share common molecular features and the contractile phenotype. To therapeutically counteract organ dysfunction caused by myofibroblasts it is helpful to understand the molecular pathways that commonly regulate their evolution from different precursor cells.

3.3.1  Mechanical Stress Regulates Myofibroblast Development The physiological role of myofibroblast activity is to restore the mechanical integrity of an injured tissue by secreting and organizing new ECM, a process that is precisely controlled by mechanical signals, such as ECM stiffness (Hinz 2009b). What is a ‘stiff’ ECM and how does ECM stiffness develop during tissue repair and remodeling? The provisional ECM laid down after acute tissue injury, e.g. the fibrin clot of dermal wounds, is estimated to be very compliant with a Young’s modulus of 10 to 1000 Pa. Under comparable conditions in vitro, such as growth on very soft two-dimensional polyacrylamide gels and in three-dimensional soft collagen gels, development of stress fibers by fibroblasts is suppressed. Fibroblasts without stress fibers form only very small and immature adhesions with the ECM

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that are called focal complexes or nascent adhesions (Tamariz and Grinnell 2002; Yeung et al. 2005). The proto-myofibroblast phenotype is only produced on stiffer culture substrates exhibiting an elastic modulus of at least 3000 Pa; these cells form α-SMA-negative stress fibers that terminate in mature focal adhesions (Janmey et al. 2009; Yeung et al. 2005). A stiffness of ~18000 Pa has been measured in 7-day-old rat wound granulation tissue which is mainly populated by proto-myofibroblasts. Even stiffer culture substrates with a Young’s modulus of ~20000  Pa and higher are required to permit further myofibroblast differentiation. Expression of α-SMA in stress fibers on stiff substrates is associated with the formation of particularly large focal adhesions (Goffin et al. 2006; Wells 2005). Corresponding to the threshold stiffness for myofibroblast differentiation in vitro, fibrotic tissues and contracting wound granulation tissue were shown to exhibit a stiffness of 25000–50000 Pa (Hinz 2009c). The high contractile activity of myofibroblasts is instrumental in gradually increasing ECM stiffness and stresses, which in turn enhances their differentiation state and level of contraction. This mechanical feed forward loop is considered as one of the most important elements in the development of chronic contractures (Hinz 2009b; Tomasek et al. 2002). The level of stress and/or stress changes are further discussed as important signals for myofibroblast survival. Mechanically stressing dermal wounds in mice causes hypertrophic scarring, presumably by decreasing the rate of apoptotic myofibroblasts (Aarabi et al. 2007). On the other hand, increased apoptotic figures have been reported for fibroblasts in stress-released collagen gels (Grinnell et  al. 1999) and experimentally relaxed wounds (Carlson et  al. 2003). Stress release also leads to a reduction of α-SMA and myofibroblast contractile features in vivo and in vitro (Goffin et al. 2006; Hinz et al. 2001a; Hinz et al. 2001b). The mechanical responsiveness of myofibroblasts and their role in stiffening the stroma ECM have important implications for tumor-stroma interaction and tumor progression. It is a well known fact that the cancer-associated stroma is stiffer than the surrounding normal soft connective tissue (Andersen et al. 2009; Beacham and Cukierman 2005; Butcher et al. 2009; Kumar and Weaver 2009; Paszek et al. 2005). This fact is clinically exploited to detect tumors by palpation and/or elastography techniques (Garra 2007; Glaser et al. 2006; Khaled et al. 2004; Khaled et al. 2006). It has been suggested that small changes in tissue stiffness arising during the inflammatory response precede and drive tissue contractures and fibrotic reactions; this has been demonstrated for the liver (Georges et al. 2007). One possible source of this ‘pre-stiffening’ in the tumor environment may be interstitial fluid pressure (Heldin et al. 2004). Using three-dimensional fibroblast collagen culture it has been shown that even very low interstitial fluid flow can lead to ECM and fibroblast alignment and subsequent myofibroblast differentiation (Ng et al. 2005). Enhanced tissue stiffness is not only a diagnostic tool for tumor detection but also appears to increase the risk and invasiveness of tumors. For breast cancers, a clear correlation exists between the degree of mammographic densities (compact and stiff tissue) and the risk of cancer formation (Boyd et al. 2007; Wolfe 1976). Recent studies demonstrated that substrate stiffness directly governs the behavior and differentiation of murine mammary epithelial cells. On culture substrates that mimic the softness

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of normal mammary gland tissue (150 kPa), these cells form polarized acini surrounded by an intact basement membrane. This structure resembling the in vivo structure of the normal breast ducts is increasingly disturbed with increasing substrate stiffness. On substrates with a stiffness of 5000 Pa, reproducing the mechanical conditions of the tumor stroma, lumen formation is inhibited and no surrounding basement membrane is established. As a consequence of the higher substrate stiffness, epithelial cells lose cell–cell adherens junctions and cell polarity to attain a mesenchymal phenotype with distinct stress fibers and increased migratory activity (Paszek et al. 2005). Very recent studies added to these findings that physiologically soft ECM suppresses the mitogenic activity of epithelial (and mesenchymal) cells; stiffer substrates induce mitogenesis (Klein et al. 2009; Winer et al. 2009). Hence, the stiff ECM generated by the fibroblast/myofibroblast remodeling activities not only impacts the stromal compartment but drives epithelial cells into an invasive and proliferative phenotype, at least in culture. What are the mechanisms and intracellular signaling pathways through which tension controls myofibroblast differentiation? Firstly, stress appears to directly modulate α-SMA gene transcription. Application of force at sites of integrin adhesions, formed with collagen-coated magnetite beads up-regulates the promoter activity of α-SMA in cardiac fibroblasts and osteoblasts. This effect involves binding of serum response factor to the CArGB box in the α-SMA promoter (Wang et al. 2003; Wang et  al. 2002). More recently, it has been shown that stress-regulated activity of the α-SMA promoter further requires Rho/Rho kinase activity and involves translocation of the myocardin-related transcription factor MRTF A/MAL to the nucleus (Zhao et al. 2007). It is noteworthy that mechanical stress alone is generally not sufficient to induce myofibroblast differentiation in the absence of active TGFβ1 (Hinz et al. 2001a, b). Interestingly, mechanical stress and TGFβ1 also collaborate to up-regulate expression of collagen, another hallmark of the myofibroblast (Lindahl et al. 2002). Secondly, the localization of cytosolic α-SMA to stress fibers is directly controlled by the level of stress. Culture experiments revealed that transferring differentiated lung myofibroblasts from stiff plastic culture dishes (Young’s modulus of MPa) to soft silicone substrates (≤16000 kPa) results in the selective dislocation of α-SMA from persisting β-cytoplasmic actin stress fibers within hours (Fig. 3.2). A similar effect is achieved by relaxing differentiated myofibroblasts with Rho kinase inhibitors on stiff culture substrates (Goffin et al. 2006). This function of α-SMA as a mechano-sensitive/responsive protein is still mysterious but suggests a mechanosensitive element within the contractile bundles that specifically binds to α-SMA. The nature of this hypothetical binding partner is unclear; its existence however is supported by the fact that cytoplasmic delivery of the α-SMA specific N-terminal sequence Ac-EEED as a ‘competitive’ peptide selectively removes α-SMA from stress fibers (Chaponnier et  al. 1995; Clement et al. 2005; Hinz et al. 2002). Proteins like zyxin, myosin and α-actinin have been demonstrated to exhibit a similar dependence of their location in stress fibers on mechanical load (Colombelli et al. 2009; Hervy et al. 2006; Peterson et al. 2004). Moreover, stress fibers under tension have been shown to reveal cryptic cysteine residues detected by in vivo labeling (Johnson et al. 2007).

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3.3.2  M  echano-Chemical Control of Myofibroblasts—A Spotlight on TGFβ1 TGFβ1 is the most potent myofibroblast-inducing factor and one of the strongest pro-fibrotic cytokines presently known. TGFβ1 exerts its pro-fibrotic activities by mediating the inflammatory response, causing excessive ECM production, decreasing synthesis of matrix metalloproteinases (MMPs), increasing the synthesis of tissue inhibitors of MMPs (TIMPs), and finally by inducing myofibroblast differentiation (Desmouliere et al. 1993; Grainger 2007; Hinz 2007; Leask and Abraham 2004; Ruiz-Ortega et al. 2007; Taipale et al. 1998). TGFβ1 normally acts to inhibit proliferation of epithelial cells, but it may stimulate proliferation of fibroblasts of dermal and renal origins (Giannouli and Kletsas 2006; Strutz et al. 2001). The fact that cancer cells become insensitive to the growth-arresting action of TGFβ1, together with its pro-angiogenic effects and its potential to induce EMT of cancer cells, also allocates TGFβ1 a central role in tumor development (Bierie and Moses 2006; Pardali and Moustakas 2007; Siegel and Massague 2003). TGFβ1 also has beneficial functions in assuring homeostasis of adult tissues by controlling proliferation of epithelial cells, endothelial cells, immune cells and fibroblasts (Feng and Derynck 2005; ten Dijke and Arthur 2007; Wakefield and Stuelten 2007). Hence, global inhibition of TGFβ1 is considered problematic as an anti-fibrotic or antitumorigenic strategy with many uncontrollable side-effects (Akhurst and Derynck 2001; Varga and Pasche 2009). On the other hand, the complex and diverse mechanisms leading to the activation of latent TGFβ1 potentially provide cell-specific means for TGFβ1 inhibition. Myofibroblasts themselves secrete biologically latent TGFβ1 in complex with the latency associated peptide (LAP). LAP and TGFβ1 form the large latent complex together with the latent TGFβ1 binding protein-1 (LTBP-1). LTBP-1 binds to proteins in the ECM and there provides a reservoir of latent TGFβ (Annes et al. 2004; Jenkins 2008; Wipff and Hinz 2008). The different mechanisms of how cells can dissociate and thus activate TGFβ1 from the latent complex are subject of comprehensive recent reviews (Jenkins 2008; Sheppard 2005; Wipff and Hinz 2008). Here, we will briefly discuss how myofibroblast mechanics play a role in latent TGFβ1 activation. The LAP portion of the ECM-bound latent TGFβ1 provides binding sites for a variety of myofibroblast integrins, including αvβ5, αvβ3, αvβ8, and α8β1 which intracellularly connect to the contractile cytoskeleton (Sheppard 2005; Wipff and Hinz 2008). Stress applied to integrin αvβ5, either by stretching the ECM or by inducing cell contraction renders the active TGFβ1 available for its cell-membrane-bound receptor, possibly by a conformational change in the latent complex (Wipff et al. 2007) (Fig. 3.4). A similar mechanism appears to be in place for epithelial cells to activate TGFβ1 via αvβ6 integrin (Jenkins et al. 2006). In fact, αvβ6 was the first integrin shown to mediate latent TGFβ1 activation in a process that requires polymerized actin (Munger et al. 1999). Importantly, mechanical activation of TGFβ1 by myofibroblasts is not operational in the context of a compliant ECM with Young’s modulus of ≤5000  kPa (Wipff et  al. 2007).

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Fig. 3.4   A schematic model of latent transforming growth factor β1 (TGFβ1) activation by cell traction. Integrin binding to a specific RGD site in the latency associated peptide (LAP) portion transmits intracellular force to the large latent complex. In the context of a soft extracellular matrix (ECM) (unloaded springs) cell pulling will simply drag the latent complex. When bound to a remodeled stiff ECM (loaded springs) integrin-mediated force exertion can trigger a conformation change in the LAP and make TGFβ1 available for its receptor. (Reprinted with permission from Hinz 2009b)

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This is explained by a model in which the cell pulling force transmitted to LAP is counteracted by anchoring the latent complex via the LTBP-1 to a mechanically resistant ECM (Hinz 2009c; Tenney and Discher 2009; Wells and Discher 2008; Wipff and Hinz 2008) (Fig. 3.4). Consistently, deletion of the ECM-binding sequence from LTBP-1 abolishes latent TGFβ1 activation via integrins as shown for αvβ6 integrin (Annes et al. 2004). It is noteworthy that the threshold ECM stiffness for latent TGFβ1 activation is lower than the minimal stiffness required for expression and incorporation of α-SMA into stress fibers. Hence, mechanical activation of TGFβ1 can provide a first control point in the progression of tissue remodeling by translating the level of ECM stiffness (organization) into a profibrotic signal.

3.3.3  More Factors That Stimulate Myofibroblast Differentiation A detailed discussion of the intracellular signaling molecules, cytokines, and ECM proteins that have been reported to modulate myofibroblast differentiation and α-SMA expression will exceed the scope of this chapter and the reader is referred to recent reviews (Hinz 2007; Horowitz and Thannickal 2006; Schurch et al. 2007; Wynn 2007, 2008). Most factors that induce myofibroblast differentiation without involving paracrine effects from other cell types act in synergy with TGFβ1 signaling (Desmouliere et al. 1993; Ronnov-Jessen and Petersen 1993). These include connective tissue growth factor (Shi-Wen et al. 2008), interleukin (IL)-6 (Gallucci et al. 2006), Fizz1 (found in inflammatory zone) (Liu et al. 2004), galectin-3 (Henderson et al. 2006), osteopontin (Lenga et al. 2008; Mori et al. 2008), endothelin-1, (Jain et  al. 2007; Shi-Wen et  al. 2007), angiotensin II (Rosenkranz 2004; Uhal et al. 2007), thrombin (Bogatkevich et al. 2003), possibly semaphorin 7A (Kang et al. 2007), nerve growth factor (Micera et al. 2001) and cleavage of the urokinase receptor (Bernstein et al. 2007). Myofibroblast differentiation is further promoted by adhesion to specific ECM proteins including collagen type VI (Naugle et  al. 2006), tenascin-C (De Wever et al. 2004; Tamaoki et al. 2005) and most importantly cellular ED-A FN (Serini et al. 1998). Another important stimulating factor for myofibroblast differentiation appears to be the production of reactive oxygen species by NADPH oxidases (NOX) in fibroblastic cells (Shen et al. 2006). NOX are transmembrane proteins that regulate intracellular redox signaling by reducing extracellular molecular oxygen to superoxide, generating downstream reactive oxygen species (Bedard and Krause 2007; Griffith et al. 2009). The predominant NOX isoform in fibroblasts is NOX4, which has been shown to mediate TGFβ1induced conversion of cardiac fibroblasts into myofibroblasts (Cucoranu et  al. 2005). NOX4 is further involved in the development of lung fibrosis and ablation of NOX4 in lung fibroblasts suppresses myofibroblast differentiation (Hecker et al. 2009).

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3.3.4  Factors That Inhibit Myofibroblast Differentiation The discovery of factors that suppress myofibroblast development is mainly driven by the search for new therapies to treat organ fibrosis (Brown et al. 2005; Friedman 2004a; Gharaee-Kermani et al. 2007; Horowitz and Thannickal 2006; Scotton and Chambers 2007; Trojanowska and Varga 2007). Growth factors that antagonize myofibroblast development in culture and in animal models have been described, including IL-1 (Kanangat et al. 2006; Shephard et al. 2004), tumor necrosis factor-α (Goldberg et al. 2007; Saika et al. 2006), TGFβ3 (Shah et al. 1995) and interferon-γ (Desmouliere et al. 1992). At present however, none of the countless factors that are implied in normal and pathological wound healing has been successfully targeted to significantly improve the tissue repair process in clinical applications. Another possibility to suppress development of the myofibroblast phenotype is to interfere with the mechanical feedback loop of high contractile activity and ECM stiffening that induces and maintains the fibrogenic cell character (Wipff et al. 2007) (Fig. 3.2). It is difficult to imagine how one could reduce the stiffness of a scar tissue in a controlled manner. However, interfering with the mechanisms through which myofibroblasts perceive extracellular stress is another option. It has been shown that the level of substrate rigidity determines the size and molecular composition of cell-ECM focal adhesions that perceive and communicate extracellular mechanical signals to the cytoskeleton, leading to specific gene expression (Bershadsky et al. 2003; Ingber 2003). By artificially reducing the adhesion area available for cell attachment using microcontact printing on rigid surfaces it is possible to ‘simulate’ a soft environment for myofibroblasts. As a consequence, these cells lose α-SMA expression in stress fibers and contractile capacity similar to culture on soft substrates (Goffin et al. 2006). Finally, the level of myofibroblast intracellular tension can be specifically and directly reduced by targeting α-SMA positive stress fibers using a competitive peptide strategy. Inhibitition of myofibroblast contraction by long term administration of the α-SMA fusion peptide results in decreased collagen production and finally disappearance of the myofibroblast (Clement et  al. 2005; Hinz et al. 2002).

3.4 The Myofibroblast in the Stroma Reaction to Tumors 3.4.1  Activities of the Myofibroblast in the Tumor Environment The tumor stroma is a connective tissue composed of cells and ECM, containing vessels and nerves. The stroma plays equivalent roles in the host reaction or immunological defense against the tumor and in enhancing tumor progression. Morphologically, the tumor stroma gloves tumor strands, follows tumor progression over time and frequently replaces the surrounding normal connective tissue. The tumor stroma contains permanent cells, notably fibroblasts and myofibroblasts,

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and transient visitors, such as the leukocytes involved in the immune response. Fibroblasts and myofibroblasts, also called CAF or tumor-associated fibroblasts, represent very important components of the tumor stroma. Recently, studies have demonstrated a prognostic significance for these cells which could also represent new therapeutic targets (Albini and Sporn 2007; Gonda et al. 2010; Hawinkels et al. 2009; Ostman and Augsten 2009; Pietras et al. 2003). Different subpopulations of CAFs participate in forming the tumor stroma. Markers such as fibroblast-activated protein, fibroblast-specific protein-1 (FSP1/S100A4), neural-glial antigen-2, and platelet-derived growth factor (PDGF)-β receptor have been used to characterize these distinct subsets (Orimo and Weinberg 2007; Sugimoto et al. 2006). Among those, the α-SMA expressing myofibroblast appears to be predominantly involved in chronic tumor–stroma reactions. A great number of myofibroblasts is present in the stroma of many types of malignant tumors and are frequently localized at the front of invasion (De Wever and Mareel 2003). Here they are involved in connective tissue remodeling by producing ECM components. Moreover, they synthesize not only proteases to degrade ECM or basement membrane such as MMPs, but also their inhibitor TIMPs. Depending on the tumor type, the tumor stroma can be inconspicuous, as shown in the main forms of malignant melanoma or hepatocellular carcinoma, or desmoplastic as in scirrhous breast carcinoma. The composition of the tumor stroma strongly resembles the granulation tissue of healing wounds. Thus, it has been suggested that the host stroma reaction promoted by the tumor cells represents a ‘never healing wound response’ (Dvorak 1986). However, tumor growth induces abnormal development of the stroma reaction leading to excessive scarring (Schafer and Werner 2008). Indeed, the stroma reaction presents features rather similar to those of skin hypertrophic scars observed after acute and deep injury such as severe burning. It is important to emphasize that the presence of fibrotic lesions, containing myofibroblasts significantly increases the risk of cancer in many tissues, including lung (Daniels and Jett 2005), liver (Bissell 2001), and breast (Radisky and Przybylo 2008). Women with dense (fibrotic) breast tissue which is linked to a substantial increase in stromal collagen deposition, have a four- to six-fold increased risk of developing breast cancer (Boyd et al. 1998).

