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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References Anastasiadis AG, Bemis DL, Stisser BC, Salomon L, Ghafar MA, Buttyan R (2003) Tumor cell hypoxia and the hypoxia-response signaling system as a target for prostate cancer therapy. Curr Drug Targets 4(3):191–196 Araujo J, Logothetis C (2009) Targeting Src signaling in metastatic bone disease. Int J Cancer 124(1):1–6 Armant DR (2005) Blastocysts don’t go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells. Dev Biol 280(2):260–280 Azad MB, Chen Y, Gibson SB (2009) Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid Redox Signal 11(4):777–790 Bander NH et  al (2005) Phase I trial of 177-lutetium-labeled J591, a monoclonal antibody to prostate-specific membrane antigen, in patients with androgen-independent prostate cancer. J Clin Oncol 23:4591–4601 Birchmeier C, Birchmeier W, Brand-Saberi B (1996) Epithelial-mesenchymal transitions in cancer progression. Acta Anat (Basel) 156(3):217–226 Bonaccorsi L, Muratori M, Marchiani S, Forti G, Baldi E (2006) The androgen receptor and prostate cancer invasion. Mol Cell Endocrinol 246(1–2):157–162 Buckle CH, Neville-Webbe HL, Croucher PI, Lawson MA (2010) Targeting RANK/RANKL in the treatment of solid tumours and myeloma. Curr Pharm Des 16(11):1272–1283 Chantrain CF, Feron O, Marbaix E, Declerck YA (2008) Bone marrow microenvironment and tumor progression. Cancer Microenviron 1(1):23–35 Chiarugi P, Giannoni E (2005) Anchorage-dependent cell growth: tyrosine kinases and phosphatases meet redox regulation. Antioxid Redox Signal 7(5–6):578–592 Cho HJ, Lee CS, Kwon YW et al (2010) Induction of pluripotent stem cells from adult somatic cells by protein-based reprogramming without genetic manipulation. Blood 116(3):386–395 Chung LW (1993) Implications of stromal-epithelial interaction in human prostate cancer growth, progression and differentiation. Semin Cancer Biol 4(3):183–192 Chung LW (1995) The role of stromal-epithelial interaction in normal and malignant growth. Cancer Surv 23:33–42 Chung LW, Baseman A, Assikis V, Zhau HE (2005) Molecular insights into prostate cancer progression: the missing link of tumor microenvironment. J Urol 173(1):10–20 Chung LW, Huang WC, Sung SY et  al (2006) Stromal-epithelial interaction in prostate cancer progression. Clin Genitourin Cancer 5(2):162–170 Cooper CR, Chay CH, Gendernalik JD et al (2003) Stromal factors involved in prostate carcinoma metastasis to bone. Cancer 97(Suppl 3):739–747 Cress AE, Mohla S (2004) Therapeutic targeting of prostate cancer. Cancer Biol Ther 3(10):1028– 1030 Cushing MC, Mariner PD, Liao JT, Sims EA, Anseth KS (2008) Fibroblast growth factor represses Smad-mediated myofibroblast activation in aortic valvular interstitial cells. Faseb J 22(6):1769–1777 Dakhova O, Ozen M, Creighton CJ et al (2009) Global gene expression analysis of reactive stroma in prostate cancer. Clin Cancer Res 15(12):3979–3989 David KA et al (2006) Clinical utility of radiolabeled antibodies in prostate cancer. Clin Genitourin Cancer 4:249–256 Desgrosellier JS, Cheresh DA (2010) Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 10(1):9–22 Franco OE, Shaw AK, Strand DW, Hayward SW (2010) Cancer associated fibroblasts in cancer pathogenesis. Semin Cell Dev Biol 21(1):33–39 Friedman R, Gelfand T, Weiss DW, Doljanski F (1984) Patterns of fibronectin deposition in normal and neoplastic fibroblasts and mammary tissue. Int J Tissue React 6(4):291–301 Garber K (2005) IGF-1: old growth factor shines as new drug target. J Natl Cancer Inst 97(11):790– 792

16

L. W. K. Chung

Garber K (2009) Companies waver in efforts to target transforming growth factor in cancer. J Natl Cancer Inst 101(24):1664–1667 Garin-Chesa P, Old LJ, Rettig WJ (1990) Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc Natl Acad Sci U S A 87(18):7235–7239 Gibbs JB (2000) Anticancer drug targets: growth factors and growth factor signaling. J Clin Invest 105(1):9–13 Hill R, Song Y, Cardiff RD, Van Dyke T (2005) Selective evolution of stromal mesenchyme with p53 loss in response to epithelial tumorigenesis. Cell 123(6):1001–1011 Hsieh CL, Kubo H, Chung LW (2004) Gene therapy for prostate cancer bone metastasis. Gene therapy targeting bone metastasis. Cancer Treat Res 118:231–290 Huang WC, Wu D, Xie Z et al (2006) Beta2-microglobulin is a signaling and growth-promoting factor for human prostate cancer bone metastasis. Cancer Res 66(18):9108–9116 Hugo H, Ackland ML, Blick T et al (2007) Epithelial–mesenchymal and mesenchymal–epithelial transitions in carcinoma progression. J Cell Physiol 213(2):374–383 Ingber DE (2008) Can cancer be reversed by engineering the tumor microenvironment? Semin Cancer Biol 18(5):356–364 Ishikawa K, Koshikawa N, Takenaga K, Nakada K, Hayashi J (2008) Reversible regulation of metastasis by ROS-generating mtDNA mutations. Mitochondrion 8(4):339–344 Jaganathan BG, Ruester B, Dressel L et al (2007) Rho inhibition induces migration of mesenchymal stromal cells. Stem Cells 25(8):1966–1974 Jiang YG, Luo Y, He DL et al (2007) Role of Wnt/beta-catenin signaling pathway in epithelialmesenchymal transition of human prostate cancer induced by hypoxia-inducible factor-1alpha. Int J Urol 14(11):1034–1039 Josson S, Matsuoka Y, Chung LW, Zhau HE, Wang, R (2010) Tumor-stroma co-evolution in prostate cancer progression and metastasis. Semin Cell Dev Biol 21(1):26–32 Jung T, Castellana D, Klingbeil P et al (2009) CD44v6 dependence of premetastatic niche preparation by exosomes. Neoplasia 11(10):1093–1105 Kabashima A, Higuchi H, Takaishi H et al (2009) Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion. Int J Cancer 124(12):2771–2779 Kaewpila S, Venkataraman S, Buettner GR, Oberley LW (2008) Manganese superoxide dismutase modulates hypoxia-inducible factor-1 alpha induction via superoxide. Cancer Res 68(8):2781– 2788 Kang BH, Altieri DC (2009) Compartmentalized cancer drug discovery targeting mitochondrial Hsp90 chaperones. Oncogene 28(42):3681–3688 Kaplan RN, Riba RD, Zacharoulis S et al (2005) VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438(7069):820–827 Kaplan RN, Psaila B, Lyden D (2007) Niche-to-niche migration of bone-marrow-derived cells. Trends Mol Med 13(2):72–81 Kargozaran H, Yuan SY, Breslin JW et al (2007) A role for endothelial-derived matrix metalloproteinase-2 in breast cancer cell transmigration across the endothelial-basement membrane barrier. Clin Exp Metastasis 24(7):495–502 Karnoub AE, Dash AB, Vo AP et al (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449(7162):557–563 Keller ET (2002) The role of osteoclastic activity in prostate cancer skeletal metastases. Drugs Today (Barc) 38(2):91–102 Kennard S, Liu H, Lilly B (2008) Transforming growth factor-beta (TGF-1) down-regulates Notch3 in fibroblasts to pro0mote smooth muscle gene expression. J Biol Chem 283(3):1324–1333 Kikuta K, Masamune A, Satoh M, Suzuki N, Satoh K, Shimosegawa T (2006) Hydrogen peroxide activates activator protein-1 and mitogen-activated protein kinases in pancreatic stellate cells. Mol Cell Biochem 291(1–2):11–20 Klymkowsky MW, Savagner P (2009) Epithelial-mesenchymal transition: a cancer researcher’s conceptual friend and foe. Am J Pathol 174(5):1588–1593

1  Critical Roles of Stromal Fibroblasts in the Cancer Microenvironments

17

Kopetz ES, Nelson JB, Carducci MA (2002) Endothelin-1 as a target for therapeutic intervention in prostate cancer. Invest New Drugs 20(2):173–182 Kubo H, Gardner TA, Wada Y et al (2003) Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther 14(3):227–241 Lalich M et al (2007) Endothelin receptor antagonists in cancer therapy. Cancer Invest 25(8):785– 794 Leber MF, Efferth T (2009) Molecular principles of cancer invasion and metastasis (review). Int J Oncol 34(4):881–895 Liegibel UM, Sommer U, Tomakidi P et al (2002) Concerted action of androgens and mechanical strain shifts bone metabolism from high turnover into an osteoanabolic mode. J Exp Med 196(10):1387–1392 Malins DC, Anderson KM, Jaruga P et  al (2006) Oxidative changes in the DNA of stroma and epithelium from the female breast: potential implications for breast cancer. Cell Cycle 5(15):1629–1632 Martin FT, Dwyer RM, Kelly J et  al (2010) Potential role of mesenchymal stem cells (MSCs) in the breast tumour microenvironment: stimulation of epithelial to mesenchymal transition (EMT). Breast Cancer Res Treat 124:317–326 McKeithen D, Graham T, Chung LW, Odero-Marah V (2010) Snail transcription factor regulates neuroendocrine differentiation in LNCaP prostate cancer cells. Prostate 70(9):982–992 McNeal JE (1978) Origin and evolution of benign prostatic enlargement. Invest Urol 15(4):340– 345 Milowsky MI et al (2004) Phase I trial of 90Y-Labeled anti-PSMA monoclonal antibody J591 for androgen-independent prostate cancer. J Clin Oncol 22:2522–2531 Mizutani K, Sud S, McGregor NA et al (2009) The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment. Neoplasia 11(11):1235–1242 Moen I, Oyan AM, Kalland KH et al (2009) Hyperoxic treatment induces mesenchymal-to-epithelial transition in a rat adenocarcinoma model. PLoS One 4(7):e6381 Molloy AP, Martin FT, Dwyer RM et al (2009) Mesenchymal stem cell secretion of chemokines during differentiation into osteoblasts, and their potential role in mediating interactions with breast cancer cells. Int J Cancer 124(2):326–332 Mueller MM, Fusenig NE (2004) Friends or foes—bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 4(11):839–849 Nilsson S, Larsen RH, Fossa SD et  al (2005) First clinical experience with alpha-emitting radium-223 in the treatment of skeletal metastases. Clin Cancer Res 11(12):4451–4459 Nilsson S, Franzen L, Parker C et al (2007) Bone-targeted radium-223 in symptomatic, hormonerefractory prostate cancer: a randomised, multicentre, placebo-controlled phase II study. Lancet Oncol 8(7):587–594 Okita K, Ichisaka T, Yamanaka, S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448(7151):313–317 Olapade-Olaopa EO, MacKay EH, Taub NA, Sandhu DP, Terry TR, Habib FK (1999) Malignant transformation of human prostatic epithelium is associated with the loss of androgen receptor immunoreactivity in the surrounding stroma. Clin Cancer Res 5(3):569–576 Olaso E, Salado C, Egilegor E et al (2003) Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology 37(3):674–685 Park SM, Gaur AB, Lengyel E, Peter ME (2008) The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 22(7):894–907 Pathak S, Nemeth MA, Multani AS, Thalmann GN, von Eschenbach AC, Chung LW (1997) Can cancer cells transform normal host cells into malignant cells? Br J Cancer 76(9):1134–1138 Pollard JW (2009) Trophic macrophages in development and disease. Nat Rev Immunol 9(4):259– 270

