Tumour-Associated Macrophages

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Tumour-Associated Macrophages

Toby Lawrence • Thorsten Hagemann Editors

Tumour-Associated Macrophages

Editors Toby Lawrence Centre d’Immunologie Marseille Luminy Inserm-CNRS-Universitie de Mediteranee Inflammation Biology Group Parc Scientifique de Luminy, Case 906 Marseille Cedex 9, France [email protected]

Thorsten Hagemann Centre for Cancer and Inflammation Barts Cancer Institute, Barts and the London School of Medicine and Dentistry Queen Mary University of London London, EC1M 6BQ, UK [email protected]

ISBN 978-1-4614-0661-7 e-ISBN 978-1-4614-0662-4 DOI 10.1007/978-1-4614-0662-4 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011937444 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Macrophages are tissue resident phagocytes derived from blood monocytes; they have diverse functions in development and immunity and display enormous phenotypic heterogeneity. Macrophages in different tissues have specialized and specific functions that support organ development and physiology, for example, kupffer cells in the liver filter debris from the blood and aid liver regeneration after injury, Langerhans cells in the skin are important immune sentinel cells and mediate immune surveillance, osteoclasts mediate bone morphogenesis, and microglia in the brain support the development and maintenance of neuronal networks. In response to inflammation or injury, monocytes are recruited into tissue and differentiate locally into macrophages and depending on the nature of the insult or injury these macrophages may acquire distinct phenotypes. Tumours are frequently infiltrated by large number of macrophages and in most cases this is linked with tumour progression and poor prognosis. Macrophage polarization is a poorly defined phenomenon; the mediators and mechanisms that maintain the phenotype of distinct macrophage subsets in both physiology and disease remain to be described. Based primarily on in vitro studies, two particular macrophage phenotypes have been described: “classically” activated or M1 macrophages are characterized by the production of pro-inflammatory cytokines and increased microbicidal or even tumouricidal activity. The second, “alternatively” activated or M2 macrophages, in contrast produce anti-inflammatory cytokines and are linked with angiogenesis, tissue repair, and remodeling. These polarized phenotypes have been described based on in vitro stimulation of macrophages with either interferon (IFN) g, in the case of M1 macrophages, or interleukin (IL)-4 for M2 macrophages. It still is not clear what correlates these populations have in vivo and their physiological relevance remains ambiguous. While these classifications have been useful in that they allow the functional grouping of different macrophage phenotypes, M1 macrophages being proinflammatory cells and M2 macrophages linked with trophic functions and wound healing, there are undoubtedly several intermediates between these polarized phenotypes. However, this classification is too restrictive and it is clear that the functional diversity macrophages in vivo may not be associated with these distinct phenotypic subsets. In fact, the question remains in the context of inflammation and v

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Preface

tumours if “the macrophage” merely displays functional plasticity within tissue responding to environmental cues, or distinct stable subsets of macrophages exist with specialized functions. This issue is particularly pertinent in the case of TAM; these cells often display an M2-like phenotype associated with trophic functions promoting tumour angiogenesis, invasion, and metastasis. However, TAM also often produce pro-inflammatory cytokines and have been associated with the promotion of inflammation-associated cancer. This volume provides an overview of current research on the form and function of TAM, highlighting both the mechanistic roles they play in carcinogenesis and tumour progression as well as the molecular mechanisms that control their phenotype and function. Marseille, France London, UK

Toby Lawrence Thorsten Hagemann

Contents

Part I

Form and Function

1

Macrophage Phenotype in Tumours ..................................................... Hsi-Hsien Lin and Siamon Gordon

2

Role of Tumour-Associated Macrophages in the Regulation of Angiogenesis ........................................................................................ Russell Hughes, Hsin-Yu Fang, Munitta Muthana, and Claire E. Lewis

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The Role of Tumour-Associated Macrophages in Malignant Invasion ............................................................................. Claudia Binder

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31

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Tumour-Induced Immune Suppression by Myeloid Cells................... Serena Zilio, Giacomo Desantis, Mariacristina Chioda, and Vincenzo Bronte

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5

TAM: A Moving Clinical Target ............................................................ Simon Hallam and Thorsten Hagemann

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Part II

Mechanisms of Action

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Arginine Metabolism and Tumour-Associated Macrophages............. Melissa Phillips and Peter W. Szlosarek

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Indoleamine 2,3-Dioxygenase Amino Acid Metabolism and Tumour-Associated Macrophages: Regulation in Cancer-Associated Inflammation and Immune Escape .................. George C. Prendergast, Richard Metz, Mee Young Chang, Courtney Smith, Alexander J. Muller, and Suzanne Ostrand-Rosenberg

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Contents

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Vascular Endothelial Growth Factor and Tumour-Associated Macrophages............................................................................................ 105 Christian Stockmann and Randall S. Johnson

Part III

Molecular Regulation

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TLR Signaling and Tumour-Associated Macrophages........................ 119 Oscar R. Colegio and Ruslan Medzhitov

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SHIP and Tumour-Associated Macrophages ....................................... 135 Victor W. Ho, Melisa J. Hamilton, Etsushi Kuroda, Jens Ruschmann, Frann Antignano, Vivian Lam, and Gerald Krystal

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NF-KappaB-Mediated Regulation of Tumour-Associated Macrophages: Mechanisms and Significance ....................................... 153 Antonio Sica and Alberto Mantovani