3.4.2  Myofibroblast–Tumor Crosstalk: A Fatal Affair It is generally accepted that myofibroblasts are recruited from normal connective tissue fibroblasts that migrate into the stroma and acquire contractile function. However, similarly to the situations found in pathological repair, BM-derived MSCs can be involved (Direkze et al. 2004; Ishii et al. 2003). Furthermore, the contribution of EMT from epithelial and endothelial cells has been discussed for tumor fibroblasts and myofibroblasts (Potenta et al. 2008; Radisky et al. 2007; Zeisberg et al. 2007a). All these possible precursors may represent alternative sources of myofibroblasts that are activated according to the specific situation. In particular when the size of

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the lesion increases, recruitment of local fibroblasts may be insufficient to ‘comply with the request’. This has been demonstrated in pathological repair such as liver cirrhosis (Guyot et al. 2006). What are the signals that these precursor cells perceive to become activated? Recruitment and activity of myofibroblasts directly depend on the inflammatory response. Since Dolberg and coworkers have demonstrated that tumors preferentially grow at sites of wounds in retrovirus infected chickens (Dolberg et al. 1985) numerous studies supported that most chronic diseases, including cancer, are caused by a deregulated inflammatory response (Aggarwal and Gehlot 2009). Interestingly, it has been shown that the transcriptome of the aging prostate stroma is characterized by the up-regulation of several genes that encode secreted inflammatory mediators. The levels of these mediators are sufficient to promote low-level increases in the proliferative rate of both epithelial and stroma fibroblast cell types observed in benign prostatic hypertrophy (Begley et al. 2008). The authors suggested that the same processes promote the proliferation of malignant prostate epithelial cells in the development and progression of invasive prostate cancer. Stroma-tumor interactions depend on multiple factors, including direct cell-cell contacts and paracrine signals (De Wever et al. 2008) (Fig. 3.5). Interactions between tumor cells and myofibroblasts, leukocytes or endothelial cells, participate in the remodeling of the microenvironment and contribute to tumor progression through growth, local spreading and metastasis. It is accepted that the crosstalk between carcinoma epithelial cells and adjacent stroma cell populations leads to the production of a tumor microenvironment that further promotes tumor progression. In a mouse model of prostate carcinoma, Hill and coworkers have shown that tumor cells induce up-regulation of p53 through a paracrine mechanism in stroma

Fig. 3.5   Schematic illustration of the heterologous cellular interactions during tumor development and progression

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fibroblasts and myofibroblasts, which results in decreased fibroblastic proliferation (Hill et  al. 2005). This process exerts a selective pressure that promotes the expansion of a highly proliferative subpopulation of fibroblastic cells, lacking p53 and contributing to tumor progression. Subsequently, co-evolution of the stroma compartment with selection of genetically altered cells can occur as a result of oncogenic stress in the epithelium. Again, this study illustrates the interdependent signaling which controls the stroma-epithelial regulation in cancer. As mentioned above, this type of observation underlines the importance for developing cancer therapies that target the stroma compartment as a means to prevent acceleration or possibly even suppress tumorigenesis (Hill et al. 2005). Interestingly, certain primary tumor cells can release soluble factors which mobilize specific populations of non-malignant hematopoietic cells which colonize distant organ tissue, establishing a ‘pre-metastatic niche’ (Kaplan et al. 2005; Noel et al. 2008). Subsequently, fibroblasts are co-recruited along with hematopoietic cells to establish the supportive tumor stroma (Kaplan et  al. 2006). Fibroblasts and myofibroblasts participate in the niche architecture by developing a microenvironment which favors tumor cell engraftment. In addition, the existence of cancer stem cells is currently discussed. Cancer stem cells are defined as undifferentiated cells with the ability to self-renew, differentiate to multiple lineages and initiate tumors that mimic the parent tumor (Gupta et al. 2009). The cooperation between these cancer stem cells and the connective tissue stem cells could constitute a specific niche, able to develop tumors if specific signals are delivered. Cytokines derived from tumor cells are also believed to modulate the stroma cells to induce expression of growth and angiogenic factors, which in turn provide the tumor with the necessary microenvironment for expansion and invasion. Among these cytokines, numerous studies underline the role of TGFβ1 (involved in myofibroblast generation), platelet-derived growth factor (involved in desmoplasia), vascular endothelial growth factor (involved in angiogenesis and desmoplasia), and hepatocyte growth factor (involved in tumor cell motility). Studies provide evidence for TGFβ1 as a supporting agent in tumor progression through the induction of a perpetual wound healing process in the tumor microenvironment. For example, normal pre-senescent fibroblasts and prostate stroma cells that are subcutaneously co-transplanted with prostate carcinoma cells into nude mice reduce tumor latency and accelerate tumor growth. Even when their TGFβ1 signaling is blocked, the fibroblasts and stroma cells continue to stimulate tumor initiation but no longer support tumor growth as opposed to control cells (Verona et al. 2007). Moreover, gene microarray and quantitative reverse transcription-PCR analyses showed that TGFβ1 up-regulates a selection of genes in stroma cells that are involved in tissue remodeling and wound healing (Verona et al. 2007). In addition to cytokines, MMPs are central regulators in the complex tumor ecosystem due to the high diversity of these enzymes concerning both their substrates and functions. In the majority of cancers, membrane-associated and extracellular MMPs are produced by host cells including inflammatory cells, endothelial cells, fibroblasts, and myofibroblasts (Noel et al. 2008). For example, ADAMTS-1, a disintegrin and MMP with thrombospondin motifs is expressed in a variety of carcinomas

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(Rocks et al. 2008a). Its production leads to increased secretion of TGFβ1 and IL1β, leading to the recruitment of fibroblasts and differentiation into myofibroblasts (Rocks et al. 2008b). Furthermore, the presence of specific stromal proteins such as the oncofetal FN (Nielsen et al. 2008), is a characteristic of the invasive phenotype. It has been shown by in situ hybridization that granulation tissue myofibroblasts show a FN splicing pattern consisting ED-A and ED-B, similar to that found in the embryo (Ffrench-Constant et al. 1989). As mentioned above, ED-A FN is crucial for myofibroblastic phenotype induction by TGFβ1 (Serini et al. 1998). Taken together, these data reinforce the central roles played by TGFβ1 and ECM protein interactions in stroma development and tumor growth. Tumor cell-myofibroblast interactions share common features with the epithelial-mesenchyme interactions seen in morphogenesis; the mechanisms of tumor survival and progression sometimes use processes also observed during normal embryonic development. In the next part, we will illustrate these interactions between myofibroblasts and tumors on the example of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA).

3.4.3  The Myofibroblast of HCC Compared with CCA HCC may have numerous etiologies, notably chronic hepatitis B and C viral infection or chronic alcohol abuse. In rare situations, HCC can develop in non-cirrhotic liver (Fig. 3.6a) but in most cases arises in livers showing cirrhosis (Desmouliere et al. 2004; Farazi and DePinho 2006) (Fig. 3.6b). Independently of the cause, cirrhosis is in itself a precancerous condition (Berasain et al. 2009). Depending on the degree of differentiation, the tumor is composed of large anastomosing plates and acini of tumor hepatocytes surrounded by capillaries. Except in rare forms of scirrhous or fibrolamellar HCC, the tumor stroma is scanty (Fig. 3.6). Sometimes the tumor is encapsulated but most often the tumor stroma is mixed with the fibrous stroma of the surrounding cirrhosis. At low microscopic magnification, the tumor stroma seems to be organized like the connective tissue surrounding the normal liver parenchyma. At higher magnification and on the ultrastructural level, important differences and modifications become evident. The vessels surrounding the tumor plates are not sinusoids but capillaries with a continuous basement membrane. They contain fewer Kupffer cells compared with normal liver (Liu et al. 2003) and instead exhibit numerous mast cells (Terada and Matsunaga 2000). The space between endothelial cells and the tumor hepatocytes does not contain HSCs but cells with myofibroblastic features like α-SMA (Fig. 3.6d). The ECM is remodeled with more deposition of collagen (Faouzi et al. 1999a), fibrillin-1 and de novo expression of elastin (Dubuisson et al. 2001). In the tumor stroma of the liver, myofibroblasts can be derived locally from the surrounding cirrhosis or from HSCs and/or portal fibroblasts. However, as discussed above, different possible sources have been recently discussed, notably BM-derived MSCs. Myofibroblasts are involved in the deposition (Dubuisson et  al. 2001; Faouzi et  al. 1999a; Le Bail et  al. 1999) and

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Fig. 3.6   Hepatocellular carcinoma features. Typical gross appearance of hepatocellular carcinomas undergoing on a non-cirrhotic, or b cirrhotic livers. In both types of hepatocellular carcinomas, a scanty stroma reaction is observed. In b, the non-tumor liver presents typical cirrhotic regenerative nodules. c Inside the tumor, collagneneous extracellular matrix (Masson’s trichrome histochemistry) is very modest in the stroma. d As detected by immunohistochemistry, few α-smooth muscle actin expressing myofibroblasts are localized between the tumor plates and the capillaries

in the remodeling of the ECM, synthesizing proteinases, like urokinase or MMPs (Dubuisson et  al. 2000; Monvoisin et  al. 2002; Monvoisin et  al. 1999). Interactions between malignant hepatocytes and stroma cells are complex. Tumor cells can recruit liver myofibroblasts locally or by migration from the transdifferentiated stroma cells. Numerous ways have been studied. Some growth factors or cytokines are involved in activation and recruitment of stroma cells, such as TGFβ1 or PDGF (Faouzi et al. 1999b). Recently, it was suggested that the TGFβ1/PDGF axis is crucial during hepatic tumor-stroma crosstalk, regulating both tumor growth and cancer progression (van Zijl et al. 2009). During early stages of hepatocarcinogenesis, TGFβ1 released from non-parenchymal compartments, such as immune cells, causes the transdifferentiation of HSCs and portal fibroblasts to myofibroblasts, which then secrete TGFβ1 and PDGF. TGFβ1 induces EMT of neoplastic hepatocytes in cooperation with the mitogen-activated protein kinase signaling, and at the same time stimulates autocrine production of PDGF, which maintains EMT. The resulting malignant hepatocytes produce both TGFβ1 and PDGF, which in turn stimulate the microenvironment to further generate and attract hepatic myofibroblasts.

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Stroma myofibroblasts can also enhance tumor invasiveness, notably by expression of hepatocyte growth factor (Neaud et al. 1997; Neaud et al. 2000) or by secretion of MMPs (Yamamoto et al. 1997). In addition, expression of monocyte chemoattractant protein-1 (MCP-1 or CCL-2) by tumor myofibroblasts can attract more monocytes into the tumor leading to increased fibroblast activation and may also play a role in activating migration of tumor cells (Dagouassat et al. 2009). Recently, as potential mechanism of the tumorigenic effects of HSCs, Ammann and coworkers identified activation of NFκB and extracellular-regulated kinase in HCC cells, two signaling cascades that play a crucial role in HCC progression (Amann et al. 2009). Because good animal models are lacking, the pathogenesis of the stroma reaction in human HCC remains unclear. In most models that have been proposed to study HCC development, a stroma reaction is practically absent. Recently, using a model associating diethylnitrosamine exposure and N-nitrosomorpholine treatment, Taras and coworkers have observed that HCC cells were surrounded by a stroma reaction resembling that observed in humans. Consequently, this model has been used to test different drugs able to interfere with lung metastatic processes of HCC (Taras et al. 2006; Taras et al. 2007). To model CCA in animal models, thioacetamide has been used, accurately mimicking the multi-stage progression of human CCA (Jan et al. 2004; Yeh et al. 2004). CCA is a cancer of the biliary epithelium and is less common than HCC. Worldwide, CCA is the second most common primary hepatic malignancy. The tumor is composed of tubular adenocarcinoma set in an abundant fibrous stroma (Fig. 3.7a). Three main forms are recognized in accordance with the location of the tumor in the liver and in the extrahepatic bile tree: intrahepatic or peripheral CCA, hilar adenocarcinoma (Klatskin tumor) and carcinoma of the extrahepatic bile ducts. Several recent epidemiological studies have shown that the incidence and mortality rates of intrahepatic bile-duct carcinoma are increasing (Khan et al. 2008). Unlike HCC, the stroma of CCA is abundant, sclerous with sometimes calcification, can be extensive and submerges the scanty epithelial component (Fig. 3.7b). The number of myofibroblasts is elevated in the intra-tumor stroma and correlates with the degree of tumor fibrosis (Terada et al. 1996a). Surrounding the tumor, local HSCs and/or portal fibroblasts acquire the expression of α-SMA and seem to form a continuity with the intra-tumor myofibroblasts (Terada et  al. 1996a) (Fig.  3.7c). These are involved in deposition of ECM and in remodeling (Okamura et al. 2005). Stroma cells express MMP-1, MMP-2, MMP-3, MMP-9, and TIMP-1 and TIMP-2 (Terada et al. 1996b). This expression of MMPs and TIMPs is stronger in CCA with severe invasion (Terada et al. 1996b). The question arises why the stroma reaction is scanty in HCC and abundant in CCA. It is conceivable that the cells contributing to the myofibroblastic population and which participate in stroma reaction are different. Precursor cells of the myofibroblast in the liver, HSCs and portal fibroblasts, are differently distributed in the hepatic lobule (Guyot et al. 2006). HSCs resemble pericytes and are located along the sinusoids, in the Disse space between the endothelium and the hepatocytes. In contrast, portal fibroblasts are embedded in the portal tract connective tissue around portal structures such as vessels and biliary structures. Differences

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Fig. 3.7   Intrahepatic cholangiocarcinoma features. a This type of tumor shows important hyaline changes. b The tumor is composed of a typical tubule proliferation in an abundant fibrous stroma (Masson’s trichrome histochemistry). c By immunohistochemistry, the tumor stroma contains numerous α-smooth muscle actin-expressing myofibroblasts

have been reported between these two fibrogenic cell populations, concerning the mechanisms leading to myofibroblastic differentiation, activation and ‘deactivation’ (Guyot et  al. 2006). For example, rat portal fibroblasts and HSCs differ in CD95-mediated apoptosis and response to tumor necrosis factor-α (Saile et al. 2002). Insulin-like growth factor-1 induces DNA synthesis and apoptosis in rat HSCs but DNA synthesis and proliferation in rat portal fibroblasts (Saile et al. 2004). Fibrotic liver remodeling was studied in culture of precision-cut liver slices derived from fibrotic liver: it was shown that after carbon tetrachloride treatment, a proportion of myofibroblasts derived from HSCs seems to dedifferentiate while in bile duct ligation model, myofibroblasts derived from portal fibroblasts disappear by apoptosis (Guyot et al. 2007). It is now widely accepted that the different types of lesions leading to liver fibrosis, e.g. lesions caused by alcohol abuse and viral hepatitis, involve specific fibrogenic cell subpopulations (Desmouliere 2007).

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Concomitantly, the stroma reaction surrounding different liver cancers also involve specific fibrogenic subpopulations. We suggest that HSC-derived myofibroblasts and portal fibroblast-derived myofibroblasts are involved in the stroma reactions encountered in HCC and CCA, respectively. The biological and biochemical characterization of these cells is essential if we are to understand the interactions between tumor cells and stroma myofibroblasts. For example, as mentioned above, HSCs and portal fibroblasts differ in their proliferative and apoptotic capacity, at least in vitro. All this information is required for the development of treatments specifically and efficiently targeting the cells responsible for the development of the stroma reaction. In conclusion, liver fibrosis and cirrhosis generate activated myofibroblasts that are crucial for HCC development, underlining the fact that the presence of fibrotic lesions may increase the risk of cancer. These myofibroblasts provoke malignant hepatocytes to undergo EMT at the tumor edge and thus render them capable to invade and metastasize. It is clear that stroma myofibroblasts, derived from HSCs or from portal fibroblasts in the liver, promote tumor progression. Stroma–myofibroblast interactions represent an interesting tumor differentiation-independent target for therapy of cancers, particularly for HCC and CCA which are highly aggressive cancers.

3.5 Conclusions and Perspectives It is now well established that the myofibroblast plays an important role in developmental processes, several types of response to injury and fibrotic phenomena. As discussed above, the participation of this cell in tumoral phenomena has become more and more evident in more recent years, implying that epithelial-stroma interactions are crucial in cancer biology. This represents a switch in the present paradigm, which considers epithelial cells exclusively responsible for cancer evolution. Many aspects of cancer-stroma interactions however, remain open and should be thoroughly investigated: 1. The participation of mechanical forces in wound healing and fibrosis has been demonstrated. It will be important to clarify the role of force development in tumor-stroma interactions and in cancer cell biology. 2. It appears clear that cancer stroma contains a heterogeneous fibroblast, and possibly myofibroblast, population. The characterization of the specific features of these cells should enhance our understanding of their biological behavior. 3. The interactions between epithelial and fibroblastic cells also take place also through soluble factors produced by both cell categories. Advances in the identification of such factors should be instrumental for the elucidation of these phenomena. Progress along these and other lines should not only advance our knowledge of tumor biology, but also allow new and fruitful approaches to therapeutic strategies.

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Acknowledgements  The work of BH is supported by grants from the Swiss National Science Foundation (#3100A0-113733/1 and #3200–067254), from the GEBERT RÜF STIFTUNG, and from the Connaught Funding Program. The work of AD was supported in part by a grant from the University of Limoges (Contrat Renforcé Recherche). The work of IAD was supported in part by a grant from the Australian Academy of Science and a visiting fellowship at the University of Limoges. We are very grateful to Paulette Bioulac-Sage, and Sébastien Lepreux (Service d’Anatomie Pathologique, Hôpital Pellegrin, CHU Bordeaux, France) for the illustrations of pathological cases.