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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.

References Aarabi S, Bhatt KA, Shi Y, Paterno J, Chang EI, Loh SA, Holmes JW, Longaker MT, Yee H, Gurtner GC (2007) Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis. Faseb J 21:3250–3261 Abe R, Donnelly SC, Peng T, Bucala R, Metz CN (2001) Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 166:7556–7562 Aggarwal BB, Gehlot P (2009) Inflammation and cancer: how friendly is the relationship for cancer patients? Curr Opin Pharmacol 9:351–369 Akhurst RJ, Derynck R (2001) TGF-beta signaling in cancer—a double-edged sword. Trends Cell Biol 11:S44–51 Albini A, Sporn MB (2007) The tumour microenvironment as a target for chemoprevention. Nat Rev Cancer 7:139–147 Amann T, Bataille F, Spruss T, Muhlbauer M, Gabele E, Scholmerich J, Kiefer P, Bosserhoff AK, Hellerbrand C (2009) Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma. Cancer Sci 100:646–653 Andersen ES, Christensen PB, Weis N (2009) Transient elastography for liver fibrosis diagnosis. Eur J Intern Med 20:339–342 Anderson JM, Rodriguez A, Chang DT (2008) Foreign body reaction to biomaterials. Semin Immunol 20:86–100 Andersson-Sjoland A, de Alba CG, Nihlberg K, Becerril C, Ramirez R, Pardo A, WestergrenThorsson G, Selman M (2008) Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol 40:2129–2140 Annes JP, Chen Y, Munger JS, Rifkin DB (2004) Integrin {alpha}V{beta}6-mediated activation of latent TGF-{beta} requires the latent TGF-{beta} binding protein-1. J Cell Biol 165:723–734 Ariyan S, Enriquez R, Krizek TJ (1978) Wound contraction and fibrocontractive disorders. Arch Surg 113:1034–1046 Armulik A, Abramsson A, Betsholtz C (2005) Endothelial/pericyte interactions. Circ Res 97:512– 523 Atiyeh BS, Costagliola M, Hayek SN (2005) Keloid or hypertrophic scar: the controversy: review of the literature. Ann Plast Surg 54:676–680 Bataller R, Brenner DA (2005) Liver fibrosis. J Clin Invest 115:209–218 Baudino TA, Carver W, Giles W, Borg TK (2006) Cardiac fibroblasts: friend or foe? Am J Physiol Heart Circ Physiol 291:H1015–1026 Beacham DA, Cukierman E (2005) Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol 15:329–341 Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313 Begley LA, Kasina S, MacDonald J, Macoska JA (2008) The inflammatory microenvironment of the aging prostate facilitates cellular proliferation and hypertrophy. Cytokine 43:194–199 Bellini A, Mattoli S (2007) The role of the fibrocyte, a bone marrow-derived mesenchymal progenitor, in reactive and reparative fibroses. Lab Invest 87:858–870

62

B. Hinz et al.

Benzonana G, Skalli O, Gabbiani G (1988) Correlation between the distribution of smooth muscle or non muscle myosins and alpha-smooth muscle actin in normal and pathological soft tissues. Cell Motil Cytoskeleton 11:260–274 Berasain C, Castillo J, Perugorria MJ, Latasa MU, Prieto J, Avila MA (2009) Inflammation and liver cancer: new molecular links. Ann N Y Acad Sci 1155:206–221 Bernstein AM, Twining SS, Warejcka DJ, Tall E, Masur SK (2007) Urokinase receptor cleavage: a crucial step in fibroblast-to-myofibroblast differentiation. Mol Biol Cell 18:2716–2727 Bershadsky AD, Balaban NQ, Geiger B (2003) Adhesion-dependent cell mechanosensitivity. Annu Rev Cell Dev Biol 19:677–695 Bhowmick NA, Moses HL (2005) Tumor-stroma interactions. Curr Opin Genet Dev 15:97–101 Bianco P, Riminucci M, Gronthos S, Robey PG (2001) Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 19:180–192 Bierie B, Moses HL (2006) Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer. Nat Rev Cancer 6:506–520 Bissell DM (2001) Chronic liver injury, TGF-beta, and cancer. Exp Mol Med 33:179–190 Bochaton-Piallat ML, Gabbiani G (2006) Smooth muscle cell: a key cell for plaque vulnerability regulation? Circ Res 98:448–449 Bogatkevich GS, Tourkina E, Abrams CS, Harley RA, Silver RM, Ludwicka-Bradley A (2003) Contractile activity and smooth muscle alpha-actin organization in thrombin-induced human lung myofibroblasts. Am J Physiol Lung Cell Mol Physiol 285:L334–343 Bonanno E, Ercoli L, Missori P, Rocchi G, Spagnoli LG (1994) Homogeneous stromal cell population from normal human adult bone marrow expressing alpha-smooth muscle actin filaments. Lab Invest 71:308–315 Boyd NF, Lockwood GA, Byng JW, Tritchler DL, Yaffe MJ (1998) Mammographic densities and breast cancer risk. Cancer Epidemiol Biomarkers Prev 7:1133–1144 Boyd NF, Guo H, Martin LJ, Sun L, Stone J, Fishell E, Jong RA, Hislop G, Chiarelli A, Minkin S, Yaffe MJ (2007) Mammographic density and the risk and detection of breast cancer. N Engl J Med 356:227–236 Brittan M et al (2002) Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut 50:752–757 Brittan M et al (2005) A regenerative role for bone marrow following experimental colitis: contribution to neovasculogenesis and myofibroblasts. Gastroenterology 128:1984–1995 Brown RD, Ambler SK, Mitchell MD, Long CS (2005) The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol 45:657–687 Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A (1994) Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med 1:71–81 Butcher DT, Alliston T, Weaver VM (2009) A tense situation: forcing tumour progression. Nat Rev Cancer 9:108–122 Cai D, Marty-Roix R, Hsu HP, Spector M (2001) Lapine and canine bone marrow stromal cells contain smooth muscle actin and contract a collagen-glycosaminoglycan matrix. Tissue Eng 7:829–841 Caplan AI (2007) Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol 213:341–347 Carlson MA, Longaker MT, Thompson JS (2003) Wound splinting regulates granulation tissue survival. J Surg Res 110:304–309 Chambers RC, Leoni P, Kaminski N, Laurent GJ, Heller RA (2003) Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching. Am J Pathol 162:533–546 Chaponnier C, Goethals M, Janmey PA, Gabbiani F, Gabbiani G, Vandekerckhove J (1995) The specific NH2-terminal sequence Ac-EEED of alpha-smooth muscle actin plays a role in polymerization in vitro and in vivo. J Cell Biol 130:887–895 Charbord P, Lerat H, Newton I, Tamayo E, Gown AM, Singer JW, Herve P (1990) The cytoskeleton of stromal cells from human bone marrow cultures resembles that of cultured smooth muscle cells. Exp Hematol 18:276–282