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Role of Hypoxia-Inducible Transcription Factors in TAM Function ..................................................................................... 167 Nadine Rohwer and Thorsten Cramer

Index ................................................................................................................. 183

Contributors

Frann Antignano The Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, BC V5Z 1L3, Canada Claudia Binder Department of Hematology/Oncology, University of Göttingen, Göttingen 37099, Germany Vincenzo Bronte Verona University Hospital and Department of Pathology, Immunology Section, Piazzale L.A. Scuro 10, 37134 Verona, Italy Mee Young Chang Lankenau Institute for Medical Research, Wynnewood, PA, USA Mariacristina Chioda Istituto Oncologico Veneto (IOV), IRCCS, Via Gattamelata 64, 35128 Padova, Italy Oscar R. Colegio Department of Immunobiology, Howard Hughes Medical Institute, Yale University, New Haven, CT, USA Thorsten Cramer Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie and Molekulares Krebsforschungszentrum, Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany Giacomo Desantis Department of Oncology and Surgical Sciences, University of Padova, Via Gattamelata 64, 35128 Padova, Italy Hsin-Yu Fang Academic Unit of Inflammation & Tumour Targeting, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK Siamon Gordon Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK

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Contributors

Thorsten Hagemann Centre for Cancer and Inflammation, Barts Cancer Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK Simon Hallam Centre for Cancer and Inflammation, Barts Cancer Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK Melisa J. Hamilton The Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, BC V5Z 1L3, Canada Victor W. Ho The Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, BC V5Z 1L3, Canada Russell Hughes Academic Unit of Inflammation & Tumour Targeting, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK Randall S. Johnson Molecular Biology Section, Division of Biology, University of California, San Diego, CA 92093, USA Gerald Krystal The Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, BC V5Z 1L3, Canada Etsushi Kuroda The Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, BC V5Z 1L3, Canada Vivian Lam The Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, BC V5Z 1L3, Canada Claire E. Lewis Academic Unit of Inflammation & Tumour Targeting, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK Hsi-Hsien Lin Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, Taiwan Alberto Mantovani Istituto Clinico Humanitas IRCCS, via Manzoni 56, 20089 Rozzano, Italy Department of Translational Medicine, University of Milan, Milan, Italy Ruslan Medzhitov Department of Immunobiology, Howard Hughes Medical Institute, Yale University, New Haven, CT, USA Richard Metz New Link Genetics Corporation, Ames, IA, USA Alexander J. Muller Lankenau Institute for Medical Research, Wynnewood, PA, USA Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA Munitta Muthana Academic Unit of Inflammation & Tumour Targeting, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK

Contributors

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Suzanne Ostrand-Rosenberg Department of Biological Sciences, University of Maryland at Baltimore, Baltimore, MD, USA Melissa Phillips Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and The London School of Medicine, Queen Mary College, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK George C. Prendergast Lankenau Institute for Medical Research, Wynnewood, PA, USA Department of Pathology, Anatomy & Cell Biology, Jefferson Medical School, Philadelphia, PA, USA Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA Nadine Rohwer Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie and Molekulares Krebsforschungszentrum, Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany Jens Ruschmann The Terry Fox Laboratory, B.C. Cancer Agency, Vancouver, BC V5Z 1L3, Canada Antonio Sica Istituto Clinico Humanitas IRCCS, via Manzoni 56, 20089 Rozzano, Italy DiSCAFF, University of Piemonte Orientale A. Avogadro, 28100 Novara, Italy Courtney Smith Lankenau Institute for Medical Research, Wynnewood, PA, USA Christian Stockmann Molecular Biology Section, Division of Biology, University of California, San Diego, CA 92093, USA Institut für Physiologie, University of Duisburg-Essen, Duisburg-Essen, Germany Peter W. Szlosarek Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and The London School of Medicine, Queen Mary College, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK Serena Zilio Department of Oncology and Surgical Sciences, University of Padova, Via Gattamelata 64, 35128 Padova, Italy Istituto Oncologico Veneto (IOV), IRCCS, Via Gattamelata 64, 35128 Padova, Italy

Part I

Form and Function

Chapter 1

Macrophage Phenotype in Tumours Hsi-Hsien Lin and Siamon Gordon

Introduction Monocytes and macrophages are a prominent component of the host response to, and manipulation by, tumour cells (Gordon and Martinez 2010; Mantovani et al. 2008). Together with other myeloid and lymphoid cells, they influence tumour development, both positively and negatively. Although the factors that determine outcome of the host–tumour relationship are not well understood, many tumours recruit immature myelomonocytic cells, block their differentiation, subvert their cytotoxicity, suppress lymphoid effector cells, and induce peripheral tolerance. In addition, they mimic and utilise macrophage functions to enhance growth, produce a stroma and promote angiogenesis, local invasion of their micro-environment and metastasis (Qian and Pollard 2010). In particular, the uptake of apoptotic tumour cells can suppress anti-tumour inflammatory responses by TGF-beta and prostaglandins. The macrophage growth factor CSF-1 stimulates macrophage recruitment and modulates its phenotype, limiting the activation of cytotoxic effector functions; Interleukin-4 and -13, acting through common and specific receptors, induce a trophic, alternative M2 activation phenotype, distinct from cytotoxic M1, classically activated (Interferon-gamma-dependent) macrophages (Reviewed by Gordon and Martinez 2010). Interleukin-10 is a potent deactivator of macrophage inflammatory properties, whereas TGF-beta, another deactivator, promotes fibrosis and vascular remodelling.