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Chapter 4

The Role of Myofibroblasts in Communicating Tumor Ecosystems Olivier De Wever, Astrid De Boeck, Pieter Demetter, Marc Mareel and Marc Bracke

4.1 Introduction To explore the transition from non-invasive to invasive forms of cancer we consider cancer as a systemic disease with a tumor as an ecosystem operating inside a living organism. Central in tumor ecosystems is the idea that cancer cells and tumorassociated host cells are continuously communicating and non-redundant changes in a single element inevitably alters the organization of the whole system (De Wever and Mareel 2003; Mareel et al. 2009). The present review focuses on the prominent role of myofibroblasts as host cells in the tumor ecosystem. When analyzing this, we will consider a global communication between local and distant ecosystems situated inside the human body, namely the primary tumor, the bone marrow, the circulation, the sites of lymph node and of distant metastases and the nervous system. When oncogenic alterations change the epithelium of a tissue during cancer development, the stroma inevitably changes also. The corresponding robust myofibroblast reaction activates an invasive growth program in cancer cells making a tumor an alien entity invading the body (De Wever et al. 2008; De Wever and Mareel, 2003; Mareel et al. 2009). This invasive growth program implicates a number of cellular activities, namely: homotypic cell-cell adhesion; heterotypic cell-cell and cell-matrix adhesion; proteolysis; migration; ectopic survival and growth (Mareel et  al. 2009). New experimental models have facilitated the direct observation of the multifaceted invasion process. Novel insights into tumor ecosystems raise the question whether myofibroblasts have a critical impact on various therapeutic strategies, and should be considered as effective targets for therapy, next to or as an alternative for the cancer cells. We must take into consideration that next to myofibroblasts, tumor ecosystems contain a plethora of cells that work in concert such as mesenchymal progenitor cells, tumor associated macrophages, mast cells, neutrophils, endothelial cells, and bacteria (Joyce and Pollard 2009; Possemiers et al. 2009; Wels et al. 2008b). O. De Wever () Laboratory of Experimental Cancer Research, Department of Radiotherapy and Experimental Cancer Research, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium e-mail: [email protected] M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_4, © Springer Science+Business Media B.V. 2011

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4.2 Definition and Origin of Myofibroblasts Identity and expression profiles of tumor-associated fibroblasts are only partially known, and it is likely that several currently unrecognized subtypes exist (Chang et al. 2002; De Wever et al. 2008). Most studies of tumor stroma consider it as a relatively uniform entity, and the contribution of inter-patient fibroblast variability to the biological and clinical heterogeneity of tumors is only beginning to be recognized and understood (Allinen et al. 2004; Beck et al. 2008). We believe that there exist distinct types of fibroblasts with distinct reaction patterns that affect invasive tumor growth in different ways. The spectrum of phenotypic entities ranges from the non-contractile fibroblast to the contractile myofibroblast with a number of intermediate phenotypes having been described (reviewed in Eyden 2005). One type that recently attracted a lot of attention is the myofibroblast. Myofibroblasts are large spindle-shaped cells defined by indented nuclei, α-smooth muscle actin containing stress fibers and well-developed cell-matrix interactions (fibronexus). Unfortunately, there is no myofibroblast-specific immunocytochemical marker and characterisation is based on a combination of positive markers such as α-SMA, γ-SMA, desmin, vimentin, prolyl-4 hydroxylase and negative markers such as cytokeratin, CD31, CD34 and smoothelin (De Wever et  al. 2008). Besides these cytoplasmic and membrane markers, stromal myofibroblasts are characterised by an elevated production of ECM components, ECM remodelling enzymes, growth factors, cytokines and chemokines creating a growth promoting environment (De Wever et al. 2008; Tuxhorn et al. 2002). The expression of α-SMA is considered to be the main biochemical marker of myofibroblastic differentiation in controlled in vitro systems. Understanding the origin and molecular events for the generation of tumorassociated myofibroblasts is still a matter of debate. Tumor-associated myofibroblasts are thought to arise from several mobilised cell types (Wels et  al. 2008a) including migratory neighbours such as tissue-resident mesenchymal stem cells (MSCs) (Muehlberg et al. 2009) or tissue resident fibroblasts (Rønnov-Jessen et al. 1995), and distant invaders such as bone marrow-derived MSCs (Direkze et al. 2004; Ishii et al. 2003) and fibrocytes (Direkze et al. 2004; Ishii et al. 2003; Mori et al. 2005). These findings demonstrate a high degree of plasticity and a diversity of origins of myofibroblast precursor populations. This diversity may also explain the heterogeneous phenotypic characterization of myofibroblast populations observed within tumors.

4.3 Experimental Ecosystems of Invasion: Implication of Myofibroblasts To advance the discovery and development of effective anti-invasive therapies, it will be necessary to develop theoretical and experimental (cell culture and mouse) models that not only recapitulate the tumor ecosystem but also provide detailed mechanistic information of how target activity and therapeutic response influence

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tumor invasion. Dynamic cell imaging, in association with multiparametric image analysis applied to physiologically relevant models, can help achieve these goals. Covering image analysis, be it only a minor part of, is beyond the scope of the present review (Carragher 2009; Lang et al. 2006). We will elaborate on various theoretical and experimental invasion models that take into account the dynamic communication between cancer cells and their environment, more exactly the tumor-associated myofibroblast. Today, there are compelling signs of renewed interest in mathematical modeling of cancer progression and a new discipline emerged: integrative mathematical oncology (Anderson and Quaranta 2008). Several groups demonstrated that invasion is a predictable process governed by biophysical laws (Anderson and Quaranta 2008; Anderson et al. 2006; Bearer et al. 2009; Frieboes et al. 2006). Multiscale mathematical modeling builds a quantitative representation of a tumor slice, on a two-dimensional graphical grid (lattice). Each cell is accounted for in the lattice and its behaviour (proliferation, migration and death) is tracked on the basis of mathematical functions and this generates a computer simulation of tumor evolution. In essence, the shape and size of the tumor emerges from the properties of individual cells as well as from inputs from parameters from the ecosystem such as oxygen concentration, nutrients availability, and ECM gradients. All known forms of collective cell migration (chains, strands and detached clusters) observed in experimental models and histopathology are consistently reproduced by the mathematical model. For example, a homogenous ECM distribution predicts a large tumor with smooth margins containing a dead-cell inner core and a thin rim of proliferating cells. In contrast a heterogenous ECM predicts a tumor with a dead inner core with a thin rim of proliferating cells displaying a striking, branched fingered morphology of the margins. Thus, multiscale mathematical modeling predicts that the environment determines the invasive phenotype of a tumor (Anderson et al. 2006). This is rather distinct from the conventional view that the environment is a supporting infrastructure for cancer cells (Mueller and Fusenig 2004). Furthermore, mathematical modeling can provide a rigorous, more precise approach for quantifying correlations between tumor parameters, prognosis, and treatment outcomes. A constant dialogue between mathematicians and cancer biologists will assist to frame cell culture experiments in the context of quantitative models. In our view of a tumor as a communicating ecosystem composed of both cancer cells and myofibroblasts we will provide an overview of experimental invasion models that take into account the role of myofibroblasts. Most models make use of purified matrix proteins such as acid-extracted, native collagen type I or mixtures of matrix proteins such as Matrigel. In scope of this review, we consider mainly models that use native collagen type I because tissue invasion requires cancer cells to negotiate a stromal environment dominated by cross-linked networks of collagen type I (Sabeh et al. 2009). Several groups studied invasion of cancer cells seeded on top of myofibroblast-enriched native collagen type I gels. Invasion was scored after 24 h as single cell invasion quantified by phase-contrast microscopy (De Wever et al. 2004). Myofibroblast-stimulated invasion of HCT-8 colon cancer cells into collagen type I

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Fig. 4.1   Phase contrast pictures of single HCT-8/E11 colon cancer cells seeded on collagen type I gel and cultured for 24 h in culture medium (control) or culture medium supplemented with conditioned medium derived from myofibroblasts (CMmyo). Arrows indicate invasive extensions. Scale bar = 20 µm

is characterized by a change from a round, nonmigratory morphotype to an elongated, migratory morphotype (Fig. 4.1). H&E stained paraffin sections from 14 day cocultures confirmed that myofibroblasts stimulated the invasion of colon cancer cells and squamous cell carcinoma cells (Daly et al. 2008; De Wever et al. 2004; Nystrom et al. 2005). Nystrom et al. (2005) further optimized quantification of these organotypic gels by a computer-assisted digital image analysis system that assesses invasion objectively and provides a numerical invasion index taking into account the depth and pattern of invasion. The use of these organotypic gels is not limited in vitro but can also be transplanted into the flank of nude mice (Borchers et al. 1997). A possible disadvantage of single-cell invasion assays is that it lacks structural architecture. A multicellular spheroid system is more adapted to study invasion mechanisms because it takes into account homotypic cell-cell contacts and progressive deprivation in oxygen (hypoxia), nutrients, growth factors as well as limitations in the penetration and action of drugs. Spheroids, prepared from epithelial non-invasive HCT-8/E11 colon cancer cells and placed inside native collagen type I gels, grow but remain ball shaped when followed over at least 96 hours. When these spheroids are co-cultured with primary colon tumor-derived myofibroblasts the spheroids display a less spherical morphology with a rough surface. This roughness reflects an invasive morphology and can be easily evaluated by calculating the factor shape (or inverse roundness, perimeter2/4πarea) (De Wever et al. 2010; Denys et al. 2008). Stromal myofibroblasts exert a dramatic selective force on patterning of cancer cell spheroids, which grow as an invasive mass with collective cell migration leading to fingering margins and individual migration leading to fragmentation of single and/or clusters of cells. This collective multicellular migration pattern is reminiscent of the proteolytic-dependent track clearance by a spindle shaped leader cell followed by multiple cells observed in vivo (Wolf et al. 2007). The round, amoeboid-type of migration observed with the fragmented single cells suggests a non-proteolytic reorganization of the collagen meshwork during invasion

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(Wolf et al. 2003). Consistent with this observation, primary tumor explants (thus including cancer cells and stromal myofibroblasts) cultured in a randomly organized, native collagen type I matrix realigned the collagen fibers, allowing individual cancer cells to migrate out along radially aligned fibers (Provenzano et al. 2006). An alternative approach is to directly confront breast cancer cell spheroids with tumor-derived fibroblast spheroids (Kunz-Schughart et al. 2001). Short-term and long-term confronted spheroids were histologically analysed and revealed cancer cell infiltration into the fibroblast spheroid. Furthermore, α-SMA immunohistochemistry showed accumulation of myofibroblasts around the infiltrating cancer cell nests. Using a bi-transgenic tumor model with increased stromal collagen in mouse mammary tissue, Provenzano et al. (2008) showed that collagen density and stromal collagen fiber reorganization (the so called tumor-associated collagen signature) facilitates local invasion.

4.4 Local and Distant Clinical Ecosystems of Invasion: Implication of Myofibroblasts 4.4.1  Primary Tumor The ecosystem of the primary tumor, instead of the cancer cells only, may dictate the invasive phenotype and therefore metastatic disease. Indeed, tumors with abundant α-SMA+ stromal myofibroblasts are associated with shorter disease-free survival rate for stage II and II colorectal cancer (Tsujino et  al. 2007). Although it is increasingly evident that cancer is influenced by signals emanating from the tumor stroma, little is known regarding how changes in stromal gene expression affect epithelial tumor progression. Neuroendocrine prostate tumors that arise in CR2-Tag transgenic mice and evolve through a series of stages closely mimicking those observed in human prostate cancer have been previously described (Garabedian et al. 1998). The mice are born with a normal prostate and develop prostate intraepithelial neoplasia (PIN) by 8 weeks, which progress to invasive carcinoma by 16–20 weeks. The invasive stage is accompanied by a robust, myofibroblast stromal reaction rendering the model attractive for addressing host tissue responses to invasive tumor growth. Bacac et al. (2006) performed cDNA microarray analysis of laser-microdissected stromal cells derived from PIN and invasive cancer. Human orthologs of differentially expressed genes identified in the myofibroblast reaction of invasive tumors were observed to be associated with shorter survival and recurrence-free periods in human breast and prostate cancer. Dvorak (Dvorak 1986) found the striking resemblance between many of the signaling processes involved in tumor progression and those that occur during wound healing. Its in vitro proxy, serum response of cultured fibroblasts is examined in exquisite detail by Chang et al. (2004) and Iyer et al. (1999). A “wound-response” signature was established from 50 fibroblast cultures whose expression changed after exposure to 10% serum

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and after removal of cell cycle-associated genes. Patients whose tumors expressed this “wound-response” signature have a markedly reduced overall survival and distant metastasis-free survival compared to tumors that did not express this signature (Chang et al. 2005). Interestingly, Finak et al. (2008) used laser capture microdissection to compare gene expression profiles of tumor stroma from primary breast tumors and derived prognostic signatures strongly associated with clinical outcome. The ecosystem, and especially myofibroblasts, may have a critical impact on chemotherapy response of the patient (De Wever et  al. 2008). To better understand the relationship between tumor-host interactions and the efficacy of chemotherapy, Farmer et al. (2009) have reported that a reactive stromal gene expression signature present in estrogen receptor-negative breast tumors predicts resistance to preoperative chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide.

4.4.2  The Bone Marrow The bone marrow can be considered as a representative site of distant metastases or as a distant source and depot of hematopoietic and mesenchymal precursor cells. In the present review we consider the bone not as a site for distant metastasis although it is not known whether the “source and depot” function changes when cancer cells are present. The cancer cell secretome induces changes in the primary tumor ecosystem, but also directs significant changes in the bone marrow ecosystem. An intricate vascular network and a dense mesenchymal-derived stroma cell scaffold exist within the bone marrow. The stromal matrix includes many essential growth factors, cytokines, chemokines and ECM components that regulate hematopoietic and mesenchymal stem cell proliferation and differentiation. More specifically, the bone marrow releases the hematopoietic and mesenchymal progenitor cells that prepare the niche for metastasis. McAllister et al. (2008) found that human breast cancer cells instigate the growth of otherwise-indolent cancer cells. The first, termed here an ‘‘instigator or inducer,’’ is the experimentally transformed human mammary epithelial BPLER cell line, which yields vigorously growing tumor xenografts that histopathologically resemble invasive ductal adenocarcinomas commonly encountered in breast cancer patients. These xenografts contain abundant stroma, indicating that they are capable of recruiting murine stromal cells. The second, termed here a ‘‘responder,’’ is the experimentally transformed human mammary epithelial cell line, HMLER hygro-H-rasV12 (HMLER-HR). Only 25% of the mice injected with these indolent cells form observable tumors when examined 9 weeks after implantation. In an initial experiment, instigating BPLER cancer cells were injected subcutaneously into the right flanks of nude mice, while indolent responding HMLER-HR cells were injected into the contralateral flanks of these mice. BPLER xenograft systemically instigate invasive growth of HMLERHR xenograft by incorporation of bone marrow-derived cells into the stroma of the distant, once indolent tumors. These bone marrow-derived cells are functionally activated prior to their mobilization. Secretion of osteopontin by instigating tumors

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is necessary for bone marrow-derived cell activation and the subsequent outgrowth of the distant otherwise-indolent tumors.

4.4.3  The Circulation Myofibroblasts can be recruited from circulating precursors such as MSCs and CD14+ monocytes. These monocytes are kept in an undifferentiated state by serum factors, but can be stimulated by soluble factors such as interleukin-4 and -13 (Shao et al. 2008) and by direct contact with T-lymphocytes (Abe et al. 2001). These factors drive the monocytes into the CD34+ fibrocyte stage, and subsequent activation of the chemokine receptors (CXCR4 and CCR7) of these fibrocytes leads to rapid extravasation and tumor infiltration (Bellini and Mattoli 2007). Once infiltrated, CD34+ fibrocytes may differentiate into α-SMA+ myofibroblasts which are abundant in breast invasive ductal carcinoma (Barth et al. 2002). Experiments in vitro have shown that differentiation from CD14+ monocytes to CD34+ fibrocytes can also occur spontaneously, but is delayed by a serum protein coined serum amyloid P (SAP) (Pilling et al. 2003). Like C-reactive protein, this molecule is secreted by the liver and belongs to the pentraxin family. The inhibitory effect of SAP on CD14+ monocytes is presumed to result from an interaction with the Fcγ receptors on the monocyte plasma membrane. It shows indeed high affinity for RI (CD64) and RII (CD32), but low affinity for RIII (CD16). The shared binding sites for SAP and IgG cause competition for Fcγ receptor binding and inhibition of immune-complex mediated phagocytosis by SAP (Lu et al. 2008). Yet, phagocytosis of immune complexes as such is known to inhibit differentiation of CD14+ monocytes as well (Pilling et al. 2006). Although serum concentrations of SAP appear to be fairly constant, in systemic sclerosis, a pathological condition characterized by high myofibroblast recruitment, low serum levels were found (Pilling et al. 2003, 2007; Postlethwaite et al. 2004; Tennent et  al. 2007). Interestingly, serum concentrations can be downregulated pharmacologically. Due to its palindromic structure, the drug R-1-[6-[R-2-carboxypyrrolidin-1-yl]-6-oxo-hexanoyl] pyrrolidine-2-carboxylic acid (CPHPC) can sequester two SAP molecules stoichimetrically for elimination from the circulation (Pepys et al. 2002). Further research to investigate whether SAP modulates cancer progression would be interesting.

4.4.4  Lymph Node Metastasis Ample experimental and clinical evidence supports the invasion-promoter role of myofibroblasts in primary cancers, whereas for lymph node metastasis data is scarce. Stromal reactions in metastatic lymph nodes, possibly containing myofibroblasts, have been described as: reactive and fibrotic tissue with enhanced deposition

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of vitronectin and fibronectin (Reilly and Nash 1988); desmoplasia (Tanaka et al. 2002); nodal fibrosis (Newman et al. 2003); hyaline stroma (Thariat et al. 2008). Immunohistochemical characterisation of myofibroblasts was reported in only one of these publications, concerning a metastatic lymph node from a patient with an adenocarcinoma of the uterine cervix who received preoperative chemotherapy (Tanaka et al. 2002). The glandular cancer cells were situated in the marginal and cortical sinus; they were surrounded by concentric fibrous connective tissue with spindle cells that were recognized by an antibody against α-SMA, but not against calponin, desmin or CD34. In primary tumors from colorectal cancer patients, who received neoadjuvant radiochemotherapy or not, we invariably found bundles of α-SMA-positive cells, associated with the cancer cells (De Wever et al. 2008). Such association was found also in metastatic lymph nodes. Comparison of consecutive 3-µm thick sections stained for CD34, a marker of hematopoietic precursor cells, and for α-SMA respectively shows vessel distribution and myofibroblasts tightly adhering to colorectal cancer cell islands in one-to-three concentric layers (Fig. 4.2).

Fig. 4.2   Immunohistochemical staining of lymph nodes from colorectal cancers. Paraffin sections were made from lymph nodes derived from resection specimens of colorectal cancers. Samples from two patients were stained for α-SMA (smooth muscle actin) ( panel a and c) and CD34 (blood vessel marker) ( panel b and d). Arrows show 2–3 layers of myofibroblasts surrounding a smaller cancer cell nest ( panel a) and infiltrating larger cancer cell nests ( panel c). Arrowheads show absence of blood vessels at a consecutive section as indicated by CD34 staining. Scale bar = 100 µm. (Our unpublished results)

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Possible sources of myofibroblasts in metastatic lymph nodes comprise: resident cells present in the reticulin network (Tanaka et  al. 2002); bone marrow-derived cells as observed in the pre-metastatic niche of regional lymph nodes from cancer patients (Kaplan et al. 2005). Fibrosis in metastatic lymph nodes is a factor of worse prognosis in cancer of the oral cavity (Lehn and Rapoport 1994), and in invasive ductal cancer of the breast (Hasebe et al. 1998). In single, presumably early, lymph node metastases from gastric cancer a strong correlation was found between extracapsular invasion and fibrotic foci, both signing a worse prognosis (Okamoto et al. 2008). In our colorectal cancer material, invasion of the lymph node capsule was accompagnied by thick bundles of myofibroblasts. These observations suggest that stromal cells assist cancer cells invading through the nodal capsule, possibly enhancing metastasis.