3  The Role of the Myofibroblast in Fibrosis and Cancer Progression

63

Chiappara G, Gagliardo R, Siena A, Bonsignore MR, Bousquet J, Bonsignore G, Vignola AM (2001) Airway remodelling in the pathogenesis of asthma. Curr Opin Allergy Clin Immunol 1:85–93 Chilosi M, Poletti V, Zamo A, Lestani M, Montagna L, Piccoli P, Pedron S, Bertaso M, Scarpa A, Murer B, Cancellieri A, Maestro R, Semenzato G, Doglioni C (2003) Aberrant Wnt/betacatenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol 162:1495–1502 Christen T, Verin V, Bochaton-Piallat M, Popowski Y, Ramaekers F, Debruyne P, Camenzind E, van Eys G, Gabbiani G (2001) Mechanisms of neointima formation and remodeling in the porcine coronary artery. Circulation 103:882–888 Clement S, Hinz B, Dugina V, Gabbiani G, Chaponnier C (2005) The N-terminal Ac-EEED sequence plays a role in {alpha}-smooth-muscle actin incorporation into stress fibers. J Cell Sci 118:1395–1404 Coleman DJ, Sharpe DT, Naylor IL, Chander CL, Cross SE (1993) The role of the contractile fibroblast in the capsules around tissue expanders and implants. Br J Plast Surg 46:547–556 Colombelli J, Besser A, Kress H, Reynaud EG, Girard P, Caussinus E, Haselmann U, Small JV, Schwarz US, Stelzer EH (2009) Mechanosensing in actin stress fibers revealed by a close correlation between force and protein localization. J Cell Sci 122:1665–1679 Comut AA, Shortkroff S, Zhang X, Spector M (2000) Association of fibroblast orientation around titanium in vitro with expression of a muscle actin. Biomaterials 21:1887–1896 Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, Sorescu D (2005) NAD(P)H oxidase 4 mediates transforming growth factor-beta1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circ Res 97:900–907 Dagouassat M, Suffee N, Hlawaty H, Haddad O, Charni F, Laguillier C, Vassy R, Martin L, Schischmanoff PO, Gattegno L, Oudar O, Sutton A, Charnaux N (2009) Monocyte chemoattractant protein-1 (MCP-1)/CCL2 secreted by hepatic myofibroblasts promotes migration and invasion of human hepatoma cells. Int J Cancer 126(5):1095–1108 Daniels CE, Jett JR (2005) Does interstitial lung disease predispose to lung cancer? Curr Opin Pulm Med 11:431–437 Darby I, Skalli O, Gabbiani G (1990) Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 63:21–29 De Wever O, Mareel M (2002) Role of myofibroblasts at the invasion front. Biol Chem 383:55–67 De Wever O, Mareel M (2003) Role of tissue stroma in cancer cell invasion. J Pathol 200:429– 447 De Wever O, Nguyen QD, Van Hoorde L, Bracke M, Bruyneel E, Gespach C, Mareel M (2004) Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac. Faseb J 18:1016–1018 De Wever O, Demetter P, Mareel M, Bracke M (2008) Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer 123:2229–2238 Desmouliere A (2007) Hepatic stellate cells: the only cells involved in liver fibrogenesis? A dogma challenged. Gastroenterology 132:2059–2062 Desmouliere A, Rubbia-Brandt L, Abdiu A, Walz T, Macieira-Coelho A, Gabbiani G (1992) Alpha-smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by gamma-interferon. Exp Cell Res 201:64–73 Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G (1993) Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122:103–111 Desmouliere A, Redard M, Darby I, Gabbiani G (1995) Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol 146:56–66 Desmouliere A, Darby IA, Gabbiani G (2003) Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest 83:1689– 1707 Desmouliere A, Guyot C, Gabbiani G (2004) The stroma reaction myofibroblast: a key player in the control of tumor cell behavior. Int J Dev Biol 48:509–517 Desmouliere A, Chaponnier C, Gabbiani G (2005) Tissue repair, contraction, and the myofibroblast. Wound Repair Regen 13:7–12

64

B. Hinz et al.

di Bonzo LV et al (2008) Human mesenchymal stem cells as a two-edged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential. Gut 57:223–231 Direkze NC, Forbes SJ, Brittan M, Hunt T, Jeffery R, Preston SL, Poulsom R, Hodivala-Dilke K, Alison MR, Wright NA (2003) Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice. Stem Cells 21:514–520 Direkze NC, Hodivala-Dilke K, Jeffery R, Hunt T, Poulsom R, Oukrif D, Alison MR, Wright NA (2004) Bone marrow contribution to tumor-associated myofibroblasts and fibroblasts. Cancer Res 64:8492–8495 Dolberg DS, Hollingsworth R, Hertle M, Bissell MJ (1985) Wounding and its role in RSV-mediated tumor formation. Science 230:676–678 Dubuisson L, Monvoisin A, Nielsen BS, Le Bail B, Bioulac-Sage P, Rosenbaum J (2000) Expression and cellular localization of the urokinase-type plasminogen activator and its receptor in human hepatocellular carcinoma. J Pathol 190:190–195 Dubuisson L, Lepreux S, Bioulac-Sage P, Balabaud C, Costa AM, Rosenbaum J, Desmouliere A (2001) Expression and cellular localization of fibrillin-1 in normal and pathological human liver. J Hepatol 34:514–522 Dugina V, Fontao L, Chaponnier C, Vasiliev J, Gabbiani G (2001) Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors. J Cell Sci 114:3285–3296 Dvorak HF (1986) Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 315:1650–1659 Eyden B (2007) The myofibroblast: a study of normal, reactive and neoplastic tissues, with an emphasis on ultrastructure. J Submicrosc Cytol Pathol:7–166 Eyden B (2008) The myofibroblast: phenotypic characterization as a prerequisite to understanding its functions in translational medicine. J Cell Mol Med 12:22–37 Faouzi S, Le Bail B, Neaud V, Boussarie L, Saric J, Bioulac-Sage P, Balabaud C, Rosenbaum J (1999a) Myofibroblasts are responsible for collagen synthesis in the stroma of human hepatocellular carcinoma: an in vivo and in vitro study. J Hepatol 30:275–284 Faouzi S, Lepreux S, Bedin C, Dubuisson L, Balabaud C, Bioulac-Sage P, Desmouliere A, Rosenbaum J (1999b) Activation of cultured rat hepatic stellate cells by tumoral hepatocytes. Lab Invest 79:485–493 Farazi PA, DePinho RA (2006) Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 6:674–687 Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, Isik F (2004) Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair. Stem Cells 22:812–822 Feng XH, Derynck R (2005) Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol 21:659–693 Ffrench-Constant C, Van de Water L, Dvorak HF, Hynes RO (1989) Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat. J Cell Biol 109:903–914 Forbes SJ, Russo FP, Rey V, Burra P, Rugge M, Wright NA, Alison MR (2004) A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis. Gastroenterology 126:955–963 Frank ED, Warren L (1981) Aortic smooth muscle cells contain vimentin instead of desmin. Proc Natl Acad Sci U S A 78:3020–3024 Frid MG, Brunetti JA, Burke DL, Carpenter TC, Davie NJ, Reeves JT, Roedersheimer MT, van Rooijen N, Stenmark KR (2006) Hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage. Am J Pathol 168:659–669 Friedman SL (2004a) Mechanisms of disease: mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol 1:98–105 Friedman SL (2004b) Stellate cells: a moving target in hepatic fibrogenesis. Hepatology 40:1041– 1043

3  The Role of the Myofibroblast in Fibrosis and Cancer Progression

65

Fujita J, Mori M, Kawada H, Ieda Y, Tsuma M, Matsuzaki Y, Kawaguchi H, Yagi T, Yuasa S, Endo J, Hotta T, Ogawa S, Okano H, Yozu R, Ando K, Fukuda K (2007) Administration of granulocyte colony-stimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair. Stem Cells 25:2750–2759 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 Gabbiani G, Schmid E, Winter S, Chaponnier C, de Ckhastonay C, Vandekerckhove J, Weber K, Franke WW (1981) Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin. Proc Natl Acad Sci U S A 78:298–302 Gallucci RM, Lee EG, Tomasek JJ (2006) IL-6 modulates alpha-smooth muscle actin expression in dermal fibroblasts from IL-6-deficient mice. J Invest Dermatol 126:561–568 Garra BS (2007) Imaging and estimation of tissue elasticity by ultrasound. Ultrasound Q 23:255– 268 Georges PC, Hui JJ, Gombos Z, McCormick ME, Wang AY, Uemura M, Mick R, Janmey PA, Furth EE, Wells RG (2007) Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis. Am J Physiol Gastrointest Liver Physiol 293:G1147–1154 Gharaee-Kermani M, Hu B, Thannickal VJ, Phan SH, Gyetko MR (2007) Current and emerging drugs for idiopathic pulmonary fibrosis. Expert Opin Emerg Drugs 12:627–646 Giannouli CC, Kletsas D (2006) TGF-beta regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2. Cell Signal 18:1417–1429 Glaser KJ, Felmlee JP, Manduca A, Kannan Mariappan Y, Ehman RL (2006) Stiffness-weighted magnetic resonance imaging. Magn Reson Med 55:59–67 Goffin JM, Pittet P, Csucs G, Lussi JW, Meister JJ, Hinz B (2006) Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers. J Cell Biol 172:259–268 Goldberg MT, Han YP, Yan C, Shaw MC, Garner WL (2007) TNF-alpha suppresses alpha-smooth muscle actin expression in human dermal fibroblasts: an implication for abnormal wound healing. J Invest Dermatol 127:2645–2655 Gonda TA, Varro A, Wang TC, Tycko B (2010) Molecular biology of cancer-associated fibroblasts: can these cells be targeted in anti-cancer therapy? Semin Cell Dev Biol 21:2–10 Grainger DJ (2007) TGF-beta and atherosclerosis in man. Cardiovasc Res 74:213–222 Gressner AM, Weiskirchen R (2006) Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 10:76–99 Griffith B, Pendyala S, Hecker L, Lee PJ, Natarajan V, Thannickal VJ (2009) NOX enzymes and pulmonary disease. Antioxid Redox Signal 11:2505–2516 Grinnell F, Zhu M, Carlson MA, Abrams JM (1999) Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue. Exp Cell Res 248:608–619 Gupta PB, Chaffer CL, Weinberg RA (2009) Cancer stem cells: mirage or reality? Nat Med 15:1010–1012 Gurtner GC, Werner S, Barrandon Y, Longaker MT (2008) Wound repair and regeneration. Nature 453:314–321 Guyot C, Combe C, Balabaud C, Bioulac-Sage P, Desmoulière A (2007) Fibrogenic cell fate during fibrotic tissue remodelling observed in rat and human cultured liver slices. J Hepatol 46:142–150 Guyot C, Lepreux S, Combe C, Doudnikoff E, Bioulac-Sage P, Balabaud C, Desmouliere A (2006) Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved. Int J Biochem Cell Biol 38:135–151 Hall B, Dembinski J, Sasser AK, Studeny M, Andreeff M, Marini F (2007) Mesenchymal stem cells in cancer: tumor-associated fibroblasts and cell-based delivery vehicles. Int J Hematol 86:8–16