H.-H. Lin Department of Microbiology and Immunology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, Taiwan S. Gordon (*) Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK e-mail: [email protected] T. Lawrence and T. Hagemann (eds.), Tumour-Associated Macrophages, DOI 10.1007/978-1-4614-0662-4_1, © Springer Science+Business Media, LLC 2012

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H.-H. Lin and S. Gordon

A wide range of chemokines such as MCP-1, often produced by tumour cells, attract mononuclear and myeloid cells. TNF-alpha has also been implicated in tumourigenesis (Mantovani et al. 2008). Monocyte-macrophages express a wide range of plasma membrane receptors which govern their response to chemokines, cytokines, growth factors and other tumour- and host-derived ligands (Taylor et al. 2005). Other membrane molecules regulate cellular responses to diverse agonists, inhibiting or enhancing macrophage effector mechanisms. These molecules provide useful markers for the presence, characterisation and possible functions of tumour-associated monocyte/macrophages, and targets for therapeutic intervention. In this review, we present a range of possible molecular markers for in situ characterisation, with special reference to the EGF-TM7 family of myeloid G protein-coupled receptors (GPCRs) with large extracellular domains. Their potential is reviewed in the context of macrophage heterogeneity and plasticity (Auffray et al. 2009; Gordon and Taylor 2005) and the experimental analysis of macrophage phenotype in tumours.

Macrophage Heterogeneity in Tumours Some of the earliest studies on the presence and possible role of macrophages in tumours were undertaken by Evans and Alexander, Mantovani, Pollard and their collaborators (Mantovani et al. 2008; Qian and Pollard 2010). The topic received renewed impetus in recent years with the work of Bronte (Peranzoni et al. 2010) and Gabrilovitch (Gabrilovich and Nagaraj 2009) and their groups. Important contributions came from Balkwill (Mantovani et al. 2008), Lewis (Coffelt et al. 2009), Karin (Grivennikov et al. 2010) and Coussens (Coussens and Werb 2002), Rosenberg (Domachowske et al. 2000) and Joyce (Joyce and Pollard 2009). A great deal of confusion has resulted from myeloid cell heterogeneity and terms such as TAMs (tumour-associated macrophages) and MDSC (myeloid-derived suppessor cells) are currently in wide use. The former embraces cells with macrophagerestricted markers such as F4/80 and alternative activation markers such as Arginase-1 (Gordon and Martinez 2010); the latter term includes cells with immature monocytic phenotype (Gr-1 low) and granulocyte characteristics (Gr-1 high). Mononuclear phagocyte heterogeneity associated with stages of differentiation and activation status gives rise to considerable plasticity within and among cell populations. Studies by Geissmann (Geissmann et al. 2010) and Jung (Varol et al. 2009) have utilised the fractalkine receptor, in combination with other chemokine receptors, to define precursors of tissue macrophages during development, adult life, physiologically and in various inflammatory and pathologic states. Studies by Nussenzweig (Dudziak et al. 2007), Merad (Merad 2010; Merad and Manz 2009) and their colleagues have helped to clarify the origins and population dynamics of myeloid dendritic cells, vis-à-vis monocyte/macrophages. Their fluorescence and transgenic methods will be useful to trace precursors of myelomonocytic cells in mouse tumours.

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Macrophage Phenotype in Tumours

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Tumours are obviously heterogeneous themselves, not only in their ability to invade (benign or malignant), but also in their micro-environment (lung, liver, bone and lymph nodes), origin (epithelial, mesenchymal and haemopoietic), vascularisation, within individual tumours as well as among different primary or secondary tumour populations. Other differences pertain as tumours induce matrix synthesis and catabolism, undergo hypoxia, apoptosis and necrosis. The concomitant presence of CD4+, CD8+ lymphocytes, FoxP3 positive suppressor cells, as well as innate lymphoid cells (NKT and NK cells) modulates myeloid cells, reciprocally. Tumour cells themselves often express characteristic properties of leukocytes that can contribute to their migration and invasion. Macrophages can also be tolerogenic and contribute to lymphocyte suppression by cell contact or secretory products. Dendritic cell maturation and antigen presentation can also be subverted by tumour- or other myeloid-derived products. Apart from the above considerations, many difficulties hinder experimental analysis of macrophage phenotype in tumours. Ideally, one should study naturally occurring tumours in situ, rather than transplantable models. Isolation of myeloid cells, especially macrophages, is difficult and prone to artefact, particularly if FACS analysis is not combined with immunocytochemistry in situ. The use of oncogenic transgenes, e.g. by Hanahan and colleagues (Hanahan 1989) made it possible to synchronise defined stages of experimental tumours. Mouse models do not necessarily replicate human tumours, often studied at late stages, or after chemotherapy and irradiation. Finally, macrophage markers used in the mouse and human may differ markedly between species. The interactions between macrophages and tumour cells result in novel gene expression profiles in both cell types, only partially reproduced during co-cultivation in vitro. Microarray and proteomic analyses, while powerful indicators of signatures, e.g. of type 1 interferon activation pathways, need refinement. The traditional methods of morphologic, diagnostic pathology are undergoing rapid advances, but have not yet progressed to interpret function at the single-cell level sufficiently.