4.4.5  Distant Metastasis In distant metastasis, like in lymph node metastases from human epithelial cancers analysis of tumor-associated myofibroblasts is scarce. In the scenario put forward by Vidal-Vanacloche (Vidal-Vanaclocha 2008) for the establishment of the premetastatic niche in the liver, tumor-infiltrating stromal cells appear in the last phase, after formation of an avascular metastasis. Endothelial cell migration towards avascular metastasis occurs only at high density of myofibroblasts. The latter cells come from three sources: hepatic stellate cells transiting to myofibroblasts in response to paracrine factors from cancer cells, from tumor-activated hepatic sinusoidal endothelial cells and from Kuppfer cells. Several studies of hepatic metastasis of B16 melanoma have shown that factors secreted by melanoma cells appear to activate hepatic stellate cells to a myofibroblast-like state associated with SMA expression and cytoskeletal changes (Olaso et al. 1997). A study by Schürch et al. (1981) found presence of myofibroblasts at the invasion front of the primary colon cancer site and in the liver metastasis. Lung and brain metastases from ASPL (alveolar softpart sarcoma, characterized by a 17; X, chromosomal translocation resulting in an ASLP-TFE3 fusion gene) are composed of small islands of cancer cells surrounded by fine strands of stroma (Genin et al. 2008). The nuclei of the cancer cells are clearly TFE-positive, whereas the stromal cells are α-SMA-positive. This distinguishes the cancer cells from the myofibroblasts, at least in the lung metastasis, though in the brain metastasis the distinction through these markers is less clear. In another scenario, considering the lung as the site of distant metastasis, enhanced expression of fibronectin, an ECM glycoprotein, and an increase in PDGFR-expressing cells were localized to premetastatic sites early in tumor progression, prior to the arrival of bone marrow-derived hematopoietic progenitor cells (Kaplan et al. 2005). Furthermore, lysyl oxidase (LOX) accumulates at premetastatic niches in target organs where it modifies the ECM by crosslinking collagen fibrils to make it more receptive for CD11b+ myeloid cell infiltration (Erler et al. 2009). Thus, the cancer

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cell-secretome prepares a premetastatic niche and distant myofibroblast recruitment to support the ectopic survival and growth of incoming cancer cells.

4.4.6  Nervous System Myofibroblasts may also be implicated in pain, for many patients the first sign of cancer. Local cancer cell infiltration and growth can cause nociceptor (a primary afferent sensory neuron) stimulation by mechanical injury or compression. Various factors implicated in invasive cancer growth can also sensitize or directly excite nociceptors without compression. These factors include prostaglandins, endothelins, IL-1 and -6, EGF, TGF-beta, and PDGF. Chronic cancer pain can cause significant alterations in the distant central nervous system. This is believed to underlie the phenomenon of central sensitization—an increased responsiveness of spinal-cord neurons that are involved in the transmission of pain. Confocal imaging of glial fibrillary acidic protein (GFAP) expression in the spinal cord showed an increase in the number of astrocytes on the right side of the spinal cord, which received sensory innervation from the tumor-bearing bone. The left side of the spinal cord, which was not transmitting painful stimuli to the brain, had fewer astrocytes (Mantyh et al. 2002).

4.5 Therapeutic Targeting of Ecosystems: Turning the Tide of the Vicious Cancer Progression Cycle? Understanding the role of myofibroblast in local and distant tumor ecosystems may open the door for new strategies which target the management of myofibroblasts and therefore new possibilities for pharmaceutical design. An intriguing question is: can cancer be reversed by engineering the tumor ecosystem? Recent insights make clear that inhibition of metastasis requires targeting of both the metastasizing cell and its supportive ecosystem (Wels et al. 2008b). Recognition that the preparatory changes in the premetastatic environment occur very early in tumorigenesis suggests that anti-metastatic agents must be used together with the primary therapy. Furthermore, therapy that focuses on the dynamic nature of tumors and their supportive cells, including myofibroblasts, may be critical to preventing and reversing tumor progression. We will quote one example that recently attracted a lot of attention. Halofuginone, which at present is being evaluated in clinical trials (de Jonge et al. 2006), inhibits the fibroblast-to-myofibroblast transition in the tumor microenvironment (Sheffer et al. 2007) by inhibiting Smad3 phosphorylation downstream of TGFβ signaling (McGaha et al. 2002; Yee et al. 2006). Halofuginone may synergize with low doses of chemotherapy in achieving a significant antitumoral effect, avoiding the need of a high dose of chemotherapy and its toxicity without impairing treatment efficacy (Sheffer et al. 2007).

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4.6 Conclusions and Perspectives New evidence is emerging on how supporting host cells composing the tumor ecosystem promote invasive growth and metastasis. The underlying mechanisms of how a cancer cell alters its local and distant ecosystem are also receiving more attention. Dissecting these individual pieces will provide valuable insights and direct future investigations. The ecology of a metastatic tumor includes genetically altered cancer cells and their heterotypic interactions with host cells and their extracellular matrix network. The importance of the myofibroblast in the tumor ecosystem is more and more recognized since the development of appropriate methods to experimentally determine its value. Focus on the dynamic exchange of cells from local and distant environments, to the invasive front of the tumor, to the premetastatic and metastatic niches can provide novel strategies for successfully targeting these processes. Acknowledgements  This work was supported by Fund for Scientific Research-Flanders (Brussels, Belgium), O. De Wever is a post-doctoral researcher and A. De Boeck is a doctoral researcher supported by Fund for Scientific Research-Flanders.

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Chapter 5

Tumor Vessel Associated-Pericytes Arne Bartol, Anna M. Laib and Hellmut G. Augustin

5.1 Introduction Tumor growth and metastasis are critically dependent on the growth of blood vessels (angiogenesis). The identification of the master inductor of the angiogenic cascade VEGF (vascular endothelial growth factor) in 1989 has within 15 years led to the development and clinical implementation of VEGF neutralizing and VEGF receptor activation blocking drugs. These drugs establish a novel paradigm in antitumor drug development since they are the first clinically effective and approved anti-stroma tumor drugs. Thus, despite the limited efficacy of the first generation anti-angiogenic therapies, VEGF/VEGFR blocking compounds have within few years emerged among the highest revenues yielding anti-cancer drugs. VEGF receptors are almost exclusively expressed by endothelial cells (EC) which are consequently the primary target of VEGF. It is for this reason that the field of angiogenesis research has developed in the nineteen eighties and nineteen nineties as an endothelial cell-centric discipline. Clearly, the endothelial cell is at the heart of the angiogenic cascade. Yet, this reductionist, EC-centered view largely ignores that new capillary networks form in close interaction with surrounding cells and that the recruitment of peri-endothelial mural cells (pericytes, smooth muscle cells) constitutes a critical step during vascular morphogenesis. This has changed in the last decade when the study of vascular maturation processes received more attention and mechanistic experiments on molecules of the TGFß family, PDGF molecules and the angiopoietins yielded insights into the crosstalk between EC and peri-endothelial mural cells. Today, the mechanistic and molecular study of pericyte structure and function is a rapidly growing area of ongoing biomedical research and it is increasingly recognized that pericyte-mediate vessel maturation A. Bartol () Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), 69120 Heidelberg, Germany e-mail: [email protected] Medical Faculty Mannheim, Vascular Biology and Tumor Angiogenesis, Heidelberg University, 68167 Mannheim, Germany M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_5, © Springer Science+Business Media B.V. 2011

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Fig. 5.1   Overview of pericyte physiology. The capillary lumen is lined by a single layer of endothelial cells. Separated by the basement membrane, capillary EC are covered by pericytes that stretch out with long dendrite-like processes to physically contact EC. The image summarizes the most established markers of pericytes, some of the key regulators of pericyte function, the established physiological functions of pericytes and the roles of pericytes in pathology. MSC mesenchymal stem cells, AMD age-related macular degeneration

is a critical determinant of the therapeutic window of anti-angiogenic therapies (Jain 2005). Historically, pericytes have first been described by Charles Rouget in 1873 and the term pericyte was introduced by Zimmerman in 1923 (Rouget 1873; Zimmermann 1923). Pericytes are the mural cells of capillaries and postcapillary venules. They share the basement membrane with EC and they contribute to basement membrane synthesis. Pericytes display dendrite-like cell extensions which envelop capillaries and enable them to establish cell-cell contacts with multiple EC. The anatomical vicinity between pericytes and EC enables pericytes to sense signals in the environment and to integrate them to orchestrate EC signaling (Gerhardt and Betsholtz 2003). Consequently, pericytes control endothelial functions, including proliferation, differentiation and permeability and they are key mediators of microvascular homeostasis. The most established markers of pericytes, some of the key regulators of pericyte function, the established physiological functions of pericytes and the roles of pericytes in pathology are summarized in Fig. 5.1.

5.2 Heterogeneity of Pericytes 5.2.1  Lineage and Origin of Pericytes During embryogenesis, vessel development occurs by the de novo formation of blood vessels from mesodermal precursor cells that are specified to form angio-

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Fig. 5.2   a Wild type mouse retina vessel (P6) stained with BS-I lectin is covered by pericytes. Staining for α-SMA ( red) and NG2 ( green) identifies pericytes that are positive for both markers ( white arrows). Scale bar 10  µm. b Electron microscopic analysis of a representative tracheal blood vessel that is covered by pericytes. One pericyte is attached to the endothelium of the blood vessel. Scale bar, 12,7  µm. P pericyte, EC endothelial cell, E erythrocyte, N nucleus. (Images kindly provided by Dr. Junhao Hu, Heidelberg)

blasts and further differentiate into EC (vasculogenesis). Pericytes arise during vasculogenic formation of a primordial vascular network from mesodermal progenitor cells. Vessels of the CNS and the cardiac tract vessels recruit pericyte precursors from the neural crest (Creazzo et al. 1998; Ema and Rossant 2003). Both, EC and pericytes may even originate from the same precursor cell during vasculogenesis. Mesodermal VEGFR-2-positive angioblasts appear to give rise to EC following VEGF stimulation or to pericytes following PDGF-B (platelet-derived growth factor-ß) stimulation, respectively (Carmeliet and Luttun 2001; Yamashita et al. 2000). The origin of pericytes during adult angiogenesis remains unclear, but it is believed that pericytes differentiate from tissue- or bone-marrow stem cell derivates. The contribution of bone-marrow derived pluripotent stem cells during physiological or pathological angiogenesis has been discussed controversially. On one hand, bonemarrow derived pluripotent stem cells are recruited to the periendothelial space near blood vessels (Rajantie et al. 2004). In turn, endothelial-to-mesenchymal transition (EndMT) has been reported to give rise to the differentiation of perivascular cells (Armstrong and Bischoff 2004; Zeisberg et al. 2008). During EndMT, EC lose cellcell junctions and endothelial markers (e.g. CD31) and transdifferentiate to acquire a highly motile and invasive phenotype. Simultaneously, these cells express mesenchymal markers such as fibroblast-specific protein 1 (FSP1) and alpha-smoothmuscle actin (α-SMA) (Zeisberg et al. 2007). Pericytes share common characteristics with mesenchymal stem cells. Remarkably, pericytes are able to differentiate into osteocytes, chondrocytes and adipocytes (Crisan et al. 2008), thereby supporting the hypothesis that the vasculature and more

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precisely pericytes themselves constitute a perivascular stem cell niche providing a source for mesenchymal stem cells (da Silva Meirelles et al. 2008).

5.2.2  Molecular Diversity of Pericytes The molecular repertoire of pericytes is incompletely characterized. Yet, a number of reliable markers of pericytes have been established in recent years (Fig. 5.2): (1) Alpha-smooth-muscle actin (α-SMA) and desmin are contractile microfilaments and intermediate filaments, respectively; (2) Regulator of G-protein signaling 5 (Rgs5) belongs to the RGS family and inhibits signaling from G-protein-coupled receptors by increasing intrinsic GTPase activity of activated Gα proteins (Hollinger and Hepler 2002). Tumors grown in Rgs5-deficient mice, for example, show a significant reduction in tumor hypoxia and vessel leakiness due to an increase of mature pericytes in the tumor vasculature (Hamzah et  al. 2008). However, Rgs5 also marks multiple other cell types in tumors and the reliable detection of Rgs5 is limited due to the lack of specific antibodies; (3) Platelet-derived growth factor receptor beta (PDGFRß), a tyrosine-kinase receptor, is one of the best characterized markers expressed by pericytes. PDGFRß signaling plays a key role in the recruitment of pericytes to newly formed blood vessels (Fig. 5.3); (4) Neuron-glial 2 (NG2) is a heparan sulfate transmembrane proteoglycan which binds growth factors (e.g. FGF2) and potentiates growth factor signaling (Goretzki et al. 1999). NG2 can interact with the integrin signaling pathway and contributes to critical processes such as cell survival, motility and proliferation (Fig. 5.4) (Stallcup and Huang 2008); (5) the transmembrane cell surface receptor Endosialin is expressed by pericytes during embryogenesis and tumorigenesis but is absent in pericytes of mature vasculature (Bagley et al. 2008; Christian et al. 2008); (6) the pericyte cell surface expressed enzymes aminopeptidase A and N (Kunz et al. 1994) are involved in the metabolic pathway of angiotensin II and III and control arterial blood pressure (Fournie-Zaluski et al. 2004). There is some degree of pericyte marker selectivity, but most pericyte markers are also expressed by other cell types including fibroblasts, astrocytes and tumor cells. The expression of α-SMA is highly dynamic and differs between different vascular beds, developmental stages or species (Gerhardt and Betsholtz 2003). α-SMA is not only expressed by skin pericytes but is also strongly upregulated during tumor angiogenesis (Abramsson et al. 2002; Morikawa et al. 2002). Comparing different species, α-SMA is, for example, detectable in chicken brain pericytes but absent in mouse brain pericytes (Gerhardt et al. 2000; Hellstrom et al. 1999). The heterogeneity of pericyte marker and phenotyptic behavior is also reflected in the observation that the ratio of pericytes to EC varies highly between species, different tissues or different vessel types. The ratio of pericytes to EC is increased in the retina compared to skeletal muscle (Shepro and Morel 1993) and it varies between quiescent vessels and tumor associated vessels (Wakui et  al. 1997).

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Fig. 5.3   Schematic overview of several key factors regulating a) the recruitment of pericytes and b) migration of pericytes along newly formed blood vessels. a) Heparin binding EGF-like growth factor (HB-EGF) and hepatocyte growth factor (HGF) expressed by endothelial cells (EC) are potential inducers for the recruitment of pericytes. Sphingosine-1-phosphate (S1P) released from erythrocytes signals through SP1R1 on endothelial cells thereby strengthening EC-pericyte cell–cell contacts. b) During angiogenesis, formation of new blood vessels is mainly driven by VEGF stimulation. The angiogenic sprout guided by a tip cell, which is not covered by pericytes, forms filopodias and abundantly expresses VEGFR2 which sensitizes EC to VEGF secreted by the surrounding tissue. Tip cell release PDGF-B which in turn binds to HSPG within the extracellular matrix (ECM). A spatial and temporal PDGF-B gradient along EC following the tip cell, the so called stalk cells, promotes pericytes proliferation and migration towards the leading edge. PDGFB signaling is mediated by PDGFRß expressed by pericytes. BM basement membrane

5.3 Signaling Pathways in Pericytes 5.3.1  TGF-ß—The Role of a Multifunctional Cytokine Transforming growth factor ß (TGF-ß) is a multifunctional cytokine that is expressed by various cell types including EC and pericytes. Genetic deletion of key components of the TGF-ß signaling pathway in mice results in cardiovascular defects and embryonic lethality (Li et  al. 1999; Oshima et  al. 1996). Co-culture experiments

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Fig. 5.4   Signaling pathways involved in EC and pericyte crosstalk. Ang-1/2, PDGF-B, S1P and TGF-ß signaling are illustrated. In addition, NG2 can bind to α3β1 integrin complexes on the EC surface resulting in enhanced β1 integrin signaling (Fukushi et  al. 2004). Black arrows imply links to major signal transduction pathways and cellular functions. Negative feedback signaling is indicated in red

with EC and pericytes revealed that pericytes or EC alone secrete TGF-ß as a latent inactive form. Latent TGF-ß is activated by plasminogen activator following the establishment of EC-pericyte cell–cell contacts (Wakui et  al. 1997). TGF-ß activates two distinct signaling pathways in EC through the ALK1/Smad1/5 or ALK5/ Smad2/3 pathways, which induce EC proliferation or differentiation, respectively (Fig. 5.4) (Goumans et al. 2002). Endoglin, a co-receptor for TGF-ß, is expressed by proliferating EC and promotes ALK1 signaling driving the TGF-ß response towards proliferation (Lebrin et al. 2004). Depending on the concentrations of TGF-ß and the level of ALK1 expression, TGF-ß acts either pro- or anti-angiogenic (Goumans et al. 2002). TGF-ß is a multifunctional cytokine and thereby induces the differentiation of pericytes from precursor mesenchymal cells (Chambers et al. 2003; Ding et al. 2004). Pericytes that are stimulated with TGF-ß express α-SMA whereas pericytes in the absence of TGF-ß express NG2 and desmin (Song et al. 2005).

5.3.2  P  DGFB-PDGFRß—The Recruitment and Expansion Pathway The PDGF family consists of four ligands (PDGF-A, PDGF-B, PDGF-C and PDGF-D) and two members of receptor tyrosine kinases (RTK), PDGFRα and

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PDGFRß. Studies in mice lacking PDGF-B or PDGFR-ß demonstrated that loss of either ligand or receptor causes embryonic death due to vascular defects (Leveen et al. 1994; Soriano 1994). PDGF-B/PDGFRß signaling elicits the recruitment of pericytes to the leading edge of newly formed blood vessels. During angiogenesis, EC of the tip cell region of sprouting vessels express PDGF-B. Expression of PDGF-B is induced by several factors including hypoxia, thrombin and different growth factors (Heldin and Westermark 1999). One key property of PDGF-B is a retention motif, composed of a stretch of positively charged amino acid residues at the C-terminus (Ostman et al. 1991). This motif allows binding of PDGF-B to heparan sulphate proteoglycans (HSPG) in the extracellular matrix which in turn establishes a gradient of PDGF-B presentation around angiogenic sprouts (Fig. 5.3). PDGF-B is temporally and spatially differentially expressed. Abundant PDGF-B expression is found in the tip cell of sprouting vessels in response to VEGF-A, whereas expression decreases in the adjacent EC, the so called stalk cells. This temporal and spatial PDGF-B gradient induces the coverage of stalk cells with newly recruited pericytes. Pericytes express PDGFRß and sense PDGF-B which results in the recruitment to newly formed blood vessels in a paracrine manner. PDGFRß expression is induced by factors including FGF2, TGF-ß or TNFα (Heldin and Westermark 1999). Binding of PDGF-B to PDGFRß triggers downstream signaling pathways including the Ras-MAPK pathway, the PI3K pathway, the FAK and PLCγ signaling cascades. Activation of these downstream targets increases actin reorganization, cell motility, proliferation and inhibit apoptosis in pericytes (Fig. 5.4) (Hu et al. 1995; Kundra et al. 1994; Seger and Krebs 1995). Interestingly, a positive feedback loop between VEGF-C/VEGFR3 and PDGF-B/PDGFRß regulates lymphangiogenesis and lymphatic maturation (Onimaru et al. 2009). In contrast, the forkhead transcription factor gene Foxc2 is thought to maintain the pericyte-free lymphatic capillary network by suppressing PDGF-B expression (Petrova et al. 2004). Additionally, VEGF acts as an inhibitor of blood vessel maturation by negatively regulating the pericyte coverage of nascent blood vessels. This is supposed to be mediated in pericytes by dimerization of VEGFR-2 and PDGFRß in response to VEGF stimulation resulting in suppression of PDGFRß signaling (Greenberg et al. 2008).