66

B. Hinz et al.

Hao H, Gabbiani G, Camenzind E, Bacchetta M, Virmani R, Bochaton-Piallat ML (2006) Phenotypic modulation of intima and media smooth muscle cells in fatal cases of coronary artery lesion. Arterioscler Thromb Vasc Biol 26:326–332 Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 113:243–252 Haudek SB, Xia Y, Huebener P, Lee JM, Carlson S, Crawford JR, Pilling D, Gomer RH, Trial J, Frangogiannis NG, Entman ML (2006) Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice. Proc Natl Acad Sci U S A 103:18284–18289 Hawinkels LJ, Verspaget HW, van der Reijden JJ, van der Zon JM, Verheijen JH, Hommes DW, Lamers CB, Sier CF (2009) Active TGF-beta1 correlates with myofibroblasts and malignancy in the colorectal adenoma-carcinoma sequence. Cancer Sci 100:663–670 He Q, Wan C, Li G (2007) Concise review: multipotent mesenchymal stromal cells in blood. Stem Cells 25:69–77 Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC, Pennathur S, Martinez FJ, Thannickal VJ (2009) NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat Med 15:1077–1081 Heldin CH, Rubin K, Pietras K, Ostman A (2004) High interstitial fluid pressure—an obstacle in cancer therapy. Nat Rev Cancer 4:806–813 Henderson NC, Mackinnon AC, Farnworth SL, Poirier F, Russo FP, Iredale JP, Haslett C, Simpson KJ, Sethi T (2006) Galectin-3 regulates myofibroblast activation and hepatic fibrosis. Proc Natl Acad Sci U S A 103:5060–5065 Hervy M, Hoffman L, Beckerle MC (2006) From the membrane to the nucleus and back again: bifunctional focal adhesion proteins. Curr Opin Cell Biol 18:524–532 Hill R, Song Y, Cardiff RD, Van Dyke T (2005) Selective evolution of stromal mesenchyme with p53 loss in response to epithelial tumorigenesis. Cell 123:1001–1011 Hinz B (2007) Formation and function of the myofibroblast during tissue repair. J Invest Dermatol 127:526–537 Hinz B (2009a) The myofibroblast—friend or foe in regenerative medicine? In: Ralphs CAJ (ed) Regenerative medicine and biomaterials for the repair of connective tissues. Woodhead, Cambridge (in press) Hinz B (2009b) The myofibroblast: paradigm for a mechanically active cell. J Biomech 43(1):146– 155 Hinz B (2009c) Tissue stiffness, latent TGF-beta1 activation, and mechanical signal transduction: implications for the pathogenesis and treatment of fibrosis. Curr Rheumatol Rep 11:120–126 Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C (2001a) Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 12:2730–2741 Hinz B, Mastrangelo D, Iselin CE, Chaponnier C, Gabbiani G (2001b) Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation. Am J Pathol 159:1009–1020 Hinz B, Gabbiani G, Chaponnier C (2002) The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo. J Cell Biol 157:657–663 Hinz B, Pittet P, Smith-Clerc J, Chaponnier C, Meister JJ (2004) Myofibroblast development is characterized by specific cell–cell adherens junctions. Mol Biol Cell 15:4310–4320 Hinz B, Phan SH, Thannickal VJ, Galli A, Bochaton-Piallat ML, Gabbiani G (2007) The myofibroblast: one function, multiple origins. Am J Pathol 170:1807–1816 Horowitz JC, Thannickal VJ (2006) Idiopathic pulmonary fibrosis: new concepts in pathogenesis and implications for drug therapy. Treat Respir Med 5:325–342 Hughes CC (2008) Endothelial-stromal interactions in angiogenesis. Curr Opin Hematol 15:204– 209 Hung SC, Deng WP, Yang WK, Liu RS, Lee CC, Su TC, Lin RJ, Yang DM, Chang CW, Chen WH, Wei HJ, Gelovani JG (2005) Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging. Clin Cancer Res 11:7749–7756

3  The Role of the Myofibroblast in Fibrosis and Cancer Progression

67

Ingber DE (2003) Tensegrity II. How structural networks influence cellular information processing networks. J Cell Sci 116:1397–1408 Iredale JP (2007) Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest 117:539–548 Ishii G, Sangai T, Oda T, Aoyagi Y, Hasebe T, Kanomata N, Endoh Y, Okumura C, Okuhara Y, Magae J, Emura M, Ochiya T, Ochiai A (2003) Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction. Biochem Biophys Res Commun 309:232–240 Ishii G, Sangai T, Sugiyama K, Ito T, Hasebe T, Endoh Y, Magae J, Ochiai A (2005) In vivo characterization of bone marrow-derived fibroblasts recruited into fibrotic lesions. Stem Cells 23:699–706 Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110:341–350 Jain R, Shaul PW, Borok Z, Willis BC (2007) Endothelin-1 induces alveolar epithelial-mesenchymal transition through endothelin type A receptor-mediated production of TGF-beta1. Am J Respir Cell Mol Biol 37:38–47 Jan YY, Yeh TS, Yeh JN, Yang HR, Chen MF (2004) Expression of epidermal growth factor receptor, apomucins, matrix metalloproteinases, and p53 in rat and human cholangiocarcinoma: appraisal of an animal model of cholangiocarcinoma. Ann Surg 240:89–94 Janmey PA, Winer JP, Murray ME, Wen Q (2009) The hard life of soft cells. Cell Motil Cytoskeleton 66:597–605 Jenkins G (2008) The role of proteases in transforming growth factor-beta activation. Int J Biochem Cell Biol 40:1068–1078 Jenkins RG, Su X, Su G, Scotton CJ, Camerer E, Laurent GJ, Davis GE, Chambers RC, Matthay MA, Sheppard D (2006) Ligation of protease-activated receptor 1 enhances alpha(v) beta6 integrin-dependent TGF-beta activation and promotes acute lung injury. J Clin Invest 116:1606–1614 Johnson CP, Tang HY, Carag C, Speicher DW, Discher DE (2007) Forced unfolding of proteins within cells. Science 317:663–666 Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6:392–401 Kanangat S, Postlethwaite AE, Higgins GC, Hasty KA (2006) Novel functions of intracellular IL1ra in human dermal fibroblasts: implications in the pathogenesis of fibrosis. J Invest Dermatol 126:756–765 Kang HR, Lee CG, Homer RJ, Elias JA (2007) Semaphorin 7A plays a critical role in TGF-beta1induced pulmonary fibrosis. J Exp Med 204:1083–1093 Kaplan RN, Riba RD, Zacharoulis S, Bramley AH, Vincent L, Costa C, MacDonald DD, Jin DK, Shido K, Kerns SA, Zhu Z, Hicklin D, Wu Y, Port JL, Altorki N, Port ER, Ruggero D, Shmelkov SV, Jensen KK, Rafii S, Lyden D (2005) VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438:820–827 Kaplan RN, Psaila B, Lyden D (2006) Bone marrow cells in the ‘pre-metastatic niche’: within bone and beyond. Cancer Metastasis Rev 25:521–529 Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, Richardson AL, Polyak K, Tubo R, Weinberg RA (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449:557–563 Khakoo AY, Pati S, Anderson SA, Reid W, Elshal MF, Rovira II, Nguyen AT, Malide D, Combs CA, Hall G, Zhang J, Raffeld M, Rogers TB, Stetler-Stevenson W, Frank JA, Reitz M, Finkel T (2006) Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med 203:1235–1247 Khaled W, Reichling S, Bruhns OT, Boese H, Baumann M, Monkman G, Egersdoerfer S, Klein D, Tunayar A, Freimuth H, Lorenz A, Pessavento A, Ermert H (2004) Palpation imaging using a haptic system for virtual reality applications in medicine. Stud Health Technol Inform 98:147–153 Khaled W, Reichling S, Bruhns OT, Ermert H (2006) Ultrasonic strain imaging and reconstructive elastography for biological tissue. Ultrasonics 44 Suppl 1:e199–202

68

B. Hinz et al.

Khan SA, Toledano MB, Taylor-Robinson SD (2008) Epidemiology, risk factors, and pathogenesis of cholangiocarcinoma. HPB (Oxford) 10:77–82 Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, Brumwell AN, Sheppard D, Chapman HA (2006) Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A 103:13180–13185 Kinner B, Gerstenfeld LC, Einhorn TA, Spector M (2002a) Expression of smooth muscle actin in connective tissue cells participating in fracture healing in a murine model. Bone 30:738– 745 Kinner B, Zaleskas JM, Spector M (2002b) Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res 278:72–83 Kisseleva T, Uchinami H, Feirt N, Quintana-Bustamante O, Segovia JC, Schwabe RF, Brenner DA (2006) Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis. J Hepatol 45:429–438 Kizu A, Medici D, Kalluri R (2009) Endothelial-mesenchymal transition as a novel mechanism for generating myofibroblasts during diabetic nephropathy. Am J Pathol 175(4):1371–1373 Klein EA, Yin L, Kothapalli D, Castagnino P, Byfield FJ, Xu T, Levental I, Hawthorne E, Janmey PA, Assoian RK (2009) Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening. Curr Biol 19:1511–1518 Komarova S, Kawakami Y, Stoff-Khalili MA, Curiel DT, Pereboeva L (2006) Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses. Mol Cancer Ther 5:755–766 Kulasekaran P, Scavone CA, Rogers DS, Arenberg DA, Thannickal VJ, Horowitz JC (2009) Endothelin-1 and transforming growth factor-beta1 independently induce fibroblast resistance to apoptosis via AKT activation. Am J Respir Cell Mol Biol 41:484–493 Kumar S, Weaver VM (2009) Mechanics, malignancy, and metastasis: the force journey of a tumor cell. Cancer Metastasis Rev 28:113–127 Lan, HY (2003) Tubular epithelial-myofibroblast transdifferentiation mechanisms in proximal tubule cells. Curr Opin Nephrol Hypertens 12:25–29 Larson DM, Fujiwara K, Alexander RW, Gimbrone MA Jr (1984) Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression. J Cell Biol 99:1582–1589 Le Bail B, Faouzi S, Boussarie L, Guirouilh J, Blanc JF, Carles J, Bioulac-Sage P, Balabaud C, Rosenbaum J (1999) Osteonectin/SPARC is overexpressed in human hepatocellular carcinoma. J Pathol 189:46–52 Leask A, Abraham DJ (2004) TGF-beta signaling and the fibrotic response. Faseb J 18:816–827 Lenga Y, Koh A, Perera AS, McCulloch CA, Sodek J, Zohar R (2008) Osteopontin expression is required for myofibroblast differentiation. Circ Res 102:319–327 Li AG, Quinn MJ, Siddiqui Y, Wood MD, Federiuk IF, Duman HM, Ward WK (2007a) Elevation of transforming growth factor beta (TGFbeta) and its downstream mediators in subcutaneous foreign body capsule tissue. J Biomed Mater Res A 82:498–508 Li Z, Dranoff JA, Chan EP, Uemura M, Sevigny J, Wells RG (2007b) Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture. Hepatology 46:1246–1256 Li J, Qu X, Bertram JF (2009) Endothelial-myofibroblast transition contributes to the early development of diabetic renal interstitial fibrosis in streptozotocin-induced diabetic mice. Am J Pathol 175(4):1380–1388 Lindahl GE, Chambers RC, Papakrivopoulou J, Dawson SJ, Jacobsen MC, Bishop JE, Laurent GJ (2002) Activation of fibroblast procollagen alpha 1(I) transcription by mechanical strain is transforming growth factor-beta-dependent and involves increased binding of CCAAT-binding factor (CBF/NF-Y) at the proximal promoter. J Biol Chem 277:6153–6161 Liu K, He X, Lei XZ, Zhao LS, Tang H, Liu L, Lei BJ (2003) Pathomorphological study on location and distribution of Kupffer cells in hepatocellular carcinoma. World J Gastroenterol 9:1946–1949