Membrane Markers for Macrophages in Tumours Given the above caveats, we present a list of validated and candidate antigen markers to define macrophage heterogeneity in tumours (Table 1.1). We feel that the present focus reported in the literature is too narrow, that FACS analysis of isolated macrophages is insufficient and that whilst antigens are reasonably well-defined in the mouse, markers for human antigens are limited and not sufficiently characterised. Monoclonal and polyclonal antibodies for FACS and western blotting are not necessarily suitable for immunocytochemistry. Tissue preservation, antigen stability and antibody staining need to be optimised for each epitope. Table 1.1 includes members of a range of molecular families, varying in cell specificity. Markers include opsonic and non-opsonic phagocytic receptors, lectins and scavenger receptors, as well as cytokine receptors and other differentiation antigens, with some functional correlates.

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Table 1.1 Selected membrane markers for macrophages in tumours Molecule Property Comment F4/80 EGF-TM7/adhesion-GPCR Peripheral tolerance, MI subpopulation CD97 EGF-TM7/adhesion-GPCR Myeloid, other cells EMR2 EGF-TM7/adhesion-GPCR Human, not mouse, aberrant in breast cancers CD68 LAMP family Pan-MI and DC, some tumours Gr-1 Ly-6 family PMN, immature monocytes Polymorphic, PMN, immature monocytes 7/4 Ly-6 family (Rosas et al. 2010) Siglec-1 IgSF Sialyl-ligand, e.g. Muc-1 CD163 SRCR family Glucocorticoid, IL-10 induced CD200/CD200R IgSF Receptor/ligand pair, negative regulator FcR IgSF Activatory/inhibitory CR3 Beta-2 integrin Opsonic and non-opsonic phagocytosis Adhesion SR-A SRCR family Clearance apoptotic cells, CSF-1 upregn MARCO SRCR family Adhesion, induced via TLRs CD36 Bispanner SR-B Ox-LDL, Apoptotic, Thrombospondin R MR C-type lectin Alternative activation marker Dectin-1 C-type lectin-like Beta-glucan R, ITAM-like domain Dectin-2 C-type lectin-like Subset macrophages, Mannose-ligand TLRs Leucine-rich repeat Sensor exogenous, host ligands IL-4/13 R Cytokine R Common, specific R, alternative activation CSF-1R Receptor tyrosine kinase fms GM-CSF R Haemopoietic R Fc-GMCSF chimeric ligand (Rosas et al. 2007) CX3CR1 GPCR Membrane bound fractalkine R CCR2 GPCR MCP-1 ligand

Curiously, in some cases, e.g. CD68, non-haemopoietic tumour cells are able to express leukocyte markers ectopically. Giant cells and hybrids arising from fusion of tumour cells and macrophages provide another mechanism for aberrant marker expression. Some of these markers have been utilised in inflammatory and infectious models in the mouse but not in tumours. The need for co-localisation and double/multiple labelling, so useful in FACS, is more difficult to achieve in immunohistochemistry, which also lacks quantitation. Laser capture microscopy and tissue arrays may overcome some of these difficulties.

EGF-TM7 Receptors and Tumour-Associated Macrophages The mouse differentiation antigen F4/80 is a well-characterised marker for mouse macrophages and has been implicated in peripheral tolerance in a non-tumour model (see below for references). The mouse and human antigen CD97 is not only associated

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with myeloid cell differentiation and activation, but has also been studied in a variety of tumour settings in vivo. The closely related antigen EMR2 provides a sensitive marker for macrophage identification in human tissues. We review the common and selective characteristics of these molecules in detail, in relation to tissue specificity and as potential markers of macrophage heterogeneity and function in tumour–host interactions.

Common Characteristics of the EGF-TM7 Receptors F4/80, EMR2 and CD97 all belong to the group of EGF-TM7 molecules that make up the second largest GPCR sub-family in man, the adhesion-GPCRs (Fig. 1.1) (McKnight and Gordon 1996; McKnight and Gordon 1998; Stacey et al. 2000; Yona et al. 2008a; Bjarnadottir et al. 2004; Bjarnadottir et al. 2007). The EGF-TM7 receptors share many common characteristics in protein structure, cellular function

E E E E E

E

E

E E

E E

E

E

E

E

E E

F4/80

Expression

EGF-like domains GPS proteolysis Ligand(s)

Function(s)

EMR2

Monocytes Resident tissue macrophages (Kupffer cells, microglia, etc.) Immature dendritic cells Eosinophils TAMs 7

Monocytes Macrophages Dendritic cells Neutrophils

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No Unknown

Yes Chondroitin sulfate

Induction of Peripheral tolerance

Priming of neutrophils

CD97

Lymphocytes Monocytes Macrophages Dendritic cells Neutrophils Smooth muscle cells Tumour cells 5 Yes CD55, Chondroitin sulfate Integrins (A5B1/AvB3 ) Migration and homeostasis of PMNs, HSC mobilization, CD4+ T cell co-stimulation, Angiogenesis, Tumour invasion

Fig. 1.1 Characteristics of F4/80, EMR2 and CD97. The three receptors are represented schematically. The EGF-like (E) motifs are shown as triangles, the GPS motif as a triangle with two disulfide bonds and the 7TM domain is represented by seven cylinders