5.3.3  Angiopoietin-Tie System—Maintenance of Pericytes The angiopoietin-Tie system is an endothelial cell specific signaling pathway. There are four angiopoietin ligands: Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Angiopoietin-3 (Ang-3) and Angiopoietin-4 (Ang-4). The best characterized ligands are Ang-1 and Ang-2. Both ligands bind Tie2, a receptor tyrosine kinase which is expressed by EC. Ang-1 is expressed and secreted by perivascular cells and activates Tie2 in a paracrine manner (Davis et al. 1996; Sato et al. 1995). The activation of Tie2 results in vessel assembly and maturation by promoting survival signals in EC and regulating the recruitment of pericytes. In contrast, Ang-2 is expressed by EC themselves and acts context dependently as an autocrine antagonist of Ang-1/ Tie2 signaling (Fig. 5.4), thereby mediating vessel destabilization. In the presence of

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growth factors (e.g. VEGF), Ang-2 promotes angiogenesis, whereas in the absence of growth factors, Ang-2 leads to the regression of newly formed blood vessels. Analyses of Ang-1- or Tie2-deficient mice revealed an abnormal vascular structure and a reduction of pericyte coverage (Dumont et al. 1994; Sato et al. 1995; Suri et al. 1996). Therefore, increasing evidence suggests that the angiopoietin-Tie system is not only involved in the control of endothelial cell homeostasis but also in the crosstalk between EC and pericytes. Elevated Ang-2 levels resulted in a diabetic rat model in the loss of pericytes from microvessels. Similar effects have been observed following the injection of recombinant Ang-2 directly into the retina (Hammes et al. 2004). The molecular mechanisms explaining these findings are not fully understood. Experiments in a pulmonary hypertension animal models suggested a link between Ang-1 and Serotonin in the context of smooth muscle cell proliferation around small vessels (Sullivan et al. 2003). The two best characterized candidates in angiopoietin-dependent pericyte recruitment are heparin binding EGF-like growth factor (HB-EGF) and hepatocyte growth factors (HGF). Both factors are upregulated in EC after Ang-1 stimulation and induce migration of smooth-muscle cells (SMC) (Iivanainen et al. 2003; Kobayashi et al. 2006). However, HB-EGF-deficient mice only display cardiovascular defects characterized by enlarged hearts and hypertrophic cardiomyocytes (Iwamoto et al. 2003; Jackson et al. 2003). Furthermore, antibodies against PDGFRß inhibit the recruitment of pericytes to newly formed vessels in the mouse retina (Uemura et al. 2002). The absence of pericytes resulted in retinal edema and hemorrage which is rescued by the injection of recombinant Ang-1, demonstrating that Ang-1 is required to prevent retinal defects (Uemura et al. 2002). Pericytes are an important source of Ang-1 (Sundberg et al. 2002; Suri et al. 1996) and loss of pericytes consequently acts negatively on Ang-1 signaling. Simultaneously, the loss of pericytes and reduced Ang-1 signaling may be responsible for the antagonistic function of Ang-2 on vessel maturation.

5.3.4  S  1P, Notch, Ephrin—Increasing the Complexity   of Endothelial Cell-Pericyte Crosstalk Sphingosine-1-phosphate (S1P) is a lipid mediator processed from membrane phospholipids. S1P binds to several G-protein coupled receptors (S1P1to5) and plays a role in cell migration, proliferation and survival (Allende and Proia 2002). Although S1P1 is a widely expressed receptor in vascular and non-vascular cells, EC specific deletion of s1p1 recapitulates vascular defects including abnormal pericyte coverage of vessels which has been observed in s1p1-deficient mice (Allende et al. 2004; Liu et  al. 2000). The observed s1p1-deficient phenotype may explain why S1P1 signaling acts upstream of Rac leading to relocation of N-cadherin and thereby enhancing cell-cell contacts between EC and pericytes (Fig. 5.4) (Paik et al. 2004). Notch signaling affects several vascular processes, including arterio-venous differentiation and regulation of vessel branching (Lawson et  al. 2001; Sainson

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et al. 2005). CADASIL (OMIM no. 125310) is a stroke-like syndrome caused by mutations in the human Notch3 gene resulting in aberrant vascular smooth muscle cell maturation (Domenga et al. 2004; Kalimo et al. 2002). Notch signaling is linked to PDGF signaling because PDGFRß is a Notch target gene. Activation of Notch leads to an increase in PDGFRß expression, Dysregulation of Notch perturbs differentiation and migration of smooth-muscle cells (Jin et al. 2008). The family of erythropoietin-producing hepatoma receptor tyrosine kinases (EphB) and their cell-surface anchored ephrin ligands act in the regulation of arterio-venous blood vessel identity (Adams 2003). Ephrin B2 and EphB are expressed by EC and pericytes. The ephrin signaling cascade is activated upon cell-cell contacts of pericytes with EC exerting forward and reverse signaling. Therefore ephrinB2 plays a pivotal role of endothelial-pericyte assembly during postnatal vascular remodelling (Salvucci et al. 2009).

5.4 Physiological Functions of Pericytes 5.4.1  Microvessel Permeability In general, the presence of attached pericytes to the endothelium is a hallmark of a quiescent microvasculature which prevents vascular leakage and reduces the permeability of EC. Pericytes are in close contact with EC forming the vessel wall and mediating vascular permeability by reducing the abluminal surface area of endothelial cells (Murphy and Wagner 1994). Permeability is dependent on intra- and intercellular processes, in which pericyte contractility alters endothelial cell-cell junctions and modifies the capacity of intercellular transport (Buchanan and Wagner 1990; Cuevas et  al. 1984). Pericytes in the brain are part of the blood-brain barrier (BBB), protecting the brain against toxic compounds of the circulation. Brain pericytes express the enzyme aminopeptidase N which is responsible for the degradation of various neuropeptides. Neuropeptides with vasoactive properties are involved in altering vascular permeability (Kunz et al. 1995). Moreover, pericytes are capable of increasing permeability by production and release of VEGF (Kim et al. 1998; Lonigro et al. 1996).

5.4.2  Regulation of Angiogenesis The angiogenic cascade is tightly controlled by distinct signaling pathways and the interaction between EC and pericytes. One hallmark of vessel maturation is the attachment of pericytes around newly formed blood vessels (Jain 2003; Paik et al. 2004). Induction of angiogenesis by VEGF switch the quiescent endothelium to an

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activated state accompanied by distinct changes of pericyte morphology and behavior, e.g. shortening of membrane extensions, increase of cytoplasmic volume and induction of pericyte proliferation (Diaz-Flores et al. 1992). Critical steps during angiogenesis are regulated by both EC and pericytes including (1) enzymatic degradation and remodeling of the basement membrane (Davis and Senger 2005); (2) regulation of EC migration, proliferation and branching (Carmeliet 2003; Orlidge and D’Amore 1987; Sato and Rifkin 1989); (3) secretion of VEGF and Ang-1 by pericytes promotes EC survival (Benjamin et al. 1998; Reinmuth et al. 2001); (4) pericyte invasion into surrounding tissues (Ozerdem and Stallcup 2003).

5.4.3  P  ericytes During Immunological and Regenerative Processes Pericytes play a major role in maintaining the blood-brain barrier (BBB). Brain pericytes are able to adopt macrophage-like functions (including the expression of macrophage specific markers) such as the uptake of small molecules by pinocytosis. This observation suggests that brain pericytes share physiological properties with macrophages and provide an immunological defence mechanism (Thomas 1999). As previously discussed, pericytes can give rise to different cell types including chondrocytes and adipocytes suggesting that pericytes are a source of mesenchymal stem cells (Crisan et al. 2008). Human dermal skin pericytes have the capacity to promote epithelial cell proliferation by modifying the ECM microenvironment (Paquet-Fifield et al. 2009). These findings implicate that pericytes play an important role in tissue regeneration.

5.5 Pericytes in the Tumor Vasculature The tumor vascular network is formed by angiogenesis. New vessels grow from pre-existing ones by sprouting, bridging and intussusception of EC followed by incomplete maturation (Hanahan and Folkman 1996). The tumor vasculature integrates heterogeneous precursor cells (Furuya and Yonemitsu 2008). Several progenitor subpopulations have been identified in tumor neovessels. Bone marrow-derived endothelial progenitor cells (EPCs), tissue-derived EPCs (Bruno et al. 2006; Zengin et al. 2006) and hematopoietic stem cells (HSCs) (Hattori et al. 2002; Lyden et al. 2001) may transdifferentiate into tumor-associated endothelial cells (TEC). In addition, mesenchymal stem cells (MSCs) and immature myelomonocytic cells expressing endothelial markers potentially have TEC commitment. It has been shown that bone marrow-derived circulating cells (RBCCs) are recruited to sites of ongoing neovascularization (Grunewald et al. 2006). Furthermore, Tie2-expressing mesenchymal progenitors are thought to be able to differentiate into tumor pericytes (De Palma et al. 2005).

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5.5.1  The Abnormalities of the Tumor Vasculature The tumor vasculature differs from normal blood vessels in several biological and structural characteristics. Whereas a physiologically growing neovasculature matures rapidly and coordinately to establish blood flow, the tumor vasculature is highly abnormal, irregularly shaped, tortuous, and largely lacks the normal hierarchical structure of arterioles, capillaries and venules (Baluk et al. 2005). Intratumoral pericytes associated with tumor vessels are loosely attached to the EC layer resulting in gaps between the two cells types. The basement membrane may be incomplete. Pericytes have a sporadic distribution in tumors compared to corresponding normal tissues (Gee et al. 2003; Morikawa et al. 2002). There is hitherto no unique marker available to identify pericytes in pathological conditions such as cancer. The degree of pericyte coverage in vessels of different tumor types ranges from high (Schlingemann et al. 1990, 1991; Wesseling et al. 1995) to little or no coverage (Benjamin et al. 1999; Eberhard et al. 2000; Johnson and Bruce 1997). To some extent, this may be explained by the differential expression of pericyte markers in different tumors. Morikawa et  al. (2002) were the first to show that pericytes in tumors are ultrastructurally different from pericytes under normal, physiological conditions. They uniformly express α-SMA and desmin in capillary sized tumor vessels in contrast to pericytes in capillaries of normal tissues. These authors reported distinctly different morphologies of pericytes in three different tumor models, suggesting a high heterogeneity of pericyte morphology and coverage in different tumor types and different tumor stages (Morikawa et al. 2002). Stromal cells that are positive for αSMA and desmin but not associated with blood vessels are abundant in many tumor types. These cells are usually defined as myofibroblasts and it has been shown that co-culture of fibroblasts with tumor cells induces the transdifferentiation from fibroblasts into myofibroblasts (Powell et al. 1999). The stromal (mesenchymal) origin of pericytes is well documented (Nehls and Drenckhahn 1993) and myofibroblasts are thought to be able to differentiate into pericyte-like cells that have been proposed to guide endothelial sprouts within tumors (Brown et al. 2001; Morikawa et al. 2002). For decades, only the expression of α-SMA or desmin have been tested and absent immunoreactivity was interpreted to reflect the absence of pericytes (Benjamin et al. 1999; Eberhard et al. 2000; Lach et al. 1999; Rangdaeng and Truong 1991).

5.5.2  Pericyte Recruitment to the Tumor Vasculature Under physiological conditions (e.g. embryonic development), pericytes are recruited around vascular EC and subsequently modulated by four different pathways: (1) PDGFB/PDGFRß, (2) S1P/endothelial differentiation gene-1 (EDG-1), (3) Ang-1/ Tie2 and (4) TGF-ß1/activin-like kinase receptor (ALK5) (Jain 2003). Several lines of evidence suggest that aberrant pericyte behavior may be due to imbalanced endothelial-cell/pericyte signaling circuits and/or a limited pool of recruitable pericytes (Abramsson et al. 2002) as well as a lack of proper maturation (Folkman 1985; Gee

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et al. 2003). Pericyte abnormalities in tumors have been shown to be associated with alterations in PDGF signaling (Abramsson et al. 2002, 2003; Furuhashi et al. 2004). PDGF and PDGF receptor gain- or loss-of-function studies have demonstrated a important roles for PDGF-B/PDGFRß signaling in pericyte recruitment to tumor vessels (Abramsson et al. 2003; Betsholtz 2004). Pericyte recruitment is dependent on PDGF-B expression by tumor endothelial cells (TEC) and PDGF-B acts in a paracrine manner on tumor pericytes expressing PDGFRß (Abramsson et al. 2002; Bergers et al. 2003). Ang-1 and Ang-2 regulate vascular maturation and integrity during development. The elevated expression of Ang-2 in tumors has long been recognized. Yet, whereas VEGF is highly expressed by tumor cells in most tumors, Ang-2 is predominately expressed by tumor endothelial cells (Stratmann et al. 1998; Zhang et al. 2003). Tumors grown in Ang-2-deficient mice show an altered pattern of mural cell recruitment and different maturation of tumor blood vessels (Nasarre et al. 2009).

5.5.3  Therapy Tumor blood vessels are the target of anti-angiogenic tumor therapy. The use of different angiogenesis inhibitors (Jain 2005; Kashiwagi et al. 2008; Sennino et al. 2007) has demonstrated that anti-angiogenic therapy not only induces vessel regression or retardation of vessel growth. Instead it also induces normalization of tumor blood vessels—a concept that reflect the transition of an irregular formed tumor vasculature to a more physiological vasculature to improve the availability of drugs and oxygen to disease areas, and thus improve e.g. combination therapy (Baluk et al. 2005; Inai et al. 2004; Jain 2005). Pericyte coverage of intratumoral blood vessels reduces VEGF-A dependency and pericyte recruitment to tumor vessels is mediated by PDGFB/PDGFRß signaling. Consequently, the combined inhibition of VEGF signaling in the endothelium and PDGFRβ signaling in pericytes is a promising combinatorial approach for antiangiogenic tumor therapy, targeting both EC and associated pericytes (Bergers et al. 2003; Saharinen and Alitalo 2003). Indeed, Bergers et al. have provided evidence for the improved efficacy of dual VEGFR plus PDGFR RTK inhibition compared to single treatment. These authors demonstrated that this combination treatment is effective in early- and late-stage tumors (Bergers et al. 2003; Saharinen and Alitalo 2003). The therapeutic effects are due to several cooperative mechanisms affecting the tumor cells directely as well as the EC compartment and the EC–pericyte interaction by paracrine effects. The signaling between different cell compartments in tumors plays a critical role in anti-cancer therapy using radiation. Radiation therapy induces the expression of VEGF in tumor cells, VEGFR-2 in the tumor EC, PDGF in EC and PDGFR in fibroblasts (Abdollahi et al. 2003; Li et al. 2006). Radiotherapy combined with VEGF and PDGF inhibition exerted the most prominent anti-angiogenic and anti-tumorigenic effect in prostate carcinoma and glioblastoma models (Timke et al. 2008).

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The angiopoietin ligands (Ang-1 and Ang-2) and their receptor Tie2 regulate vascular maturation and integrity during vascular development suggesting that their blockade might affect tumor growth and vessel normalization. However, due to the limited availability of selective angiopoietin inhibitors, their effects have not been studied extensively. Selectively blocking of Ang-2 by antibodies and peptibodies (peptide-Fc fusion proteins) had potent anti-angiogenic and anti-tumor effects in several tumor xenograft models (Oliner et al. 2004; Sarraf-Yazdi et al. 2008). Inhibition of Ang-1 expression by antisense RNA in a stably transfected tumor cell line similarly resulted in reduced tumor growth and angiogenesis (Shim et  al. 2001). Selective inhibitors (peptibodies) against Ang-1 and Ang-2 led to more potent antiangiogenic effects than the use of each inhibitor alone. Tumor vessel normalization as seen after Ang-2 inhibition was not observed (Falcon et al. 2009). Drugs targeting epidermal growth factor receptor (EGFR), such as the small molecular weight tyrosine kinase inhibitors gefitinib and erlotinib, as well as the anti-EGFR antibodies cetuximab and panitumumab have established anti-tumor effects and have been approved for clinical use (Lynch et al. 2004; Paez et al. 2004). In addition to the direct effect of EGFR blockade on tumor cells, it has been hypothesized that EGFR inhibition also acts anti-angiogenic. Gefitinib or cetuximab have been reported to inhibit neovascularization in several tumor xenograft models; however, it is not known whether this anti-angiogenic effect (1) is due to indirect suppression of growth factor production by tumor cells, (2) resulting from effects on non-endothelial cells within the tumor (SMCs/pericytes), or (3) has direct effects on TEC (Ciardiello et al. 2001; Morelli et al. 2006; Perrotte et al. 1999; Petit et al. 1997). In addition, relatively little is known about the consequences of EGFR targeting on tumor angiogenesis or vasculogenesis. The effect of gefitinib on tumor neovascularization has only recently been studied using a syngeneic tumor model in mice (Iivanainen et al. 2009). This study provides evidence that gefitinib acts on tumor angiogenesis via two different mechanisms. First, CD31-positive vessels were less covered by NG2-positive peri-endothelial cells and second, less BM-derived progenitor cells were recruited to the perivascular space of tumor vessels. These results suggest that the primary vascular target of gefitinib in vivo are SMCs/pericytes rather than EC. It further suggests that gefitinib treatment results in a relative reduction in the number of mature and fully functional tumor vessels (Iivanainen et al. 2009). Targeting EGFR in tumor therapy might be promising due to its selectivity for tumor angiogenesis.

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Chapter 6

The Role of Cancer-Associated Adipocytes (CAA) in the Dynamic Interaction Between the Tumor and the Host Marie-Christine Rio

6.1 Introduction It is becoming more and more evident that the tumor environment actively participates in cancer progression (Werb et al. 1996; Mueller and Fusenig 2002; DeClerck et al. 2004; Jodele et al. 2006). Adipocytes are present in multiple tissues. Some tissues have adipocyte-rich environments such as the mammary gland and bone marrow. Recent evidence suggest that adipocytes play a role in the progression of tumors. It has been shown that obesity leads to increased cancer risk and poorer patient outcome in several types of cancers (Hausman et al. 2001; Vainio et al. 2002; Calle et al. 2003; Calle and Thun 2004; Wright et al. 2007; Fair and Montgomery 2009). The reported link between adipocytes and cancer is multiple, including systemic and/or local effects. However, the study of the local function of adipocytes in tumors has just began to unfold. The nature of interplay between adipocytes and cancer cells remains at present largely unknown. Very little clear data implicate paracrine function between adipocytes and cancer cells. The present review will examine recent literature addressing the bidirectional impact of adipocytes and cancer cells, and the molecular basis that underlies the pejorative effect of adipocytes on the behavior of cancer cells. Indeed, the key determinant for cancer-associated adipocyte (CAA) effects on tumorigenesis may involve the cancer-induced molecular expression and secretory profile changes of the adipocytes in the local tumor microenvironment. The systemic effects of CAAs will also be discussed.