3  The Role of the Myofibroblast in Fibrosis and Cancer Progression

69

Liu T, Dhanasekaran SM, Jin H, Hu B, Tomlins SA, Chinnaiyan AM, Phan SH (2004) FIZZ1 stimulation of myofibroblast differentiation. Am J Pathol 164:1315–1326 Malmstrom J, Lindberg H, Lindberg C, Bratt C, Wieslander E, Delander EL, Sarnstrand B, Burns JS, Mose-Larsen P, Fey S, Marko-Varga G (2004) Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus. Mol Cell Proteomics 3:466–477 Mattoli S, Bellini A, Schmidt M (2009) The role of a human hematopoietic mesenchymal progenitor in wound healing and fibrotic diseases and implications for therapy. Curr Stem Cell Res 4:266–280 Menon LG, Picinich S, Koneru R, Gao H, Lin SY, Koneru M, Mayer-Kuckuk P, Glod J, Banerjee D (2007) Differential gene expression associated with migration of mesenchymal stem cells to conditioned medium from tumor cells or bone marrow cells. Stem Cells 25:520–528 Metz CN (2003) Fibrocytes: a unique cell population implicated in wound healing. Cell Mol Life Sci 60:1342–1350 Micera A, Vigneti E, Pickholtz D, Reich R, Pappo O, Bonini S, Maquart FX, Aloe L, Levi-Schaffer F (2001) Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair. Proc Natl Acad Sci U S A 98:6162– 6167 Minguell JJ, Erices A, Conget P (2001) Mesenchymal stem cells. Exp Biol Med (Maywood) 226:507–520 Mishra PJ, Mishra PJ, Humeniuk R, Medina DJ, Alexe G, Mesirov JP, Ganesan S, Glod JW, Banerjee D (2008) Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res 68:4331–4339 Mishra PJ, Glod JW, Banerjee D (2009) Mesenchymal stem cells: flip side of the coin. Cancer Res 69:1255–1258 Moeller A, Gilpin SE, Ask K, Cox G, Cook D, Gauldie J, Margetts PJ, Farkas L, Dobranowski J, Boylan C, O’Byrne PM, Strieter RM, Kolb M (2009) Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 179:588–594 Mollmann H et al (2006) Bone marrow-derived cells contribute to infarct remodelling. Cardiovasc Res 71:661–671 Monvoisin A, Neaud V, De Ledinghen V, Dubuisson L, Balabaud C, Bioulac-Sage P, Desmouliere A, Rosenbaum J (1999) Direct evidence that hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells is mediated by urokinase. J Hepatol 30:511–518 Monvoisin A, Bisson C, Si-Tayeb K, Balabaud C, Desmouliere A, Rosenbaum J (2002) Involvement of matrix metalloproteinase type-3 in hepatocyte growth factor-induced invasion of human hepatocellular carcinoma cells. Int J Cancer 97:157–162 Moreels M, Vandenabeele F, Dumont D, Robben J, Lambrichts I (2008) Alpha-smooth muscle actin (alpha-SMA) and nestin expression in reactive astrocytes in multiple sclerosis lesions: potential regulatory role of transforming growth factor-beta 1 (TGF-beta1). Neuropathol Appl Neurobiol 34:532–546 Mori L, Bellini A, Stacey MA, Schmidt M, Mattoli S (2005) Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Exp Cell Res 304:81–90 Mori R, Shaw TJ, Martin P (2008) Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring. J Exp Med 205:43–51 Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D (1999) The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96:319–328 Naugle JE, Olson ER, Zhang X, Mase SE, Pilati CF, Maron MB, Folkesson HG, Horne WI, Doane KJ, Meszaros JG (2006) Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling. Am J Physiol Heart Circ Physiol 290:H323–330 Neaud V, Faouzi S, Guirouilh J, Le Bail B, Balabaud C, Bioulac-Sage P, Rosenbaum J (1997) Human hepatic myofibroblasts increase invasiveness of hepatocellular carcinoma cells: evidence for a role of hepatocyte growth factor. Hepatology 26:1458–1466

70

B. Hinz et al.

Neaud V, Hisaka T, Monvoisin A, Bedin C, Balabaud C, Foster DC, Desmouliere A, Kisiel W, Rosenbaum J (2000) Paradoxical pro-invasive effect of the serine proteinase inhibitor tissue factor pathway inhibitor-2 on human hepatocellular carcinoma cells. J Biol Chem 275:35565–35569 Ng CP, Hinz B, Swartz MA (2005) Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro. J Cell Sci 118:4731–4739 Nielsen JD, Moeslund M, Wandall HH, Dabelsteen S (2008) Influences of tumor stroma on the malignant phenotype. J Oral Pathol Med 37:412–416 Ninichuk V et al (2006) Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice. Kidney Int 70:121–129 Noel A, Jost M, Maquoi E (2008) Matrix metalloproteinases at cancer tumor-host interface. Semin Cell Dev Biol 19:52–60 Okamura N, Yoshida M, Shibuya A, Sugiura H, Okayasu I, Ohbu M (2005) Cellular and stromal characteristics in the scirrhous hepatocellular carcinoma: comparison with hepatocellular carcinomas and intrahepatic cholangiocarcinomas. Pathol Int 55:724–731 Orimo A, Weinberg RA (2007) Heterogeneity of stromal fibroblasts in tumors. Cancer Biol Ther 6:618–619 Ostman A, Augsten M (2009) Cancer-associated fibroblasts and tumor growth–bystanders turning into key players. Curr Opin Genet Dev 19:67–73 Pardali K, Moustakas A (2007) Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer. Biochim Biophys Acta 1775:21–62 Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, Reinhart-King CA, Margulies SS, Dembo M, Boettiger D, Hammer DA, Weaver VM (2005) Tensional homeostasis and the malignant phenotype. Cancer Cell 8:241–254 Peled A, Zipori D, Abramsky O, Ovadia H, Shezen E (1991) Expression of alpha-smooth muscle actin in murine bone marrow stromal cells. Blood 78:304–309 Peterson LJ, Rajfur Z, Maddox AS, Freel CD, Chen Y, Edlund M, Otey C, Burridge K (2004) Simultaneous stretching and contraction of stress fibers in vivo. Mol Biol Cell 15:3497–3508 Phan, SH (2002) The myofibroblast in pulmonary fibrosis. Chest 122:286S–289S Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 114:438–446 Pietras K, Sjoblom T, Rubin K, Heldin CH, Ostman A (2003) PDGF receptors as cancer drug targets. Cancer Cell 3:439–443 Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 Potenta S, Zeisberg E, Kalluri R (2008) The role of endothelial-to-mesenchymal transition in cancer progression. Br J Cancer 99:1375–1379 Qi W, Chen X, Poronnik P, Pollock CA (2006) The renal cortical fibroblast in renal tubulointerstitial fibrosis. Int J Biochem Cell Biol 38:1–5 Quan TE, Cowper SE, Bucala R (2006) The role of circulating fibrocytes in fibrosis. Curr Rheumatol Rep 8:145–150 Radisky DC, Przybylo JA (2008) Matrix metalloproteinase-induced fibrosis and malignancy in breast and lung. Proc Am Thorac Soc 5:316–322 Radisky DC, Kenny PA, Bissell MJ (2007) Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem 101:830–839 Rajkumar VS, Shiwen X, Bostrom M, Leoni P, Muddle J, Ivarsson M, Gerdin B, Denton CP, Bou-Gharios G, Black CM, Abraham DJ (2006) Platelet-derived growth factor-beta receptor activation is essential for fibroblast and pericyte recruitment during cutaneous wound healing. Am J Pathol 169:2254–2265 Ramadori G, Saile B (2004) Portal tract fibrogenesis in the liver. Lab Invest 84:153–159 Rocks N, Paulissen G, El Hour M, Quesada F, Crahay C, Gueders M, Foidart JM, Noel A, Cataldo D (2008a) Emerging roles of ADAM and ADAMTS metalloproteinases in cancer. Biochimie 90:369–379