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and expression patterns (Stacey et al. 2000; Yona et al. 2008a). As suggested by the nomenclature, EGF-TM7 molecules are chimeric proteins composed of two major protein modules, namely the epidermal growth factor (EGF)-like motifs and the seven-span transmembrane (7TM) domains. A typical EGF-TM7 molecule contains tandem repeats of EGF-like motifs at the N terminus followed by a stalk region, which connects to a 7TM domain (McKnight and Gordon 1996; McKnight and Gordon 1998). Most of the EGF-like motifs of the EGF-TM7 molecules belong to the Ca2+binding subtype usually found in extracellular matrix proteins. Thus, Ca2+ binding is important for the cellular function of these receptors (Lin et al. 2001). The 7TM domains of the EGF-TM7 molecules share strong sequence similarity to the class B or secretin-like GPCRs. However, recent phylogenetic analyses have suggested an independent evolutionary lineage and group them with others to form the adhesionGPCR sub-family (Bjarnadottir et al. 2004; Bjarnadottir et al. 2007; Fredriksson et al. 2003). The stalk region between the EGF-like motifs and the 7TM domain usually contains numerous Ser and Thr residues and N-link glycosylation sites. Therefore, it is believed to be heavily decorated with O- and N-link glycans and is thought as a rigid structure. In addition, a highly conserved Cys-rich motif of ~50 residues located immediately upstream of the first TM region has been identified. A posttranslational auto-proteolytic reaction at this Cys-rich motif would cleave the receptor molecule into an extracellular- and 7TM-subunits (Lin et al. 2004). The specific proteolytic cleavage site is, therefore, named the GPCR proteolysis site (GPS) (Krasnoperov et al. 1999). It is thought that the EGF-TM7 receptors would bind to specific cellular ligands through the extracellular domain, especially by the EGF-like motifs, which in turn activate the 7TM domain to transmit intracellular signals. The majority of the EGF-TM7 receptors are restrictedly expressed in myeloid cells, including monocytes, macrophages, polymorphonuclear cells and dendritic cells (Stacey et al. 2000; Yona et al. 2008a). Thus, a role in innate as well as adaptive immune functions was predicted for these molecules.

F4/80 The F4/80 molecule is a ~160 kDa cell surface glycoprotein (Austyn and Gordon 1981). However, the full-length F4/80 cDNA predicted a mature protein of 904 amino acid residues with an estimated molecular weight of 99 kDa (Lin et al. 1997; McKnight et al. 1996). It was shown that the ~60 kDa difference in size was due to heavy glycosylation of the molecule as predicted by the multiple O- and N-linked glycosylation sites in the stalk region. In addition, a total of 7 EGF-like motifs, one glycosaminoglycan attachment site and an Arg-Gly-Asp (RGD) motif, were also identified in the extracellular domain of F4/80 (McKnight and Gordon 1996; McKnight and Gordon 1998; McKnight et al. 1996). Despite the presence of a typical

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GPS motif, no GPS proteolytic cleavage was identified in F4/80 because of the lack of a consensus cleavage site. Hence, unlike typical adhesion-GPCRs, the F4/80 receptor is a single-chain polypeptide. The main cellular functions of F4/80 was found to be involved in the generation of Ag-specific efferent CD8+ regulatory T (Treg) cells responsible for peripheral immune tolerance (Lin et al. 2005). During the induction of peripheral immune tolerance, it is thought that F4/80 is required for the cellular interactions among Ag-presenting cells (APC) and other immune effector cells that lead to the generation of efferent CD8+ Treg cells. In addition, it was also suggested that F4/80 is involved in macrophage-NK interaction in a Listeria monocytogenes infection model (Warschkau and Kiderlen 1999). Overall, F4/80 is believed to play an immuneregulatory role through the interaction with an unidentified cellular ligand expressed on other immune effector cells (Lin et al. 2005; van den Berg and Kraal 2005). F4/80 is one of the best surface markers for the majority of mouse tissue macrophages (Taylor et al. 2005; Gordon and Taylor 2005; Austyn and Gordon 1981). The F4/80 Ag was detected strongly in many tissue macrophage populations, including Kupffer cells in liver, red pulp macrophages in spleen, microglia cells in brain as well as other resident macrophages in bone marrow stroma, gut lamina propria, testis, kidney, lymph nodes and peritoneum. On the other hand, macrophages within T-cell areas of lymph nodes (paracortex), spleen (white pulp) and Peyer’s patches are usually negative for F4/80. Very low levels of F4/80 were expressed in some other resident tissue macrophages such as alveolar macrophages, marginal zone and subcapsular sinus macrophages in the spleen and lymph nodes (Taylor et al. 2005; McKnight and Gordon 1998). Blood monocytes, the precursors of tissue macrophages, express less F4/80 than their tissue counterparts, suggesting that the expression of F4/80 is regulated during differentiation. Furthermore, it was also noted that F4/80 expression is modulated according to the activation status of macrophages. With regards to cells of other hematopoietic origins, no reactivity was observed in lymphoid cells, neutrophils and monocyte-derived osteoclasts. Nevertheless, eosinophils were shown to also express the F4/80 Ag. Likewise, Langerhans cells, a type of dendritic cells (DC) in the epidermis, were found to be F4/80 positive, but the Ag is down-regulated upon subsequent DC maturation and migration to the draining lymph nodes (Taylor et al. 2005; McKnight and Gordon 1998). In mice, F4/80-expressing TAMs have been documented for many types of tumours, either occurred spontaneously or induced experimentally (Qian & Pollard 2010; Mantovani et al. 2002; Rabinovich et al. 2007; Umemura et al. 2008). Therefore, the presence or absence of the F4/80 reactivity has been used to determine the efficiency of macrophage ablation experiments in tumour studies. In addition, F4/80 was also detected in tumour-infiltrating MDSC. The Ag has been useful to define the cellular origin of MDSC in tumours as MDSC was initially thought to represent immature myelomonocytic cells with common monocytic and granulocytic phenotypes. It is now generally accepted that TAMs and MDSC include cells with similar phenotypes to alternatively activated macrophages, which all express F4/80 (Gordon and Martinez 2010; Umemura et al. 2008; Martinez et al. 2009; Sinha et al. 2005).