M.-C. Rio () Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104, INSERM U964, UDS, BP, 67404 Illkirch Cedex, 10142 C.U. de Strasbourg, France e-mail: [email protected] M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_6, © Springer Science+Business Media B.V. 2011

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6.2 Evidence That CAAs Favor Tumor Progression 6.2.1  In Vitro Experiments There is some in vitro evidence that environmental cues from CAAs affect tumor cell survival, proliferation, differentiation and migration/metastasis. For example, fat cells clearly increase the invasive function of squamous cell carcinoma of the larynx (Yamada et al. 1999). More recently, the relative function of pre-adipocytes and mature adipocytes on various cancer cell lines have been studied by several laboratories, but results remain controversial concerning their efficiency. Mature adipocytes, but not pre-adipocytes, promote the growth of breast cancer cells in collagen gel matrix culture through cancer-stromal cell interactions (Manabe et al. 2003). However, both pre-adipocytes and mature adipocytes have been shown to promote the proliferation of colon cancer cells. Interestingly, the proliferative effects of mature adipocytes do not occur for colon cancer cells from leptin-deficient ob/ob mice, indicating that leptin is, at least partially, responsible for this adipocyte function (Amemori et al. 2007). Moreover, pre-adipocytes show similar effects in wild type mice and ob/ob mice, showing that other factors are also involved (Amemori et al. 2007). Similarly, it was shown that adipocytes favor the proliferation of PC3 prostate cancer cells (Tokuda et al. 2003). Finally, adipocyte-derived secreted factors have been shown to promote breast cancer cell proliferation and invasion (Iyengar et al. 2003). Another study profiling gene expression of breast cancer cells treated with adipocyte culture medium showed induction of immune system- and wound healing-related genes (Kim et al. 2008).

6.2.2  Mouse Models Several years ago, it was shown that an adipocyte-rich environment facilitates SPI murine mammary carcinoma cell growth after subcutaneous injection in mice. In this case, the adipose tissue exerts an estrogen-dependent positive regulatory effect on SPI (Elliott et al. 1992). The impact of the mammary fat pad in tumor development has also been studied (reviewed by (Edwards 2000)). Adipose stromal cells stimulate the migration and invasion of estrogen receptor (ER)-negative breast cancer cells in vitro and tumor invasion in a co-transplant xenograft mouse model. This function is dependent on adipocyte-secreted IL6 (Walter et al. 2009). However, it should be noted that both adipose stromal cells and the mammary fat pad do not include only pre-adipocytes and adipocytes, but also some other cells, most notably endothelial cells, that may interfere. An in vivo coinjection system using 3T3L1 adipocytes and SUM159PT cancer cells recapitulating host-tumor interactions in primary breast tumors have demonstrated that adipocytes favor tumorigenesis (Iyengar et al. 2003).

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Fig. 6.1   CAAs at the invasive front of breast carcinomas. Hematoxylin-eosin histological examination of the invasive front of a human breast primary tumor show CAAs (lipids in white color) exhibiting reduced size at the interface of invading cancer cells. Immunostaining shows that CAAs express MMP11 ( black staining). Magnification 100×

6.2.3  Clinical Data It is obvious that adipocyte-cancer cell heterotypic cross-talk/interaction occurs in invasive carcinomas when both cell types are physically close. Histological data of human carcinomas show that dynamic desmoplastic events continuously occur at the tumor invasive front located at the periphery of primary tumors (Fig. 6.1). Numerous recent clinical studies have evaluated the prognosis value of local adipose tissue invasion by cancer cells at the tumor margin. The majority of data report a positive correlation with poor patient outcome. This is the case for breast (Kimijima et al. 2000; Yamaguchi et al. 2008), prostate (Sung et al. 2006), pancreas (Bandyopadhyay et al. 2009), kidney (Thomas et al. 2003; Cho et al. 2008; Thompson et al. 2007; Bedke et al. 2009) and colon (Puppa et al. 2007). Proteomic analysis of fattissue samples from breast tumors show alterations in the expression of proteins involved in energy metabolism and adipocyte function (Celis et al. 2005). Altogether, these data indicate that adipocyte-cancer cell cross-talk/interaction is a trivial event during the dynamic tumor progression process, and that CAAs play a pejorative role by favoring a more aggressive tumor phenotype.

6.3 CAA Morphology/Structure Major CAA changes are observed in association with cancer cell invasion of adipose tissue (Fig. 6.1). Thus, CAAs at the invasive front are smaller than those observed at a distance. This size reduction implies lipolysis and therefore modification of lipid droplets (also called lipid bodies, fat bodies or adiposomes). Accordingly, the induction of lipolysis in 3T3-L1 adipocytes causes dramatic fission of lipid droplets

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(Brasaemle et al. 2004). Lipid droplets are specialized structures that store neutral lipids (triacylglycerols and sterol esters). They recently emerged as highly dynamic organelles that play crucial roles outside of cellular energy homeostasis and lipid metabolism (Walther and Farese 2009). Indeed, several proteomic analyses have shown that, in addition, lipid droplets contain numerous proteins and might serve as a depot for proteins, to store them for future use or to sequester them until they can be degraded (Cermelli et al. 2006; Welte 2007). Moreover, the key structural lipid droplet proteins vary according to size, with Tip47 and S3–12 localized to the smallest droplets, adipophilin to intermediate droplets, and perilipin to the largest droplets (Wolins et al. 2006). Thus, given the adipocyte-cancer cell interaction, we might hypothesize that in CAAs, lipid droplets undergo fission, leading to protein alteration and/or release, that may possibly play paracrine function(s) on cancer cells.

6.4 CAA and Steroid Hormones CAAs might promote aggressive hormone-dependent cancers (breast, prostate) by increasing hormone synthesis. Indeed, it is established that breast adipose tissue is an important site of estrogen production in post-menopausal women, and elevated estrogen biosynthesis from intratumoral adipose tissue has been demonstrated (Kamat et al. 2002). This is dependent on aromatase, a key enzyme that is involved in peripheral estrogen aromatisation in the adipose tissue. This function has been previously extensively reviewed (O’Neill et  al. 1988; Bulun et  al. 2005; Suzuki et al. 2005; Fischer-Posovszky et al. 2007; Macedo et al. 2009). Some breast cancer cells themselves possess aromatase activity (Sasano et al. 2006). Moreover, several findings indicate that mature human adipocytes possess ERs, and that adipose tissue might be itself an estrogen-responsive tissue (Cooke and Naaz 2004).

6.5 CAA-Derived Growth Factors, Cytokines, Adipokines Cancer cell invasion in vivo involves multiple cell–cell and cell–matrix contacts as well as soluble factors. Adipocytes might locally provide growth support to cancer cells via the secretion of substances that may offer growth/invasion benefits. Indeed, aside from their energy-storing function, adipocytes are also active endocrine cells that produce and secrete various adipokines, growth factors and cytokines (Maeda et al. 1997). The production and secretion patterns of adipose tissue-specific proteins in normal and obesity conditions have been extensivelly studied in human and mouse (Fain et al. 2004; Rabe et al. 2008). A few studies have reported the modification of this production in peritumoral CAAs. These alterations concern several types of proteins, notably pro-inflammatory cytokines (IL6, TNFa), growth factors (IGF1, IGF-BPs), angiogenic factors (VEGF), transcriptional factors (PPARg) and adipokines (leptin and adiponectin; Vona-Davis and Rose 2007). Interestingly, most of these factors have numerous functions, and are interconnected since they often

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regulate each other directly or indirectly. Thus, TNFa is a pleiotropic factor that regulates the synthesis of aromatase expression in the adipose tissue, thus stimulating estrogen production. It also regulates the synthesis of IL6 which also induce aromatase expression. Finally, TNFa induces leptin expression. Similarly, there is increasing evidence that adiponectin and leptin, secreted by peritumoral adipose tissues in several cancers, are important (Rose et  al. 2004; Miyoshi et  al. 2006; Ishikawa et al. 2007; Schaffler et al. 2007). Leptin appears to be a positive factor for tumor development and aggressiveness, while adiponectin protects against cancers. Like TNFa, leptin exerts various functions. In vitro, it can induce cancer cell growth and mediate angiogenesis through VEGF induction. Leptin not only stimulates aromatase expression but also induces the ER pathway independently of estradiol. Furthermore, microarray studies of leptin-regulated gene expression in MCF7 human breast cancer cells suggest that leptin favors mammary tumor growth through multiple mechanisms, including cell cycle and apoptosis (Perera et al. 2008). However, it should be noted that CAA-specific molecular patterns are very difficult to study in vivo since cancer cells themselves aberrantly express and secrete adipocyte-specific proteins. Indeed, it has been shown that high intratumoral levels of leptin mRNA expression in ER-positive breast cancers are specifically involved in the growth stimulation of tumors through an autocrine mechanism (Miyoshi et al. 2006). Collectively, these data show that the potential effects of CAAs on tumor growth and progression might include cell proliferation, differentiation and apoptosis, as well as tumor angiogenesis.

6.6 CAA Microenvironment The adipocyte is a unique cell since it is encapsulated by two membranes, a plasma membrane and a basement membrane. Differentiation is associated with an increase in the secretion of basement membrane components such as laminin, proteoglycans, and type IV and VI collagens. Once cells are committed pre-adipocytes, the original ECM is remodeled, leading to mature lipid-filled adipocytes through proteinase action (Bernlohr et al. 1984; Nakajima et al. 1998; Lilla et al. 2002). Thus, adipogenesis is characterized by the conversion of a fibronectin-rich matrix to a basement membrane (Gregoire 2001; Selvarajan et al. 2001). There is some evidence of modifications of CAA ECM and ECM-related proteins.

6.6.1  MMP-11 Matrix metalloproteinase 11 (MMP-11) (also named stromelysin-3), is ectopically expressed in almost all invasive human carcinomas, and in a majority of their associated metastases (Basset et al. 1990, 1993). High MMP-11 levels are associated with poor patient clinical outcome and MMP-11 is therefore a factor of bad prognosis (Basset et al. 1997; Rio 2005). A MMP-11 tumor promoting effect has been

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demonstrated in several in vivo mouse tumorigenesis models (Masson et al. 1998; Noel et al. 2000; Andarawewa et al. 2003; Deng et al. 2005), and it has been shown that MMP-11 lowers cancer cell death through apoptosis and necrosis (Boulay et al. 2001; Andarawewa et al. 2005; Wu et al. 2001). MMP-11 is not expressed by the cancer cells themselves, but by stromal cells (Basset et al. 1997). More recently, it has been shown that invasive cancer cells induce the expression of MMP-11 in neighboring CAAs (Andarawewa et al. 2005) (Fig. 6.1). The ECM adaptation via the desmoplastic response (Mueller and Fusenig 2004; Tlsty 2001) is associated with a modification in the nature, and relative ratio, of the stromal cells; notably adipocytes disappear and fibroblast-like cells accumulate. It has been proposed that peritumoral fibroblasts might derive, at least partially, from adipocytes and/or preadipocytes through a trans-differentiation process (Bouloumie et al. 2001; Hennighausen and Robinson 2001; Meng et al. 2001). MMP-11, which in vitro negatively regulates adipogenesis by limiting pre-adipocyte differentiation and/or reversing mature adipocyte differentiation, participates in this process (Andarawewa et  al. 2005; Lijnen et al. 2002). Thus, MMP-11 and CAAs participate in a highly complex vicious tumor progression cycle orchestrated by the invasive cancer cells to support tumor progression (Motrescu and Rio 2008).

6.6.2  Collagen VI The extracellular matrix of adipose tissues has received limited attention to date, despite evidence showing that it is a functionally relevant constituent of adipose tissue physiology. Type VI collagen, a soluble ECM, is a specific member of the collagen family exhibiting predominant expression in the adipose tissue. Type VI collagen was shown to be up-regulated in adipocytes during tumorigenesis and to be critical for tumor progression (Iyengar et al. 2003). Indeed, CAA-derived type VI collagen, notably a C-terminal fragment of the alpha3 chain, affects mammary tumor progression in vivo, (Iyengar et al. 2005). Interestingly, it has recently been shown that MMP-11 cleaves the native alpha 3 chain of collagen VI. This MMP-11 collagenolytic activity is functional during fat tissue ontogenesis as well as during cancer invasive steps (Motrescu et al. 2008). These results indicate that CAA-related ECM and associated enzymes play a role during invasive steps in carcinomas, favoring local tumor progression.

6.7 CAA Systemic Effects We have seen above the local effects of CAAs on primary tumors. However, several clinical studies report altered levels of adipocyte-related factors in normal weight patients with invasive tumors. This suggests that local CAA protein expression and secretion might affect the whole individual, and possibly metastatic processes.

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Accordingly, TNFa and IL6 that are expressed by CAAs have been shown to act both locally and in a systemic way (Maccio et  al. 2009). Moreover, circulating levels of leptin increase in both gynecological and breast cancer patients whereas adiponectin levels decreased (Tessitore et al. 2004; Miyoshi et al. 2006; Vona-Davis and Rose 2007). Similar leptin and adiponectin data were reported for prostate cancer (Mistry et al. 2007). Moreover, it has been shown that leptin stimulates the production of sexual hormones. All of these studies show the difficulties in defining the relative importance of local and systemic mechanisms in influencing a range of cancer processes.

6.8 Lessons from CAAs for Elucidating the Pejorative Impact of Obesity on Cancer The data on CAAs call into question the possible similarity between blood modifications observed in cancer patients and those observed in obese people. Plasma analyses of obese people devoid of cancer show alterations in the levels of several factors that are modified in CAAs. Indeed, the cellular homeostasis and the secretory profile of hypertrophied adipocytes from obese people is altered and increasingly dysregulated compared with normal adipocytes (Bluher et  al. 2002; Guilherme et al. 2008). Thus, obesity is associated with an altered cytokine profile, characterized by a reduction in the release of anti-inflammatory cytokines and an increase in the secretion of proinflammatory cytokines. Consequently, obesity is considered an important source of chronic inflammation (Guzik et al. 2006; Hoene and Weigert 2008). Moreover, in obesity, the adipose tissue is infiltrated by an increased number of TNFa- and IL6-secreting macrophages which may be responsible for the increase of estrogen synthesis (Weisberg et al. 2003). An adipose tissue mass increases the circulating concentration of insulin (Pollak 2008). In addition, hyperinsulinemia promotes the synthesis and biological activity of insulin-like growth factor (IGF1) which regulates cell proliferation (Ezzat et al. 2008; Pollak 2008). Insulin also affects the synthesis and the bioavailability of the male and female sex steroids, including androgens, progesterone and estrogens (Fischer-Posovszky et  al. 2007). Leptin is also activated by obesity-related stimuli in adipocytes while adiponectin is decreased (Tanko et al. 2004; Calle and Thun 2004; Rabe et al. 2008). Finally, expanding adipose tissues require local active angiogenesis and neovascularisation (Hausman and Richardson 2004; Guzik et al. 2006). These data all show that part of the local and systemic molecular alterations occurring in adipose tissue of obese people are similar to those occurring in CAAs of invasive tumors, even if the mechanisms regulating their expression is sometimes different. Thus, in normal weight people who develop tumors, factors that favor tumor progression are provided by CAAs, and the stimuli to convert tumor resident adipocytes into CAAs come from invasive cancer cells. It might be hypothesized that obesity provides a “constitutively activated” local and systemic permissive environment for cancer cells.

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6.9 Concluding Remarks and Future Perspectives Collectively, all of these studies strongly support the concept that CAAs favor invasive cancer cell life in connective tissues and therefore tumor progression. CAAs function at both local and systemic levels. The CAA studies provide a molecular basis for a causal link between obesity and cancer. The main conclusions from epidemiological studies is that obesity influences both cancer risk, tumor behavior and clinical outcome (Abu-Abid et al. 2002; Schaffler et al. 2007; Fair and Montgomery 2009). These results raise two salient questions. First, might obesity-related modifications inhibit and/or reverse cancer cell or microtumor dormancy (Demicheli 2001)? Indeed, this would explain the higher risk of cancer development correlated with obesity, since it is difficult to imagine that adipocytes might lead to cell transformation per se. Second, in addition to the fact that obesity makes a hospitable environment for cancer cells and CAAs exacerbate the local reaction, it might be asked if adipocytes are involved locally in metastasis development? Indeed, the microenvironment of preferential organs for secondary tumors (ie: bone, brain, skin) often contains adipocytes (Siclari et al. 2006). This would contribute to explain the worse prognostic values associated with obesity that have been reported. Studies on the metastatic process in normal weight and obese patients should be done to answer this question. Actually, there is great interest in understanding all the mechanisms by which obesity contributes to the carcinogenic process, since obesity is continuously increasing (Kaur and Zhang 2005; Vainio et al. 2002). Improving knowledge of CAA functions might help to explain the relationship between increased BMI, and higher cancer risk and poor clinical outcome. In this context, modulating the plasticity of adipocytes and elucidating the extracellular and intracellular signaling pathways are major challenges for the future research in the field. Molecules linked to adipocyte biology might offer new therapeutic opportunities for treating cancer. Indeed, while each type of cancer is genetically and phenotypically heterogeneous, adipocytes are cells common to all cancers. Now that the adipocyte is no longer the stepchild of cancer biology, many new insights can be expected from its biochemical functions and biological roles in the coming years. Acknowledgements  We thank Susan Chan for helpful discussion. This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Ligue Nationale Française Contre le Cancer and the Comités du Haut-Rhin et du Bas-Rhin, the Fondation de France, the European Commission (FP6 LSHC-CT-2003–503297; Cancer Degradome project), the Institut National du Cancer, Fond National pour la Santé ACI 2004 Cancéropôle Grand-Est, and the “Ruban Rose” prize.

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Yamada S, Toda S, Shin T, Sugihara H (1999) Effects of stromal fibroblasts and fat cells and an environmental factor air exposure on invasion of laryngeal carcinoma (HEp-2) cells in a collagen gel invasion assay system. Arch Otolaryngol Head Neck Surg 125:424–431 Yamaguchi J, Ohtani H, Nakamura K, Shimokawa I, Kanematsu T (2008) Prognostic impact of marginal adipose tissue invasion in ductal carcinoma of the breast. Am J Clin Pathol 130:382– 388

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Part III

The Tumor ECM

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Chapter 7

Hyaluronan: A Key Microenvironmental Mediator of Tumor-Stromal Cell Interactions Naoki Itano

7.1 Tumor Microenvironment and Tumor-Associated Fibroblasts The tumor microenvironment surrounding tumor cells is composed of not only cellular compartments, such as fibroblasts, lymphocytes, macrophages, and endothelial cells, but also of bioactive substances, including growth factors and extracellular matrix (ECM) (Bissell and Radisky 2001). The tumor microenvironment varies both qualitatively and structurally among cancers, and is known to be remodeled in a stepwise fashion during tumor progression. In breast cancer, microenvironmental changes are characterized by formation of stroma containing abundant fibrous components in the intercellular space during the progression from mammary gland hyperplasia to adenoma formation and then to breast cancer (Wiseman and Werb 2002; Petersen et al. 2003). Dynamic changes in tumor stroma include the active recruitment of stromal and inflammatory cells, ECM remodeling, and increased growth factor production (Bhowmick and Moses 2005; Mantovani et  al. 2006). Such remodeling of the tumor microenvironment is known to influence the cancer cells themselves and alter their characteristics (Mueller and Fusenig 2004; Bhowmick et al. 2004). In other words, an interactive loop between cancer cells and the tumor microenvironment is considered to lead to the enhancement of malignant alterations. It thus becomes apparent that a molecular understanding of the tumor microenvironment may lead to the development of new molecular-targeted therapies to block this signaling loop (Joyce 2005). Fibroblasts that are recruited into cancer masses, called tumor-associated fibroblasts (TAFs), are the main cellular components of the stroma of many solid cancers (Kalluri and Zeisberg 2006). TAFs supply a variety of cytokines, growth factors, tissue remodeling enzymes, and ECM components, all of which modulate tumorN. Itano () Department of Molecular Oncology, Division of Molecular and Cellular Biology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, Nagano 390-8621, Japan e-mail: [email protected] M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_7, © Springer Science+Business Media B.V. 2011

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stroma interactions (Kalluri and Zeisberg 2006; Desmouliere et  al. 2004). Clinicopathological studies of various human cancers have reported that an increased amount of stroma and TAF proliferative activity were correlated with cancer malignancy and prognosis (Hasebe et al. 2000; Yazhou et al. 2004). In one report, TAFproduced growth factors, such as HGF, directly and indirectly promoted the proliferation and survival of tumor cells (Matsumoto and Nakamura 2006). TAFs are also known to vigorously produce ECM components, including collagen, proteoglycans, hyaluronan, and matrix metalloproteinase (MMP), thereby involving themselves in the remodeling of the ECM and the generation of a tumor microenvironment favorable for tumor cell proliferation and invasion.