3  The Role of the Myofibroblast in Fibrosis and Cancer Progression

71

Rocks N, Paulissen G, Quesada-Calvo F, Munaut C, Gonzalez ML, Gueders M, Hacha J, Gilles C, Foidart JM, Noel A, Cataldo DD (2008b) ADAMTS-1 metalloproteinase promotes tumor development through the induction of a stromal reaction in vivo. Cancer Res 68:9541–9550 Ronnov-Jessen L, Petersen OW (1993) Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab Invest 68:696–707 Rosenkranz S (2004) TGF-beta1 and angiotensin networking in cardiac remodeling. Cardiovasc Res 63:423–432 Roufosse CA, Direkze NC, Otto WR, Wright NA (2004) Circulating mesenchymal stem cells. Int J Biochem Cell Biol 36:585–597 Ruiz-Ortega M, Rodriguez-Vita J, Sanchez-Lopez E, Carvajal G, Egido J (2007) TGF-beta signaling in vascular fibrosis. Cardiovasc Res 74:196–206 Saika S, Ikeda K, Yamanaka O, Flanders KC, Okada Y, Miyamoto T, Kitano A, Ooshima A, Nakajima Y, Ohnishi Y, Kao WW (2006) Loss of tumor necrosis factor alpha potentiates transforming growth factor beta-mediated pathogenic tissue response during wound healing. Am J Pathol 168:1848–1860 Saile B, Matthes N, Neubauer K, Eisenbach C, El-Armouche H, Dudas J, Ramadori G (2002) Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha. Am J Physiol Gastrointest Liver Physiol 283:G435–444 Saile B, DiRocco P, Dudas J, El-Armouche H, Sebb H, Eisenbach C, Neubauer K, Ramadori G (2004) IGF-I induces DNA synthesis and apoptosis in rat liver hepatic stellate cells (HSC) but DNA synthesis and proliferation in rat liver myofibroblasts (rMF). Lab Invest 84:1037– 1049 Sakai N, Wada T, Yokoyama H, Lipp M, Ueha S, Matsushima K, Kaneko S (2006) Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates fibrocytes in renal fibrosis. Proc Natl Acad Sci U S A 103:14098–14103 Sartore S, Chiavegato A, Faggin E, Franch R, Puato M, Ausoni S, Pauletto P (2001) Contribution of adventitial fibroblasts to neointima formation and vascular remodeling: from innocent bystander to active participant. Circ Res 89:1111–1121 Sasaki M et al (2008) Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type. J Immunol 180:2581–2587 Sata M et al (2002) Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 8:403–409 Schafer M, Werner S (2008) Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol 9:628–638 Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S (2003) Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 171:380–389 Schurch W, Seemayer TA, Hinz B, Gabbiani G (2007) Myofibroblast. In: Mills SE (ed) Histology for Pathologists. Lippincott-Williams & Wilkins, Philadelphia, pp 123–164 Scotton CJ, Chambers RC (2007) Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest 132:1311–1321 Serini G, Bochaton-Piallat ML, Ropraz P, Geinoz A, Borsi L, Zardi L, Gabbiani G (1998) The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J Cell Biol 142:873–881 Shah M, Foreman DM, Ferguson MW (1995) Neutralisation of TGF-beta 1 and TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 108:985–1002 Shen WL, Gao PJ, Che ZQ, Ji KD, Yin M, Yan C, Berk BC, Zhu DL (2006) NAD(P)H oxidasederived reactive oxygen species regulate angiotensin-II induced adventitial fibroblast phenotypic differentiation. Biochem Biophys Res Commun 339:337–343 Shephard P, Martin G, Smola-Hess S, Brunner G, Krieg T, Smola H (2004) Myofibroblast differentiation is induced in keratinocyte-fibroblast co-cultures and is antagonistically regulated by endogenous transforming growth factor-beta and interleukin-1. Am J Pathol 164:2055–2066 Sheppard D (2005) Integrin-mediated activation of latent transforming growth factor beta. Cancer Metastasis Rev 24:395–402

72

B. Hinz et al.

Shi-Wen X, Renzoni EA, Kennedy L, Howat S, Chen Y, Pearson JD, Bou-Gharios G, Dashwood MR, du Bois RM, Black CM, Denton CP, Abraham DJ, Leask A (2007) Endogenous endothelin-1 signaling contributes to type I collagen and CCN2 overexpression in fibrotic fibroblasts. Matrix Biol 26:625–632 Shi-Wen X, Leask A, Abraham D (2008) Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev 19:133–144 Shibata T, Ochiai A, Gotoh M, Machinami R, Hirohashi S (1996) Simultaneous expression of cadherin-11 in signet-ring cell carcinoma and stromal cells of diffuse-type gastric cancer. Cancer Lett 99:147–153 Siegel PM, Massague J (2003) Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 3:807–821 Siggelkow W, Faridi A, Spiritus K, Klinge U, Rath W, Klosterhalfen B (2003) Histological analysis of silicone breast implant capsules and correlation with capsular contracture. Biomaterials 24:1101–1109 Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146–156 Simmons PJ, Torok-Storb B (1991) Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 78:55–62 Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G (1986) A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103:2787–2796 Strehlow D, Korn JH (1998) Biology of the scleroderma fibroblast. Curr Opin Rheumatol 10:572– 578 Strutz F, Zeisberg M, Renziehausen A, Raschke B, Becker V, van Kooten C, Muller G (2001) TGFbeta 1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2). Kidney Int 59:579–592 Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M (2002) Bone marrowderived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res 62:3603–3608 Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, Champlin RE, Andreeff M (2004) Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst 96:1593–1603 Sugimoto H, Mundel TM, Kieran MW, Kalluri R (2006) Identification of fibroblast heterogeneity in the tumor microenvironment. Cancer Biol Ther 5:1640–1646 Sundberg C, Ivarsson M, Gerdin B, Rubin K (1996) Pericytes as collagen-producing cells in excessive dermal scarring. Lab Invest 74:452–466 Suska F, Emanuelsson L, Johansson A, Tengvall P, Thomsen P (2008) Fibrous capsule formation around titanium and copper. J Biomed Mater Res A 85:888–896 Taipale J, Saharinen J, Keski-Oja J (1998) Extracellular matrix-associated transforming growth factor-beta: role in cancer cell growth and invasion. Adv Cancer Res 75:87–134 Tamaoki M, Imanaka-Yoshida K, Yokoyama K, Nishioka T, Inada H, Hiroe M, Sakakura T, Yoshida T (2005) Tenascin-C regulates recruitment of myofibroblasts during tissue repair after myocardial injury. Am J Pathol 167:71–80 Tamariz E, Grinnell F (2002) Modulation of Fibroblast Morphology and Adhesion during Collagen Matrix Remodeling. Mol Biol Cell 13:3915–3929 Taras D, Blanc JF, Rullier A, Dugot-Senant N, Laurendeau I, Bieche I, Pines M, Rosenbaum J (2006) Halofuginone suppresses the lung metastasis of chemically induced hepatocellular carcinoma in rats through MMP inhibition. Neoplasia 8:312–318 Taras D, Blanc JF, Rullier A, Dugot-Senant N, Laurendeau I, Vidaud M, Rosenbaum J (2007) Pravastatin reduces lung metastasis of rat hepatocellular carcinoma via a coordinated decrease of MMP expression and activity. J Hepatol 46:69–76 ten Dijke P, Arthur HM (2007) Extracellular control of TGFbeta signalling in vascular development and disease. Nat Rev Mol Cell Biol 8:857–869 Tenney RM, Discher DE (2009) Stem cells, microenvironment mechanics, and growth factor activation. Curr Opin Cell Biol 21:630–635

3  The Role of the Myofibroblast in Fibrosis and Cancer Progression

73

Terada T, Makimoto K, Terayama N, Suzuki Y, Nakanuma Y (1996a) Alpha-smooth muscle actinpositive stromal cells in cholangiocarcinomas, hepatocellular carcinomas and metastatic liver carcinomas. J Hepatol 24:706–712 Terada T, Okada Y, Nakanuma Y (1996b) Expression of immunoreactive matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human normal livers and primary liver tumors. Hepatology 23:1341–1344 Terada T, Matsunaga Y (2000) Increased mast cells in hepatocellular carcinoma and intrahepatic cholangiocarcinoma. J Hepatol 33:961–966 Thannickal VJ, Toews GB, White ES, Lynch JP 3rd, Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annu Rev Med 55:395–417 Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2:442–454 Tomasek JJ, Vaughan MB, Haaksma CJ (1999) Cellular structure and biology of Dupuytren’s disease. Hand Clin 15:21–34 Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechanoregulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3:349–363 Tomita K, van Bokhoven A, van Leenders GJ, Ruijter ET, Jansen CF, Bussemakers MJ, Schalken JA (2000) Cadherin switching in human prostate cancer progression. Cancer Res 60:3650–3654 Trojanowska M, Varga J (2007) Molecular pathways as novel therapeutic targets in systemic sclerosis. Curr Opin Rheumatol 19:568–573 Uhal BD, Kim JK, Li X, Molina-Molina M (2007) Angiotensin-TGF-beta 1 crosstalk in human idiopathic pulmonary fibrosis: autocrine mechanisms in myofibroblasts and macrophages. Curr Pharm Des 13:1247–1256 van Amerongen MJ et al (2008) Bone marrow-derived myofibroblasts contribute functionally to scar formation after myocardial infarction. J Pathol 214:377–386 van der Loop FT, Schaart G, Timmer ED, Ramaekers FC, van Eys GJ (1996) Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells. J Cell Biol 134:401–411 van Zijl F, Mair M, Csiszar A, Schneller D, Zulehner G, Huber H, Eferl R, Beug H, Dolznig H, Mikulits W (2009) Hepatic tumor–stroma crosstalk guides epithelial to mesenchymal transition at the tumor edge. Oncogene 28(45):4022–4033 Varcoe RL, Mikhail M, Guiffre AK, Pennings G, Vicaretti M, Hawthorne WJ, Fletcher JP, Medbury HJ (2006) The role of the fibrocyte in intimal hyperplasia. J Thromb Haemost 4:1125–1133 Varga J, Abraham D (2007) Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin Invest 117:557–567 Varga J, Pasche B (2009) Transforming growth factor beta as a therapeutic target in systemic sclerosis. Nat Rev Rheumatol 5:200–206 Verona EV, Elkahloun AG, Yang J, Bandyopadhyay A, Yeh IT, Sun LZ (2007) Transforming growth factor-beta signaling in prostate stromal cells supports prostate carcinoma growth by up-regulating stromal genes related to tissue remodeling. Cancer Res 67:5737–5746 Wada T, Sakai N, Matsushima K, Kaneko S (2007) Fibrocytes: a new insight into kidney fibrosis. Kidney Int 72:269–273 Wakefield LM, Stuelten C (2007) Keeping order in the neighborhood: new roles for TGFbeta in maintaining epithelial homeostasis. Cancer Cell 12:293–295 Wang Q, Breinan HA, Hsu HP, Spector M (2000) Healing of defects in canine articular cartilage: distribution of nonvascular alpha-smooth muscle actin-containing cells. Wound Repair Regen 8:145–158 Wang J, Su M, Fan J, Seth A, McCulloch CA (2002) Transcriptional regulation of a contractile gene by mechanical forces applied through integrins in osteoblasts. J Biol Chem 277:22889–22895 Wang J, Chen H, Seth A, McCulloch CA (2003) Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts. Am J Physiol Heart Circ Physiol 285:H1871–1881 Wang D, Park JS, Chu JS, Krakowski A, Luo K, Chen DJ, Li S (2004) Proteomic profiling of bone marrow mesenchymal stem cells upon transforming growth factor beta1 stimulation. J Biol Chem 279:43725–43734