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CD97 The full-length CD97 molecule contains a total of five EGF-like motifs. However, as a result of RNA alternative splicing, three major isoforms containing different combinations of EGF-like motifs were predicted. These include CD97(1, 2, 5), CD97(1, 2, 3, 5) and CD97(1, 2, 3, 4, 5) (Gray et al. 1996; Hamann et al. 1995). Apart from multiple O- and N-linked glycosylation sites, one RGD motif and a complete GPS motif were present in the extracellular region. Hence, CD97 was cleaved at the predicted GPS site into two subunits (Gray et al. 1996). CD97 was found to interact through its extracellular region with several cellular ligands, including CD55 (DAF) (Hamann et al. 1998; Hamann et al. 1996), dermatan sulphate (Kwakkenbos et al. 2005; Stacey et al. 2003) and D5E1/DvE3 integrins (Wang et al. 2005). Interestingly, the CD97-ligand interaction is mostly isoform-specific such that CD55 binds better to CD97(1, 2, 5), while dermatan sulphate only interacts with CD97(1, 2, 3, 4, 5) (Lin et al. 2001; Stacey et al. 2003). CD97–CD55 interaction is species-specific so that human CD97 only reacts with human CD55 but not murine CD55 (Lin et al. 2001). Through the utilisation of specific mAbs, soluble CD97 proteins, and the generation and analysis of knock-out animals, CD97 has been implicated in the cellular migration and homeostasis of polymorphonuclear cells (Kop et al. 2006; Leemans et al. 2004; Wang et al. 2007), hematopoietic stem cell/progenitor cell mobilisation (van Pel et al. 2008a; van Pel et al. 2008b), co-stimulation of CD4+ T cells (Abbott et al. 2007; Capasso et al. 2006) and angiogenesis (Wang et al. 2005). CD97 was also shown to be potentially involved in the pathogenesis of arthritis (Kop et al. 2006). CD97 was first identified as an early activation marker for T and B lymphocytes, which express low levels of CD97 in resting conditions (Gray et al. 1996; Hamann et al. 1995). On the other hand, CD97 is constitutively expressed on granulocytes and monocytes/macrophages (Jaspars et al. 2001). Immunohistochemistry staining of normal human tissues in situ has shown that CD97 is abundantly expressed in resident macrophages of most tissues (Jaspars et al. 2001). These include liver (Kupffer cells and periportal histiocytes), lung (alveolar macrophages), skin (including Langerhans cells), brain (perivascular macrophages but not microglia), kidney (mesangial cells of the glomeruli) and secondary lymphoid organs such as lymph nodes, spleen (red pulp and white pulp macrophages), tonsil and mucosa-associated lymphoid tissues (MALT). Dendritic cells in most of the lymphoid tissues also express CD97, whereas CD97-expressing lymphocytes are mostly restricted in intraepithelial and sub-epithelial locations. Lymphocytes in paracortical areas, follicles and germinal centres are mostly CD97 weak or negative. Outside lymphoid tissues, smooth muscle cells also express CD97. In addition, CD97 was often detected in many types of tumours, including thyroid, gastric, pancreatic, esophageal and colorectal carcinomas (Aust et al. 1997; Aust et al. 2002; Steinert et al. 2002). Most interestingly, stronger CD97 expression levels were usually detected at the invasion fronts of tumours, suggesting a role in tumour cell migration/invasion (Steinert et al. 2002; Galle et al. 2006; Wobus et al. 2006).