7.2 Hyaluronan Accumulation in Cancers 7.2.1  Hyaluronan Production and Cancer Prognosis Hyaluronan is the main component of the tumor microenvironment and has thus become an increasingly important target for cancer therapy. While hyaluronan is essential for the construction of normal tissue architecture, it is also known as a polysaccharide closely related to cancer (Toole 2004). The malignant transformation of cells frequently impairs regulation of hyaluronan synthesis, which leads to excessive hyaluronan production (Hamerman et al. 1965; Hopwood and Dorfman 1977). The association between a rise in hyaluronan and cancer dates back to the 1950s, when a study first reported increased hyaluronan production in a mesothelioma (Blix 1951; Truedsson 1951). Since then, hyaluronan has been accepted as a significant prognostic factor in many types of cancers, and high levels of this molecule are correlated with a poor prognosis, including overall survival (OS) and disease-free survival (DFS) (Hopwood and Dorfman 1978; Delpech et al. 1990). In several studies, the proportion of hyaluronan-positive cells and intensity of hyaluronan staining in breast cancer tissues were closely correlated with a poor survival rate and prognosis (Auvinen et al. 2000). In colorectal cancers, the 5-year survival rate of patients with a large fraction of hyaluronan-positive cancer cells was reported to be only 20% (Ropponen et al. 1998). Poorly differentiated or high-grade tumors generally have more stromal hyaluronan than well-differentiated tumors; multivariate analyses have confirmed the rate of positive staining for hyaluronan in tumor stroma to be an independent poor prognostic factor. In ovarian cancers, the hyaluronan level in tumor stroma was correlated with the degree of cancer cell undifferentiation and cancer stage, and the 5-year outlook of the disease deteriorated with increasing levels for both OS and DFS (Anttila et al. 2000). The intensity of stromal hyaluronan signaling was also associated with the poor prognosis of breast cancer patients (Auvinen et al. 2000). Thus, hyaluronan, and especially stromal hyaluronan, is an important factor determining the malignant characteristics of cancer that is expected to be more frequently used for the differential diagnosis of cancers.

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7.2.2  B  iological Significance of Hyaluronan Accumulation   in Cancer Tissues As described above, hyaluronan accumulation is associated with degree of clinical malignancy, and, therefore, has been speculated to be closely involved in cancer progression. Qualitative and quantitative changes in hyaluronan ECM are often observed during carcinogenesis and malignant progression. The biological significance of cancer cell-induced ECM remodeling is considered to lie in the formation of an environment appropriate for cancer growth, invasion, and metastasis. Indeed, cell surface hyaluronan-positive mouse melanoma cells were reported to show a significantly higher metastatic frequency than their cell surface hyaluronan-negative counterparts (Zhang et al. 1995). It appears that hyaluronan promotes changes in cell adhesiveness and motility, leading to the detachment of cancer cells from the primary lesion, invasion of the surrounding tissue, and metastasis to distant organs. In addition to this microenvironmental remodeling by the cancer cells themselves, it has been suggested that further alterations are caused by stromal fibroblasts and inflammatory cells, which are recruited into cancer tissues and contribute to the acquisition of malignant characteristics, such as increased cancer cell motility, invasiveness, and tumor angiogenesis.

7.3 Factors Determining the Properties and Functions of Hyaluronan As shown in Fig. 7.1, hyaluronan is a simple polysaccharide molecule composed of repeating disaccharide units of alternating N-acetylglucosamine and glucuronic acid. Despite its relatively simple chemical composition, hyaluronan mediates many important biological functions, including signaling during embryonic morphogenesis, cellular regeneration, and wound healing (Laurent and Fraser 1992). Hyaluronan’s diverse functions are multilaterally regulated by factors like concentration, polysaccharide chain length, and the association state of binding molecules (Itano 2008). High molecular-weight hyaluronan associates with binding molecules to form the ECM that accumulates around cells. This type of hyaluronan ECM is believed to maintain tissue structure and serve as a scaffold for cell adhesion and migration. Alternatively, oligosaccharides resulting from the catabolic activity of hyaluronidase diffuse through the tissue and bind to hyaluronan receptors on adjacent cells, thereby acting as intracellular signals (Stern et al. 2006). In order to comprehensively understand the functions of hyaluronan with its diverse modes of occurrence and association with cancer, it is necessary to first investigate the synthesis and degradation of hyaluronan along with its interaction states with binding molecules. Additionally, several regulatory factors that determine the molecular state of hyaluronan have been identified to date, which have permitted a more detailed understanding of the properties and functions of hyaluronan.

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N. Itano Hyaluronan O

O C

HO

O

CH2OH O

HO O

OH

HO

O NH O C O C O CH3

OH

CH3 O C NH O

O HO

CH2OH

O

Elongating hyaluronan

→ 4GlcA β1→3 GlcNAc β1→ 4 GlcA β1→3 GlcNAc β1→ O

HO

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CH2OH O HO O OH

O O C

NH

Hyaluronidase OH CH3 O C NH

CH3 OH HO

a

O C O

HAS

O

O HO

O CH2OH

b

UDP-GlcA UDP

UDP-GlcNAc UDP

Fig. 7.1   Diagrams illustrating the proposed reaction mechanisms of hyaluronan degradation (a) and synthesis (b). Hyaluronan is composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) β(1→4)-glucuronic acid (GlcA) β(1→3). Hyaluronidase cleaves the N-acetylglucosamine β(1→4) glycoside bonds in hyaluronan. HAS transfers both UDP-GlcNAc and UDP-GlcA substrates to elongating hyaluronan and may secrete the product through a pore made by the enzyme itself along with lipid components of the plasma membrane

7.3.1  Hyaluronan Biosynthesis Due to the peculiarities of the hyaluronan biosynthetic pathway and its mode of sugar chain elongation, the complete mechanism of hyaluronan synthesis remains elusive despite nearly 70 years passing since it was first recognized. However, the identification of hyaluronan synthase (HAS), a glyctabosyltransferase involved in hyaluronan biosynthesis, has led to the elucidation of several molecular mechanisms regulating this process (Weigel et al. 1997). Information on glycosyltransferase reactions has also been steadily accumulating since the advent of biochemical enzyme analysis (Weigel and DeAngelis 2007). Hyaluronan synthesis is catalyzed by one of three isoforms of the HAS enzyme: HAS1, HAS2, and HAS3. These isoforms differ from one another in the length of sugar chains synthesized and ECMforming ability (Itano et al. 1999). Thus, the biological significance of HAS isoforms in animal cells is presumably to synthesize functionally different hyaluronan molecules and modulate their diverse functions. Indeed, expression of each HAS gene is regulated in a different manner by various growth factors and cytokines, resulting in temporally and spatially different expression patterns (Sugiyama et al. 1998; Pienimaki et al. 2001).

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7.3.2  Hyaluronan Catabolism The dynamic turnover of hyaluronan is tightly balanced by its synthesis and degradation to maintain a specific concentration and chain length in tissues. Hyaluronan catabolism is predominantly regulated by several hyaluronidases, which are classified as endo-β-N-acetylglucosaminidases according to their hydrolytic mechanisms (Fig. 7.1). Six structurally homologous hyaluronidase genes (HYAL-1, -2, -3, -4, -P1, and PH-20) have been identified so far in mammals (Coska et al. 2001). Among them, the main tissue hyaluronidases HYAL-1 and -2 regulate hyaluronan degradation in a sequential series of catabolic reactions. The hyaluronidase enzymes differ in optimal pH; HYAL-1, -2, and -3 are acidic hyaluronidases with an optimal pH range of 6.0 or lower, and testicular hyaluronidase PH-20, with a pH optimum of around 7.0, acts as a neutral hyaluronidase. Hyaluronidases also have a different catalytic specificity. For example, whereas HYAL-2 degrades high molecularweight hyaluronan into fragments of up to 20 kDa, HYAL-1 breaks down hyaluronan into fragments of up to 800 Da. Extracellular hyaluronan is known to bind to cell surface receptors, such as CD44, and be internalized into cells by endocytosis. During this process, hyaluronan undergoes one catabolic step by HYAL-2 on cell surfaces or in endosomes. Next, the moderately degraded fragments of hyaluronan undergo further degradation by HYAL-1 in lysosomes (Harada and Takahashi 2007). Hyaluronan fragmentation may also occur through the action of free radicals (Myint et al. 1987). Since free radicals are actively generated under pathological conditions, free radical-mediated hyaluronan degradation may occur in conjunction with hyaluronidase-mediated hyaluronan degradation in subjects with cancer or chronic inflammation.

7.3.3  Hyaluronan-Binding Molecules and Signal Transduction The structure and function of the hyaluronan ECM are complexly regulated through association and combination with specific hyaluronan-binding proteins, such as chondroitin sulfate proteoglycans, link proteins, TSG-6, and inter-α-trypsin inhibitor heavy chain (also called SHAP) (Day and Prestwich 2002). For example, hyaluronan modified by SHAP strengthens the adhesiveness of lymphocytes to hyaluronan ECM via cell surface CD44, thereby promoting their recruitment into inflammatory sites (Zhuo et  al. 2006). The complex signals of various hyaluronan sugar chains are transmitted into cells through hyaluronan receptors, such as CD44 and RHAMM, and dynamically regulate the signal transduction, metabolic reactions, and gene expression required for cell proliferation and migration (Turley et al. 2002). Since CD44 interacts with Src kinase in glycosphingolipid-rich rafts, hyaluronan may activate the Src in rafts to regulate this process (Lee et al. 2008). It is also known that hyaluronan upregulates the Ras-MAP kinase and PI3 kinase-Akt pathways through CD44 to promote cell proliferation and motility.

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7.4 Mode of Occurrence and Functions of Hyaluronan in Cancer Tissues The rates of hyaluronan synthesis and degradation are much higher in cancers than in normal tissues, which produce an increase in hyaluronan ECM and excess low molecular-weight oligosaccharides. Thus, cancer cells are exposed to an extremely unusual environment where the functions of hyaluronan ECM and shorter oligosaccharides occur simultaneously. High molecular-weight hyaluronan exhibits spacefilling and water-retention properties and functions to modulate the pericellular ECM microenvironment Hyaluronan species of different sugar-chain lengths are known to have different biological effects on tumor activity (Table  7.1). For instance, whereas high molecular-weight structural hyaluronan inhibits angiogenesis, shorter oligosaccharides promote vascular endothelial cell proliferation and angiogenesis (Feinberg and Beebe 1983; West et  al. 1985; Slevin et  al. 1998). Recent studies have also reported that hyaluronan oligosaccharides act on monocytes and macrophages to differentiate them into M2 macrophages (Kuang et al. 2007). These macrophages produce Th2 cytokines and several angiogenic factors, which are believed to enhance tumorigenicity through tumor immunosuppression and promotion Table 7.1   Size-dependent functions of hyaluronan Hyaluronan length Biological functions related to cancer (mers or kDa) aggressiveness 4–6-mers Activation of NF-κB and upregulation of MMP expression 4–16-mers Induction of cytokines and chemokines Activation and maturation of DCs Suppression of PI3-kinase/Akt cell survival signals and induction of apoptosis 6–32-mers Stimulation of angiogenesis Proliferation, activation and differentiation of ECs Growth inhibition of B16F10 melanoma Suppression of PI3-kinase/Akt cell survival signals, inhibition of anchorageindependent growth and induction of apoptosis 6–36-mers Induction of CD44 cleavage and promotion of tumor cell motility 10 kDa Stimulation of fibroblast migration 40–800 kDa Induction of proinflammatory cytokines and chemokines in Mφ Induction of iNOS and COX-2 in Mφ Activation of innate immunity

Reference Fieber et al. (2004); Voelcker et al. (2008) McKee et al. (1996); Taylor et al. (2004); Termeer et al. (2002); Alaniz et al. (2006) West et al. (1985); Sattar et al. (1994); Montesano et al. (1996); Deed et al. (1997); Lokeshwar and Selzer (2000); Slevin et al. (1998); Takahashi et al. (2005); Zeng et al. (1998); Ghatak et al. (2002) Sugahara et al. (2003) Tolg et al. (2006) Noble et al. (1996); HodgeDufour et al. (1997); BeckSchimmer et al. (1998); Horton et al. (1998); McKee et al. (1997); Sun et al. (2001); Scheibner et al. (2006)

MMP Matrix metalloprotease, DC Dendritic cell, EC Endothelial cell, Mφ Macrophage

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of angiogenesis. Thus, in advanced cancer, abnormal synthesis and degradation of hyaluronan may facilitate the malignant transformation and survival of cancer cells through tumor-led microenvironmental remodeling characterized by the accumulation of hyaluronan ECM and excessive low molecular-weight hyaluronan production.

7.4.1  Increased Hyaluronan Synthesis in Cancer Cells Increased hyaluronan synthesis in cancer cells is considered to be the main cause of abnormal hyaluronan ECM accumulation. Indeed, infection of cells with an oncogenic virus causes not only abnormal cell proliferation, but also a marked increase in hyaluronan synthesis (Hamerman et  al. 1965; Hopwood and Dorfman 1977). It had long been unclear what mechanism induced excess hyaluronan production associated with malignant cell transformation until we clarified a part of the mechanism by demonstrating that HAS activity and transcription rose after transforming the fibroblast cell line with v-ras or v-src oncogenes (Itano et al. 2004). Similarly, studies using several cancer cell lines reported that HAS expression was increased in cancer cells whose ability to produce hyaluronan was enhanced (Simpson et al. 2001; Bullard et al. 2003). Furthermore, amplification of the genomic region that includes the HAS2 gene has also been found with high frequency in prostate cancer (Tsuchiya et al. 2002). Although not directly proven, HAS gene amplification may be directly linked to increased hyaluronan synthesis in cancer cells. These results suggest that abnormal hyaluronan accumulation in cancer tissue is the result of transformed cells acquiring a high hyaluronan-producing ability associated with increased HAS expression. Interestingly, some cancer cells, such as human lung and breast cancer cells, produce large amounts of hyaluronan when transplanted subcutaneously or intramuscularly despite exhibiting low levels of hyaluronan production in cultures (Knudson et al. 1989). It has also been reported that co-culture of these cancer cells with fibroblasts results in increased hyaluronan production. Thus, the interaction of cancer cells with TAFs and intratumoral stromal cells seems to be important for the accumulation of this molecule, and growth factors derived from stromal cells likely stimulate hyaluronan production in cancer cells. Indeed, proliferation signals induced by several growth factors such as EGF, TGF-β, PDGF, IGF-1, and FGF enhance hyaluronan synthesis (Heldin et  al. 1989; Suzuki et  al. 1995; Postlethwaite et al. 1989; Jacobson et al. 2000). Increased HAS expression is known to be closely linked to cancer cell proliferation, tumor formation, and malignant alteration (Yabushita et  al. 2004, 2005; Kanomata et al. 2005). Pathological analysis of clinical samples has demonstrated that the increased expression of HAS1 among the three HAS isoforms in human colorectal cancer tissues is correlated with lymph node metastasis (Yamada et al. 2004). Another study reported that when increased hyaluronan production was induced by excess HAS2 expression in human HT-1080 sarcoma cells, anchoringdependent cell proliferation and subcutaneous tumor formation were promoted in

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mice (Kosaki et al. 1999). Similar results were obtained when prostate cancer cells were forced to express HAS3 (Liu et al. 2001). In the latter case, the promotion of tumor blood vessel formation was noted as well, suggesting that hyaluronan induced angiogenesis. Analysis of HAS gene expression in prostate cancer cells with different metastatic potentials showed that the expression of HAS2 and 3 genes was increased in cells with a high metastatic potential (Simpson et al. 2002a, b). In this experiment, they found that when low-metastatic cells were transfected with the HAS2 gene to promote hyaluronan production and ECM formation, the adhesion of cancer cells to bone marrow endothelial cells was increased. Conversely, when the expression of HAS2 or HAS3 in high-metastatic cells were decreased by antisense suppression, cancel cell adhesion to endothelial cells was inhibited. Although such a close correlation between HAS gene expression and tumor formation and metastasis has been suggested, exceptions have also been observed. Enegd et al. reported that the forced expression of HAS2 in glioma cells resulted in the suppression of tumor formation, and postulated that a balance between hyaluronan degradation by hyaluronidase and synthesis by HAS was responsible for tumor formation (Enegd et  al. 2002). We also investigated the correlation between HAS2 expression and tumor formation using ras-transformed fibroblasts, and were able to determine the optimal level of HAS2 expression for tumor development (Itano et al. 2004). These observations therefore suggest that hyaluronan promotes tumor progression at a specific concentration range and sugar-chain length, but inhibits tumor formation at other concentrations or sugar-chain lengths, which is to say that the malignant characteristics of tumor cells are regulated by the fine balance between hyaluronan synthesis and degradation under the control of several enzymes.

7.4.2  E  ffects of Hyaluronan Oligosaccharides on Tumor Formation Since the time of its cloning, the expression of the hyaluronidase gene has been examined in detail in numerous cancer tissues and cell lines (Lokeshwar et al. 2001; Franzmann et al. 2003; Christopoulos et al. 2006). Pathological analysis of clinical samples has shown that HYAL-1 expression is a good prognostic indicator of prostate and bladder cancers (Lokeshwar et al. 2001), while the analysis of breast cancer cell lines has revealed that highly invasive cancer cells express high levels of HYAL-2 (Udabage et al. 2005). The association between hyaluronidase and tumor formation has been directly confirmed by the fact that HYAL-2 overexpression in mouse astrocytoma promotes the formation of tumors (Novak et al. 1999). On the other hand, similarly to HAS, hyaluronidase exhibited a completely opposite effect in some cases; a human breast cancer transplanted into immunodeficient (SCID) mice markedly regressed after intravenous administration of testicular hyaluronidase (Shuster et al. 2002), and HYAL-1 overexpression in a colorectal cancer model inhibited tumor formation (Lokeshwar et al. 2005). These conflicting results support the notion of a delicate balance between hyaluronan degradation and synthesis in tumor development.