74

B. Hinz et al.

Wells RG (2005) The role of matrix stiffness in hepatic stellate cell activation and liver fibrosis. J Clin Gastroenterol 39:S158–161 Wells RG, Discher DE (2008) Matrix Elasticity, Cytoskeletal Tension, and TGF-β: the Insoluble and Soluble Meet. Sci Signal 1:pe13 Werner S, Grose R (2003) Regulation of wound healing by growth factors and cytokines. Physiol Rev 83:835–870 Winer JP, Janmey PA, McCormick ME, Funaki M (2009) Bone marrow-derived human mesenchymal stem cells become quiescent on soft substrates but remain responsive to chemical or mechanical stimuli. Tissue Eng Part A 15:147–154 Wipff PJ, Hinz B (2008) Integrins and the activation of latent transforming growth factor beta1— an intimate relationship. Eur J Cell Biol 87:601–615 Wipff PJ, Rifkin DB, Meister JJ, Hinz B (2007) Myofibroblast contraction activates latent TGFbeta1 from the extracellular matrix. J Cell Biol 179:1311–1323 Wolfe JN (1976) Risk for breast cancer development determined by mammographic parenchymal pattern. Cancer 37:2486–2492 Wynn TA (2007) Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 117:524–529 Wynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214:199–210 Yamaguchi Y et al (2005) Bone marrow cells differentiate into wound myofibroblasts and accelerate the healing of wounds with exposed bones when combined with an occlusive dressing. Br J Dermatol 152:616–622 Yamamoto H, Itoh F, Adachi Y, Sakamoto H, Adachi M, Hinoda Y, Imai K (1997) Relation of enhanced secretion of active matrix metalloproteinases with tumor spread in human hepatocellular carcinoma. Gastroenterology 112:1290–1296 Yan X et al (2007) Injured microenvironment directly guides the differentiation of engrafted Flk1(+) mesenchymal stem cell in lung. Exp Hematol 35:1466–1475 Ye J, Yao K, Kim JC (2006) Mesenchymal stem cell transplantation in a rabbit corneal alkali burn model: engraftment and involvement in wound healing. Eye 20:482–490 Yeh CN, Maitra A, Lee KF, Jan YY, Chen MF (2004) Thioacetamide-induced intestinal-type cholangiocarcinoma in rat: an animal model recapitulating the multi-stage progression of human cholangiocarcinoma. Carcinogenesis 25:631–636 Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA (2005) Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskeleton 60:24–34 Zalewski A, Shi Y, Johnson AG (2002) Diverse origin of intimal cells: smooth muscle cells, myofibroblasts, fibroblasts, and beyond? Circ Res 91:652–655 Zeisberg M, Kalluri R (2008) Fibroblasts emerge via epithelial–mesenchymal transition in chronic kidney fibrosis. Front Biosci 13:6991–6998 Zeisberg EM, Potenta S, Xie L, Zeisberg M, Kalluri R (2007a) Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts. Cancer Res 67:10123– 10128 Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman AL, McMullen JR, Gustafsson E, Chandraker A, Yuan X, Pu WT, Roberts AB, Neilson EG, Sayegh MH, Izumo S, Kalluri R (2007b) Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 13:952–961 Zeisberg M, Yang C, Martino M, Duncan MB, Rieder F, Tanjore H, Kalluri R (2007c) Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J Biol Chem 282:23337–23347 Zeisberg EM, Potenta SE, Sugimoto H, Zeisberg M, Kalluri R (2008) Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition. J Am Soc Nephrol 19:2282–2287 Zhao XH, Laschinger C, Arora P, Szaszi K, Kapus A, McCulloch CA (2007) Force activates smooth muscle alpha-actin promoter activity through the Rho signaling pathway. J Cell Sci 120:1801–1809

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.

References Abe R, Donnelly SC, Peng T, Bucala R, Metz CN (2001) Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 166:7556–7562 Allinen M, Beroukhim R, Cai L, Brennan C, Lahti-Domenici J, Huang H, Porter D, Hu M, Chin L, Richardson A, Schnitt S, Sellers WR, Polyak K (2004) Molecular characterization of the tumor microenvironment in breast cancer. Cancer Cell 6:17–32 Anderson AR, Quaranta V (2008) Integrative mathematical oncology. Nat Rev Cancer 8:227–234 Anderson AR, Weaver AM, Cummings PT, Quaranta V (2006) Tumor morphology and phenotypic evolution driven by selective pressure from the microenvironment. Cell 127:905–915 Bacac M, Provero P, Mayran N, Stehle JC, Fusco C, Stamenkovic I (2006) A mouse stromal response to tumor invasion predicts prostate and breast cancer patient survival. PLoS One 1:e32 Barth PJ, Ebrahimsade S, Ramaswamy A, Moll R (2002) CD34+ fibrocytes in invasive ductal carcinoma, ductal carcinoma in situ, and benign breast lesions. Virchows Arch 440:298–303 Bearer EL, Lowengrub JS, Frieboes HB, Chuang YL, Jin F, Wise SM, Ferrari M, Agus DB, Cristini V (2009) Multiparameter computational modeling of tumor invasion. Cancer Res 69:4493–4501 Beck AH, Espinosa I, Gilks CB, van de Rijn M, West RB (2008) The fibromatosis signature defines a robust stromal response in breast carcinoma. Lab Invest 88:591–601 Bellini A, Mattoli S (2007) The role of the fibrocyte, a bone marrow-derived mesenchymal progenitor, in reactive and reparative fibroses. Lab Invest 87:858–870 Borchers AH, Steinbauer H, Schafer BS, Kramer M, Bowden GT, Fusenig NE (1997) Fibroblastdirected expression and localization of 92-kDa type IV collagenase along the tumor-stroma interface in an in vitro three-dimensional model of human squamous cell carcinoma. Mol Carcinog 19:258–266 Carragher NO (2009) Profiling distinct mechanisms of tumour invasion for drug discovery: imaging adhesion, signalling and matrix turnover. Clin Exp Metastasis 26:381–397 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 U S A 99:12877–12882

86

O. De Wever et al.

Chang HY, Sneddon JB, Alizadeh AA, Sood R, West RB, Montgomery K, Chi JT, van de Rijn M, Botstein D, Brown PO (2004) Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS Biol 2:E7 Chang HY, Nuyten DS, Sneddon JB, Hastie T, Tibshirani R, Sorlie T, Dai H, He YD, van’t Veer LJ, Bartelink H, van de Rijn M, Brown PO, van de Vijver MJ (2005) Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival. Proc Natl Acad Sci U S A 102:3738–3743 Daly AJ, McIlreavey L, Irwin CR (2008) Regulation of HGF and SDF-1 expression by oral fibroblasts-implications for invasion of oral cancer. Oral Oncol 44:646–651 de Jonge MJ, Dumez H, Verweij J, Yarkoni S, Snyder D, Lacombe D, Marreaud S, Yamaguchi T, Punt CJ, van Oosterom A (2006) Phase I and pharmacokinetic study of halofuginone, an oral quinazolinone derivative in patients with advanced solid tumours. Eur J Cancer 42:1768–1774 Denys H, Derycke L, Hendrix A, Westbroek W, Gheldof A, Narine K, Pauwels P, Gespach C, Bracke M, De Wever O (2008) Differential impact of TGF-beta and EGF on fibroblast differentiation and invasion reciprocally promotes colon cancer cell invasion. Cancer Lett 266:263– 274 De Wever O, Mareel M (2003) Role of tissue stroma in cancer cell invasion. J Pathol 200:429–447 De Wever O, Nguyen QD, Van Hoorde L, Bracke M, Bruyneel E, Gespach C, Mareel M (2004) Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac. FASEB J 18:1016–1018 De Wever O, Demetter P, Mareel M, Bracke M (2008) Stromal myofibroblasts are drivers of invasive cancer growth. Int J Cancer 123:2229–2238 De Wever O, Hendrix A, De Boeck A, Westbroek W, Braems G, Emami S, Sabbah M, Gespach C, Bracke M (2010) Modeling and quantification of cancer cell invasion through collagen type I matrices. Int J Dev Biol 54(5):887–896 Direkze NC, Hodivala-Dilke K, Jeffery R, Hunt T, Poulsom R, Oukrif D, Alison MR, Wright NA (2004) Bone marrow contribution to tumor-associated myofibroblasts and fibroblasts. Cancer Res 64:8492–8495 Dvorak HF (1986) Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 315:1650–1659 Erler JT, Bennewith KL, Cox TR, Lang G, Bird D, Koong A, Le QT, Giaccia AJ (2009) Hypoxiainduced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche. Cancer Cell 15:35–44 Eyden B (2005) The myofibroblast: a study of normal, reactive and neoplastic tissues, with an emphasis on ultrastructure. Part I—normal and reactive cells. J Submicroscop Cytol Pathol 37:109–204 Farmer P, Bonnefoi H, Anderle P, Cameron D, Wirapati P, Becette V, Andre S, Piccart M, Campone M, Brain E, Macgrogan G, Petit T, Jassem J, Bibeau F, Blot E, Bogaerts J, Aguet M, Bergh J, Iggo R, Delorenzi M (2009) A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat Med 15:68–74 Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, Chen H, Omeroglu G, Meterissian S, Omeroglu A, Hallett M, Park M (2008) Stromal gene expression predicts clinical outcome in breast cancer. Nat Med 14:518–527 Frieboes HB, Zheng X, Sun CH, Tromberg B, Gatenby R, Cristini V (2006) An integrated computational/experimental model of tumor invasion. Cancer Res 66:1597–1604 Garabedian EM, Humphrey PA, Gordon JI (1998) A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells. Proc Natl Acad Sci U S A 95:15382–15387 Genin O, Rechavi G, Nagler A, Ben-Itzhak O, Nazemi KJ, Pines M (2008) Myofibroblasts in pulmonary and brain metastases of alveolar soft-part sarcoma: a novel target for treatment? Neoplasia 10:940–948 Hasebe T, Tsuda H, Hirohashi S, Shimosato Y, Tsubono Y, Yamamoto H, Mukai K (1998) Fibrotic focus in infiltrating ductal carcinoma of the breast: a significant histopathological prognostic parameter for predicting the long-term survival of the patients. Breast Cancer Res Treat 49:195–208