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Specifically, in colorectal and gastric carcinomas, the strongest CD97 staining was identified in disseminated or scattered tumour cells at the invasion front of the tumour. Furthermore, a poorer clinical stage and increased lymph vessel invasion were found to be correlated positively in tumour patients with more CD97-high scattered tumour cells. In the cell level, higher CD97 expression stimulated singlecell motility, enhanced the proteolytic activity of matrix metalloproteinases and secretion of chemokines. Tumour growth in scid mice was enhanced in cells overexpressing CD97 (Steinert et al. 2002; Galle et al. 2006; Wobus et al. 2006). Moreover, CD97 was shown recently to stimulate angiogenesis through binding to D5E1 integrin on endothelial cells (Wang et al. 2005). Briefly, soluble CD97 was tested in a quantitative, directed in vivo angiogenesis assay (DIVAA). It was found that CD97 at 25 and 100 ng/ml is as efficient as bFGF in inducing blood vessel development in vivo. Furthermore, developing tumours derived from CD97expressing cells display a significantly greater vessel density than those from control CD97-negative tumour cells. The angiogenic response of CD97 is most likely mediated by the RGD motif in the stalk as it interacts with the D5E1 and DvE3 integrins. Through these interactions, CD97 promotes the adhesion, migration and invasion of human umbilical vein endothelial cells (HUVECs). Nevertheless, another ligand of CD97, chondroitin sulphate, seems to also contribute to these effects (Wang et al. 2005). A more recent study using CD97 transgenic mice, however, indicates that the role of CD97 in tumourigenesis might be more complicated than we thought earlier (Becker et al. 2010). To investigate the involvement of CD97 in colorectal carcinogenesis, Becker et al. generated transgenic mice that overexpress CD97 specifically in enterocytes (Becker et al. 2010). These animals were then subjected to azoxymethane (AOM)/dextran sodium sulphate (DSS)-induced colitis-associated tumourigenesis. Interestingly, DSS-induced colitis was reduced in transgenic mice when compared with the wild-type control. This reduction was dependent on the copy number of the CD97 transgene. Through ultrastructural and biochemical analyses, it was concluded that CD97 over-expression can enhance the structural integrity of enterocytic adherens junctions, which in turn enforce intestinal epithelial strength leading to the attenuation of experimental colitis.

EMR2 Highly similar to CD97, the full-length EMR2 molecule also contains a total of five EGF-like motifs. A total of four major isoforms, namely EMR2(1, 2), EMR2 (1, 2, 5), EMR2(1, 2, 3, 5) and EMR2(1, 2, 3, 4, 5), were predicted from spliced mRNA sequences (Lin et al. 2000). EMR2 shares ~97% sequence identity in the EGF-like domains with CD97. Interestingly, however, a highly restricted ligand binding specificity was identified for these EGF-TM7 molecules. Thus, EMR2 (1, 2, 5) was shown to bind CD55 with a tenfold weaker affinity than CD97(1, 2, 5), even though the two molecules only differ in three residues (Lin et al. 2001).

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Nevertheless, EMR2(1, 2, 3, 4, 5) and CD97(1, 2, 3, 4, 5) were found to bind dermatan sulphate equally well (Stacey et al. 2003). These results suggest potential overlapping and also unique functions for EMR2 and CD97. Phylogenetic analysis has revealed an interesting evolutionary relationship between EMR2 and CD97; although both genes are highly homologous in the human genome, the EMR2 gene is lost in rodents, while the CD97 gene is present in man and mouse. In fact, it seems that EMR2 evolved through gene conversion and duplication by combining part of CD97 and another EGF-TM7 receptor, EMR3. Thus, EMR2 is a chimeric gene evolved relatively recently, probably to meet the need of the immune system in primates (Kwakkenbos et al. 2004; Kwakkenbos et al. 2006). A role for EMR2 in regulating neutrophil activation was found recently (Yona et al. 2008b). Binding of EMR2 receptor by a specific mAb was shown to strongly enhance the inflammatory responses of neutrophils to a panel of stimuli. Interestingly, mAb treatment alone did not activate neutrophils. Hence, EMR2 receptor activation seems to have a priming effect on neutrophil activation. Expression of EMR2 is more restricted than CD97 to myeloid cells (Lin et al. 2000; Chang et al. 2007; Kwakkenbos et al. 2002). Monocytes, macrophages, neutrophils and dendritic cells have all been shown to express EMR2. Specifically, the strongest EMR2 signal was detected on CD16+ blood monocytes, macrophages and BDCA-3+ myeloid dendritic cells. No expression was ever detected in resting or activated lymphocytes. Interestingly, it was found that EMR2 is up-regulated during the differentiation and maturation of macrophages in vitro, but is downregulated during dendritic cell maturation (Chang et al. 2007). EMR2 expression in monocytes and macrophages can be up-regulated by LPS and IL-10 in an IL-10dependent manner. In some limited tissues surveyed, EMR2 was detected in certain macrophage sub-populations of skin, spleen, lung, placenta, and tonsil. Macrophages of liver and kidney do not seem to express EMR2. In inflamed tissues, EMR2 was found in sub-populations of macrophages and neutrophils. Recently, it was shown that foamy macrophages in atherosclerotic vessels and Gaucher cells in spleen express high levels of EMR2. In contrast, multiple sclerosis brain foam cells expressed little if any EMR2, but strong CD97 (van Eijk et al. 2010). Unlike CD97, EMR2 expression in tumours has not been observed much. In fact, in thyroid, gastric, pancreatic, and esophageal carcinomas, many tumour cells are CD97 positive but EMR2 negative (Aust et al. 2002). In some colorectal tumour cell lines and adenocarcinomas, alternatively spliced EMR2 mRNA transcripts were identified, but EMR2 protein expression remained low in the tumour cells (Aust et al. 2003). Within the carcinomas, certain tumour-infiltrating macrophages expressed strong surface EMR2. Interestingly, by staining breast cancer tissue sections, we recently identify strong EMR2 reactivity within tumour cells (Davies et al. unpublished results). The significance of this finding awaits further investigation, but suggests that aberrantly expressed EMR2 could be linked to certain tumour types. In conclusion, members of the myeloid-restricted adhesion-GPCRs, the EGF-TM7 receptors, could be considered as important new molecules associated with tumourigenesis. Based on the structural, functional and expressional characteristics, the

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EGF-TM7 proteins could serve as the cell surface marker of tumour-infiltrating or tumour-associated myeloid cells (macrophages and neutrophils). These receptors can also be expressed by tumour cells to facilitate the interaction with the tumour microenvironment including cells and extracellular matrix. By doing so, the EGF-TM7 receptors might promote angiogenesis and the migration and invasion of tumour cells. Finally, a potential role of the EGF-TM7 receptors in immunosuppression might provide tumour cells an opportunity to evade immune surveillance.