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7.4.3  Metabolism and Function of Stromal Hyaluronan In addition to hyaluronan-synthesizing cancer cells, TAFs and other stromal cells are considered to contribute significantly to the intratumoral accumulation of hyaluronan. In cancer tissues, fibroblasts are activated by exposure to various growth factors and cytokines, resulting in a marked increase in hyaluronan synthesis. As TGF-β can up-regulate HAS gene expression, it may be responsible for the stimulation of hyaluronan synthesis in fibroblasts. Although tumor stromal cells are considered to be non-transformed cells, fibroblasts adjacent to cancer cells exhibit a loss of heterozygosity (LOH) with high frequency (Kurose et al. 2002), suggesting that they carry genetic changes. Therefore, not only active hyaluronan production by normal fibroblasts, but also abnormal hyaluronan synthesis by genetically altered fibroblasts, may help explain the excessive accumulation of hyaluronan in tumor stroma. In prostate cancer, decreased hyaluronidase activity along with decreased hyaluronan degradation in fibroblasts has also been shown to be a cause of hyaluronan accumulation in tumor stroma (Lokeshwar et al. 2001). Considerable information has been accumulated on the function of stromal hyaluronan in cancer progression. Recently, Calabro et  al. investigated the synthesis of hyaluronan in bone marrow mesenchymal progenitor cells (bmMPCs) using bone marrow from myeloma patients, and reported that bmPMCs produced hyaluronan levels about six times higher than those from healthy donors (Calabro et al. 2002). Therefore, bone marrow stromal ECM that is rich in hyaluronan may be important for the expression of malignant characteristics in myeloma cells. Since the functions of hyaluronan are regulated in a complex manner by its molecular weight and associated molecules, hyaluronan species produced by cancer cells and TAFs may have specific individual properties and functions that differentially contribute to cancer progression. However, it remains to be fully clarified whether or not hyaluronan species exert different actions due to the lack of an appropriate experimental system.

7.5 Stromal Formation, Angiogenesis, and Lymphangiogenesis 7.5.1  I nduction of the Tumor Stroma by Hyaluronan Overproduction The proceeding discussion focuses on the complex functions of tumoral and stromal hyaluronan in cancer progression based on findings revealed by analysis of genetically modified mice. In recent years, our laboratory has successfully generated a transgenic mouse model overproducing hyaluronan in breast cancer cells to analyze the actions of tumoral hyaluronan, and we have found that the rate of tumor development and proliferation increases rapidly in hyaluronan-overproducing mice compared with a control group (Koyama et  al. 2007). Histological examination revealed breast cancer formation characterized by the induction of tumor stroma

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and marked recruitment of TAFs into tumor tissues. Very interestingly, numerous blood and lymphatic vessels were observed in or adjacent to tumor stroma (Koyama et al. 2007, 2008), which probably occurred in the context of tumoral hyaluronaninduced tumor stroma formation. We have thus far stated that the main cellular component of the tumor stroma, TAFs, promote tumor formation and progression as a modulator of the tumor microenvironment, and that hyaluronan is important in the recruitment of TAFs into tumor tissue. At the time of writing, the mechanism by which tumor-derived hyaluronan recruits TAFs to enhance tumor stroma formation remains unclear. However, two mechanisms are conceivable: (1) The extracellular accumulation of hyaluronan with its excellent water-retaining and space-filling properties is favorable for the migration of TAFs, and (2) Hyaluronan interacts with cell surface receptors, such as CD44 and RHAMM, to activate intracellular signals and promote stromal cell motility. TAFs are considered to originate both from fibroblasts migrating from surrounding tissue and from hyaluronan-recruited mesenchymal stem cells (MSCs) (Zhu et al. 2006). In addition, hyaluronan overproduction is known to promote epithelial-mesenchymal transition (EMT) (Zoltan-Jones et al. 2003), suggesting that the hyaluronan-dependent EMT of cancer cells also functions as a mechanism of TAF supply.

7.5.2  T  he Role of a Hyaluronan-Rich Tumor Microenvironment in Tumor Angiogenesis HAS overexpression in cancer cells has been reported to induce angiogenesis in xenografted tumors, and the inhibition of hyaluronan synthesis was found to markedly reduce the density of tumor blood vessels (Liu et al. 2001; Simpson et al. 2002b). Our findings with animal models have confirmed that hyaluronan overproduction markedly promotes tumor angiogenesis (Koyama et al. 2007), but excessive hyaluronan accumulation was seen to inhibit tumor angiogenesis in prostate cancer in another study (Bharadwaj et  al. 2007). These conflicting results indicate that the capacity for hyaluronan to promote or inhibit tumor angiogenesis may depend on its state. For many years, low molecular-weight hyaluronan was known to promote vascular endothelial cell proliferation and induce angiogenesis in vivo (Feinberg and Beebe 1983; West et al. 1985; Slevin et al. 1998). However, only shorter hyaluronan oligosaccharides promote tumor angiogenesis, whereas high molecularweight hyaluronan has the opposite effect (Table 7.1). In cancer tissues, the angiogenic inhibitor high molecular-weight hyaluronan and the angiogenic promoter low molecular-weight hyaluronan coexist, suggesting the importance of their balance in angiogenic processes. Interestingly, our studies have shown that, similarly to low molecular-weight hyaluronan, high molecular-weight hyaluronan may actually promote angiogenesis when associated with versican, a chondroitin sulfate proteoglycan, to form a complex (Koyama et al. 2007). In an in vivo experimental model for angiogenesis

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using Matrigel plug assays, the hyaluronan-versican complex is found to actively recruits fibroblasts and vascular endothelial cells into subcutaneously transplanted Matrigels. Since angiogenic processes are regulated by aberrant production of angiogenic factors, it was speculated that the hyaluronan-versican complex indirectly promotes angiogenesis through fibroblast-produced angiogenic factors. Indeed, in the above-described experiments with hyaluronan-overproducing breast cancer, numerous microvessels were present adjacent to tumor stroma containing abundant hyaluronan and versican. Analysis of angiogenic factor expression further revealed increases in the expression of stroma-derived factors, such as b-FGF and SDF-1 chemokines, in hyaluronan-producing tumors. The promotion of angiogenesis by stromal cell-derived factors was also clearly illustrated by another experiment using a mouse embryonic fibroblast (MEF) cell line established from vascular endothelial growth factor (VEGF)-deficient mice. Dong et al. demonstrated that fewer blood vessels were formed when cancer cells were transplanted subcutaneously in the co-presence of VEGF-deficient MEFs compared with wild-type cells (Dong et al. 2004). Furthermore, Orimo et al. reported that TAFs established from human breast cancers recruited vascular endothelial progenitor cells and promoted tumor angiogenesis in xenografted tumors through production of SDF-1 (Orimo et al. 2005). Along with our results, these studies provide compelling evidence that stromal cellderived angiogenic factors are crucial for tumor angiogenesis. Since monocytes and macrophages are the main sources of angiogenic factors in cancer tissue, the involvement of inflammatory cells may be important in promoting angiogenesis in hyaluronan-positive tumors (Lewis et al. 2006). De la Motte et al. showed that hyaluronan and its associated molecules formed cable-like ECM structures that were involved in the adhesion, and presumably recruitment, of monocytes through interactions with CD44 (de la Motte et al. 1999). Furthermore, Kuang et al. found that hyaluronan fragments differentiate monocytes into tumorassociated macrophages (TAMs), which are reportedly M2-type macrophages that actively produce angiogenic factors (Kuang et al. 2007). Thus, the hyaluronan-rich tumor microenvironment recruits effector cells, such as TAFs and TAMs, into tumor tissue, then promotes angiogenesis by inducing production of angiogenic factors (Fig. 7.2).

7.5.3  H  yaluronan-Dependent Promotion of Tumor Iymphangiogenesis Lymphangiogenesis has recently gained attention for its potential involvement in cancer dissemination and metastasis (Alitalo et al. 2005). Since lymphatic vessels in the early stages of carcinogenesis are localized in the peritumoral stroma of several human solid cancers (Leu et al. 2000; Padera et al. 2002; Williams et al. 2003), lymphangiogenesis is likely governed by complex interactions between tumor cells and stroma-based mediators. In a mouse model of hyaluronan overproduction, we have shown that lymphangiogenesis occurs in the context of tumor stroma

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3

Tumor cells HMW & LMW HA

Lymphatic vessels

2

Blood vessels

TAMs

EPCs

ECs

TAFs

Fig. 7.2   Hyaluronan mediates host-tumor cell interactions in the tumor microenvironment. During tumor progression, high-molecular-weight (HMW) hyaluronan forms part of the tumor microenvironment by linking its binding partners into macromolecular aggregates. The hyaluronan-rich tumor microenvironment accelerates the recruitment of TAFs and TAMs. The complex cellular interactions between tumor cells and stromal cells accelerate tumor angiogenesis and lymphangiogenesis by stimulating infiltration of endothelial cells (ECs) and recruitment of bone marrowderived endothelial progenitor cells (EPCs). Low-molecular-weight (LMW) hyaluronan generated by hyaluronan fragmentation simultaneously accelerates tumor angiogenesis

(Koyama et  al. 2008). TAFs were found to give rise to highly lymphatic tumors when transplanted subcutaneously with cancer cells established from breast cancer tissue, indicating that TAFs are important cellular components for tumor lymphangiogenesis. Thus, a hyaluronan-rich tumor microenvironment also seems to promote lymphangiogenesis through the recruitment of TAFs. Recent studies have revealed that stromal cells in cancer tissue promote lymphangiogenesis by producing lymphangiogenic factors. We have shown that TAFs produce lymphangiogenic factors, such as VEGF-C and -D (Koyama et al. 2008), suggesting that the increased production of such stroma-derived factors may result in increased lymphangiogenesis. Furthermore, Schoppmann et al. reported that monocytes recruited into tumor stroma differentiate into TAMs following exposure to active substances to actively produce VEGF-C and -D (Schoppmann et al. 2002). Since hyaluronan promotes the differentiation of monocytes into TAMs, it may be involved in a mechanism that promotes lymphangiogenesis in tumors. Lastly, it is possible that, in addition to the action of humoral factors, the direct interactions between hyaluronan-rich ECM and lymphatic endothelial cell-surface

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receptors contribute to lymphangiogenesis. LYVE-1, which contains a hyaluronanbinding domain, is expressed on the surface of lymphatic endothelial cells and was initially speculated to play an important role in lymphangiogenesis as a hyaluronan receptor on lymphatic endothelial cells (Jackson et al. 2001). However, an analysis of LYVE-1-deficient mice showed no gross abnormalities in lymphatic formation, and these mice did not differ from wild-type mice in their level of pathological lymphangiogenesis (Gale et  al. 2007). In the future, it should be investigated in more detail whether hyaluronan-rich ECM promotes lymphangiogenesis through reaction mechanisms with or without mediation by other receptors.

7.6 Conclusions and Future Prospects In light of recent discoveries, the formation and restructuring of the “hyaluronanrich tumor microenvironment” seems to be the main cause of cancer development and progression. The analysis of cancer cells and genetically-altered mouse models is steadily advancing, and accumulating lines of evidence are suggesting that hyaluronan overproduction in cancer cells and tumor stroma is a key event in the acquisition of malignant characteristics. Cancer tissue is composed of cancer cells and recruited host stromal cells, such as TAFs, and the microenvironment created by the interactions of these cells is closely associated with tumor proliferation and angiogenesis. In addition, the hyaluronan-rich tumor microenvironment appears to promote angiogenesis and lymphangiogenesis through tumor-stromal cell interaction, thereby synergistically accelerating tumor progression. It is our hope that further elucidation of the detailed mechanisms by which hyaluronan promotes cancer progression will lead to the development of new therapeutic strategies. Acknowledgements  I am grateful for Dr Shun’ichiro Taniguchi (Shinshu University Graduate School of Medicine) for his support. This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science.

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Chapter 8

Function of Tenascins in the Tumor Stroma Florence Brellier and Ruth Chiquet-Ehrismann

8.1 Introduction In 1983, Bourdon et  al. (1983) first described glioma-mesenchymal extracellular matrix (GMEM) antigen. Shortly thereafter it became clear that GMEM antigen, now known as tenascin-C, was expressed not only in brain tumors but also in rat mammary tumors (Chiquet-Ehrismann et al. 1986) and in human breast cancer (Mackie et al. 1987). Work that followed demonstrated that tenascin-C was, in fact, overexpressed in almost all solid tumors (for review see Orend and Chiquet-Ehrismann 2006). In addition to tenascin-C there are three other proteins that belong to the tenascin family: tenascin-R, tenascin-X and tenascin-W (for review see Chiquet-Ehrismann 2004; Tucker et al. 2006). The four tenascins fall into two groups that can be distinguished from each other by their patterns of expression. The first group includes tenascin-R and tenascin-X, which have stabile, restricted expression patterns and are present both in developing and in adult tissues. In contrast, tenascin-C and tenascin-W, making up the second group, are mostly absent in the adult organism, are regulated by the tissue microenvironment, and are highly induced in cancer stroma (Tucker and Chiquet-Ehrismann 2009). Therefore, this chapter will summarize the evidence for an important role of these two tenascins, tenascin-C and tenascin-W, in tumor stroma as well as their role in cancer progression.

8.2 Structure, Regulation and Interactions of Tenascins All tenascins are large oligomeric extracellular matrix glycoproteins built from four different building blocks. This is depicted in Fig.  8.1 for human tenascin-C and tenascin-W. The building blocks include an N-terminal oligomerization domain consisting of 3–4 heptad repeats for trimerization flanked by disulphide bridges R. Chiquet-Ehrismann () Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Maulbeerstrasse 66, 4058 Basel, Switzerland e-mail: [email protected] M. M. Mueller, N. E. Fusenig (eds.), Tumor-Associated Fibroblasts and their Matrix, The Tumor Microenvironment 4, DOI 10.1007/978-94-007-0659-0_8, © Springer Science+Business Media B.V. 2011

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c Oligomerization domain EGF-like repeat Fibronectin type 3 domain Fibrinogen-related domain

Fig. 8.1   Domain structure of tenascins. a Each tenascin subunit is built up from several different domains. In the case of human tenascin-C there are 8 constant fibronectin type 3 domains and 9 additional ones (shown below the bracket) that can be included or excluded in the mature protein in many different combinations. Tenascin-W exists as a single version of the protein. b Tenascin-C as well as tenascin-W assemble as hexameric proteins by oligomerization including covalent disulfide crosslinks in the central oligomerization domain. c Legend for the building blocks used to create the protein models

covalently crosslinking the three subunits. Furthermore, an N-terminal extension includes additional cysteines that lead to dimerization of the trimers as shown in Fig. 8.1b. This N-terminal region is followed by a series of epidermal growth factorlike repeats (EGF), fibronectin type 3 domains (FN3) and a C-terminal fibrinogenrelated domain (FReD). The number of EGF-repeats ranges from 3 in tenascin-W to 14 in tenascin-C. The number of FN3 domains also differs, with 9 FN3 domains in tenascin-W to 8 constant FN3 domains in tenascin-C where an additional 9 FN3 domains can be included based on alternative splicing. Thus, there exist many different splice variants of tenascin-C that can have different functions (see below) while tenascin-W exists as a single version of the protein.

8.2.1  Tenascin-C The chromosomal location of the tenascin-C gene is 9q33.1 (Degen and ChiquetEhrismann 2008a). Interestingly, this locus has recently been implicated in pro-

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gression of childhood ependymomas (Puget et al. 2009). Using a candidate gene strategy the authors found overexpression of two potential oncogenes, namely tenascin-C and Notch1. This is interesting, since it is known that Notch signaling induces tenascin-C expression (Sivasankaran et al. 2009). There exist many other signaling pathways that induce tenascin-C expression. These signaling pathways are initiated by the action of transforming growth factor-beta, fibroblast growth factors, platelet-derived growth factors, tumor necrosis factor-alpha or even by mechanical strain (for a recent review see Tucker and Chiquet-Ehrismann 2009). Once tenascin-C is expressed it can interact with cell surface receptors and other extracellular matrix proteins triggering cellular responses such as cell proliferation, de-adhesion and migration. One of the most important binding partners of tenascin-C that greatly influences cell physiology is fibronectin (Chiquet-Ehrismann et al. 1991). Mixed substates of fibronectin and tenascin-C can induce collagenase expression in fibroblasts (Tremble et al. 1994) as well as inhibit cell spreading by suppression of Rho activation (Wenk et  al. 2000) through blocking access of the transmembrane proteoglycan syndecan-4 to fibronectin (Huang et al. 2001; Midwood et al. 2004). Other ligands of tenascin-C are the proteoglycans neurocan and phosphacan, the latter being the extracellular domain of protein-tyrosine phosphatase-zeta/beta (Milev et  al. 1994). Furthermore, several integrins have been implicated in binding to tenascinC (Ramos et  al. 1997; Sriramarao et  al. 1993; Yokosaki et  al. 1998; Yokoyama et  al. 2000). Finally, toll-like receptor-4 (TLR4) has recently been shown to be activated by tenascin-C, inducing a signaling cascade that results in the secretion of inflammary cytokines. The FReD domain of tenascin-C was shown to be responsible for this activity (Midwood et al. 2009). In this study it was found that the presence of tenascin-C is a crucial factor mediating persistent inflammation and tissue destruction in a mouse model of arthritic joint disease. Since tumors can be seen as over-healing wounds with chronic inflammation (Schafer and Werner 2008) we speculate that stromal tenascin-C could contribute to this type of derailment in cancer.

8.2.2  Tenascin-W The gene encoding human tenascin-W is located on chromosome 1q25.1 in a tailto-tail configuration next to the tenascin-R gene (Degen and Chiquet-Ehrismann 2008b). Tenascin-W is a tightly regulated protein with even more restricted expression than tenascin-C. Its major site of expression is during bone development (Scherberich et al. 2004). Tenascin-W expression can be regulated by various factors such as bone morphogenic protein (BMP) 2 and tumor necrosis factor-alpha (Scherberich et  al. 2005). It is similarly deposited in the extracellular matrix as tenascin-C, but its interaction partners remain to be identified.

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8.3 Localization of Tenascins in Tumors 8.3.1  Tenascin-C In adult healthy tissues, tenascin-C expression is low and is mainly restricted to tendons, ligaments, and some smooth muscles. In contrast, tumors arising in many different epithelial organs such as breast, colon, prostate, kidney, pancreas, lung, skin, bladder, uterus and ovaries often express high levels of tenascin-C (for a recent review see Orend and Chiquet-Ehrismann 2006). In most epithelial cancers, the cellular source of tenascin-C is not the tumor cells themselves, but rather tumorassociated fibroblasts residing in the tumor stroma. Immunohistochemical analyses of these tumors usually reveal a fibrous network of tenascin-C enclosing unstained tumor nests. Examples of this in breast and colon carcinomas are shown in Fig. 8.2. At the invasive front, where tumor cells are not encapsulated anymore but leave the nests to invade the neighboring normal tissue, tenascin-C is often highly expressed suggesting a link between tenascin-C and tumor aggressiveness (Jahkola et al. 1996). Consistent with this observation, a majority of the studies report a correlation between expression of tenascin-C and bad prognosis (summarize