4  The Role of Myofibroblasts in Communicating Tumor Ecosystems

87

Ishii G, Sangai T, Oda T, Aoyagi Y, Hasebe T, Kanomata N, Endoh Y, Okumura C, Okuhara Y, Magae J, Emura M, Ochiya T, Ochiai A (2003) Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction. Biochem Biophys Res Commun 309:232–240 Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JC, Trent JM, Staudt LM, Hudson J Jr, Boguski MS, Lashkari D, Shalon D, Botstein D, Brown PO (1999) The transcriptional program in the response of human fibroblasts to serum. Science 283:83–87 Joyce JA, Pollard JW (2009) Microenvironmental regulation of metastasis. Nat Rev Cancer 9:239–252 Kaplan RN, Riba RD, Zacharoulis S, Bramley AH, Vincent L, Costa C, MacDonald DD, Jin DK, Shido K, Kerns SA, Zhu Z, Hicklin D, Wu Y, Port JL, Altorki N, Port ER, Ruggero D, Shmelkov SV, Jensen KK, Rafii S, Lyden D (2005) VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438:820–827 Kunz-Schughart LA, Heyder P, Schroeder J, Knuechel R (2001) A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation. Exp Cell Res 266:74–86 Lang P, Yeow K, Nichols A, Scheer A (2006) Cellular imaging in drug discovery. Nat Rev Drug Discov 5:343–356 Lehn CN, Rapoport A (1994) The desmoplastic lymph node reaction as a prognostic factor of cancer of the tongue and floor of the mouth. Sao Paulo Med J 112:591–596 Lu J, Marnell LL, Marjon KD, Mold C, Du Clos TW, Sun PD (2008) Structural recognition and functional activation of FcgammaR by innate pentraxins. Nature 456:989–992 Mantyh PW, Clohisy DR, Koltzenburg M, Hunt SP (2002) Molecular mechanisms of cancer pain. Nat Rev Cancer 2:201–209 Mareel M, Oliveira MJ, Madani I (2009) Cancer invasion and metastasis: interacting ecosystems. Virchows Arch 454:599–622 McAllister SS, Gifford AM, Greiner AL, Kelleher SP, Saelzler MP, Ince TA, Reinhardt F, Harris LN, Hylander BL, Repasky EA, Weinberg RA (2008) Systemic endocrine instigation of indolent tumor growth requires osteopontin. Cell 133:994–1005 McGaha TL, Kodera T, Spiera H, Stan AC, Pines M, Bona CA (2002) Halofuginone inhibition of COL1A2 promoter activity via a c-Jun-dependent mechanism. Arthritis Rheum 46:2748–2761 Mori L, Bellini A, Stacey MA, Schmidt M, Mattoli S (2005) Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Exp Cell Res 304:81–90 Muehlberg FL, Song YH, Krohn A, Pinilla SP, Droll LH, Leng X, Seidensticker M, Ricke J, Altman AM, Devarajan E, Liu W, Arlinghaus RB, Alt EU (2009) Tissue-resident stem cells promote breast cancer growth and metastasis. Carcinogenesis 30:589–597 Mueller MM, Fusenig NE (2004) Friends or foes—bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 4:839–849 Newman LA, Pernick NL, Adsay V, Carolin KA, Philip PA, Sipierski S, Bouwman DL, Kosir MA, White M, Visscher DW (2003) Histopathologic evidence of tumor regression in the axillary lymph nodes of patients treated with preoperative chemotherapy correlates with breast cancer outcome. Ann Surg Oncol 10:734–739 Nystrom ML, Thomas GJ, Stone M, Mackenzie IC, Hart IR, Marshall JF (2005) Development of a quantitative method to analyse tumour cell invasion in organotypic culture. J Pathol 205:468–475 Okamoto T, Tsuburaya A, Kameda Y, Yoshikawa T, Cho H, Tsuchida K, Hasegawa S, Noguchi Y (2008) Prognostic value of extracapsular invasion and fibrotic focus in single lymph node metastasis of gastric cancer. Gastric Cancer 11:160–167 Olaso E, Santisteban A, Bidaurrazaga J, Gressner AM, Rosenbaum J, Vidal-Vanaclocha F (1997) Tumor-dependent activation of rodent hepatic stellate cells during experimental melanoma metastasis. Hepatology 26:634–642 Pepys MB, Herbert J, Hutchinson WL, Tennent GA, Lachmann HJ, Gallimore JR, Lovat LB, Bartfai T, Alanine A, Hertel C, Hoffmann T, Jakob-Roetne R, Norcross RD, Kemp JA, Yamamura K, Suzuki M, Taylor GW, Murray S, Thompson D, Purvis A, Kolstoe S, Wood SP, Hawkins PN (2002) Targeted pharmacological depletion of serum amyloid P component for treatment of human amyloidosis. Nature 417:254–259

88

O. De Wever et al.

Pilling D, Buckley CD, Salmon M, Gomer RH (2003) Inhibition of fibrocyte differentiation by serum amyloid P. J Immunol 171:5537–5546 Pilling D, Tucker NM, Gomer RH (2006) Aggregated IgG inhibits the differentiation of human fibrocytes. J Leukoc Biol 79:1242–1251 Pilling D, Buckley CD, Salmon M, Gomer RH (2007) Serum amyloid P and fibrosis in systemic sclerosis: comment on the article by Tennent et al. Arthritis Rheum 56:4229; author reply 4229–4230 Possemiers S, Grootaert C, Vermeiren J, Gross G, Marzorati M, Verstraete W, van de Wiele T (2009) The intestinal environment in health and disease—recent insights on the potential of intestinal bacteria to influence human health. Curr Pharm Des 15:2051–2065 Postlethwaite AE, Shigemitsu H, Kanangat S (2004) Cellular origins of fibroblasts: possible implications for organ fibrosis in systemic sclerosis. Curr Opin Rheumatol 16:733–738 Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White JG, Keely PJ (2006) Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Med 4:38 Provenzano PP, Inman DR, Eliceiri KW, Knittel JG, Yan L, Rueden CT, White JG, Keely PJ (2008) Collagen density promotes mammary tumor initiation and progression. BMC Med 6:11 Reilly JT, Nash JR (1988) Vitronectin (serum spreading factor): its localisation in normal and fibrotic tissue. J Clin Pathol 41:1269–1272 Rønnov-Jessen L, Petersen OW, Koteliansky VE, Bissell MJ (1995) The origin of the myofibroblasts in breast-cancer—recapitulation of tumor environment in culture unravels diversity and implicates converted fibroblasts and recruited smooth. J Clin Investig 95:859–873 Sabeh F, Shimizu-Hirota R, Weiss SJ (2009) Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited. J Cell Biol 185:11–19 Schurch W, Seemayer TA, Lagace R (1981) Stromal myofibroblasts in primary invasive and metastatic carcinomas. A combined immunological, light and electron microscopic study. Virchows Arch A Pathol Anat Histol 391:125–139 Shao DD, Suresh R, Vakil V, Gomer RH, Pilling D (2008) Pivotal advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation. J Leukoc Biol 83:1323–1333 Sheffer Y, Leon O, Pinthus JH, Nagler A, Mor Y, Genin O, Iluz M, Kawada N, Yoshizato K, Pines M (2007) Inhibition of fibroblast to myofibroblast transition by halofuginone contributes to the chemotherapy-mediated antitumoral effect. Mol Cancer Ther 6:570–577 Tanaka Y, Hirokawa M, Wakatsuki S, Sano T (2002) Metastatic adenocarcinoma of the lymph node: peculiar desmoplasia mimicking intravenous metastasis. Histopathology 41:566–567 Tennent GA, Dziadzio M, Triantafillidou E, Davies P, Gallimore JR, Denton CP, Pepys MB (2007) Normal circulating serum amyloid P component concentration in systemic sclerosis. Arthritis Rheum 56:2013–2017 Thariat J, Ahamad A, El-Naggar AK, Williams MD, Holsinger FC, Glisson BS, Allen PK, Morrison WH, Weber RS, Ang KK, Garden AS (2008) Outcomes after radiotherapy for basaloid squamous cell carcinoma of the head and neck: a case-control study. Cancer 112:2698–2709 Tsujino T, Seshimo I, Yamamoto H, Ngan CY, Ezumi K, Takemasa I, Ikeda M, Sekimoto M, Matsuura N, Monden M (2007) Stromal myofibroblasts predict disease recurrence for colorectal cancer. Clin Cancer Res 13:2082–2090 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 8:2912–2923 Vidal-Vanaclocha F (2008) The prometastatic microenvironment of the liver. Cancer Microenviron 1:113–129 Wels J, Kaplan RN, Rafii S, Lyden D (2008a) Migratory neighbors and distant invaders: tumorassociated niche cells. Genes Dev 22:559–574 Wels J, Kaplan RN, Rafii S, Lyden D (2008b) Migratory neighbors and distant invaders: tumorassociated niche cells. Genes Dev 22:559–574 Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Brocker EB, Friedl P (2003) Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160:267–277

4  The Role of Myofibroblasts in Communicating Tumor Ecosystems

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Wolf K, Wu YI, Liu Y, Geiger J, Tam E, Overall C, Stack MS, Friedl P (2007) Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat Cell Biol 9:893–904 Yee KO, Connolly CM, Pines M, Lawler J (2006) Halofuginone inhibits tumor growth in the polyoma middle T antigen mouse via a thrombospondin-1 independent mechanism. Cancer Biol Ther 5:218–224

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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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