Conclusion A larger range of molecular markers is available than currently in use, to characterise macrophages within and isolated from tumours. The leap from macrophage marker to function, taking into account the relevant tumour subpopulation actually responsible for tumour progression, is considerable. A great deal of further characterisation of promising markers is needed, especially in humans, who provide a large pool of material of natural history and diversity. Studies on individual macrophage markers, membrane and otherwise, combined with biomarkers in whole blood and tissues, should aid diagnosis and possible targeting of macrophage functions in the future.

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Krasnoperov V, Bittner MA, Holz RW, Chepurny O, Petrenko AG (1999) Structural requirements for alpha-latrotoxin binding and alpha-latrotoxin-stimulated secretion. A study with calciumindependent receptor of alpha-latrotoxin (CIRL) deletion mutants. J Biol Chem 274(6): 3590–3596 Kwakkenbos MJ, Chang GW, Lin HH, Pouwels W, de Jong EC, van Lier RA, Gordon S, Hamann J (2002) The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells. J Leukoc Biol 71(5):854–862 Kwakkenbos MJ, Kop EN, Stacey M, Matmati M, Gordon S, Lin HH, Hamann J (2004) The EGF-TM7 family: a postgenomic view. Immunogenetics 55(10):655–666 Kwakkenbos MJ, Pouwels W, Matmati M, Stacey M, Lin HH, Gordon S, van Lier RA, Hamann J (2005) Expression of the largest CD97 and EMR2 isoforms on leukocytes facilitates a specific interaction with chondroitin sulfate on B cells. J Leukoc Biol 77(1):112–119 Kwakkenbos MJ, Matmati M, Madsen O, Pouwels W, Wang Y, Bontrop RE, Heidt PJ, Hoek RM, Hamann J (2006) An unusual mode of concerted evolution of the EGF-TM7 receptor chimera EMR2. FASEB J 20(14):2582–2584 Leemans JC, te Velde AA, Florquin S, Bennink RJ, de Bruin K, van Lier RA, van der Poll T, Hamann J (2004) The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense. J Immunol 172(2):1125–1131 Lin HH, Stubbs LJ, Mucenski ML (1997) Identification and characterization of a seven transmembrane hormone receptor using differential display. Genomics 41(3):301–308 Lin HH, Stacey M, Hamann J, Gordon S, McKnight AJ (2000) Human EMR2, a novel EGF-TM7 molecule on chromosome 19p13.1, is closely related to CD97. Genomics 67(2):188–200 Lin HH, Stacey M, Saxby C, Knott V, Chaudhry Y, Evans D, Gordon S, McKnight AJ, Handford P, Lea S (2001) Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: dissection of the CD97–CD55 complex. J Biol Chem 276(26):24160–24169 Lin HH, Chang GW, Davies JQ, Stacey M, Harris J, Gordon S (2004) Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif. J Biol Chem 279(30):31823–31832 Lin HH, Faunce DE, Stacey M, Terajewicz A, Nakamura T, Zhang-Hoover J, Kerley M, Mucenski ML, Gordon S, Stein-Streilein J (2005) The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance. J Exp Med 201(10):1615–1625 Mantovani A, Sozzani S, Locati M, Allavena P, Sica A (2002) Macrophage polarization: tumorassociated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23(11):549–555 Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454(7203):436–444 Martinez FO, Helming L, Gordon S (2009) Alternative activation of macrophages: an immunologic functional perspective. Annu Rev Immunol 27:451–483 McKnight AJ, Gordon S (1996) EGF-TM7: a novel subfamily of seven- transmembrane-region leukocyte cell-surface molecules. Immunol Today 17(6):283–287 McKnight AJ, Gordon S (1998) The EGF-TM7 family: unusual structures at the leukocyte surface. J Leukoc Biol 63(3):271–280 McKnight AJ, Macfarlane AJ, Dri P, Turley L, Willis AC, Gordon S (1996) Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family. J Biol Chem 271(1):486–489 Merad M (2010) PU.1 takes control of the dendritic cell lineage. Immunity 32(5):583–585 Merad M, Manz MG (2009) Dendritic cell homeostasis. Blood 113(15):3418–3427 Peranzoni E, Zilio S, Marigo I, Dolcetti L, Zanovello P, Mandruzzato S, Bronte V (2010) Myeloid-derived suppressor cell heterogeneity and subset definition. Curr Opin Immunol 22(2):238–244 Qian BZ, Pollard JW (2010) Macrophage diversity enhances tumor progression and metastasis. Cell 141(1):39–51

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Chapter 2

Role of Tumour-Associated Macrophages in the Regulation of Angiogenesis Russell Hughes, Hsin-Yu Fang, Munitta Muthana, and Claire E. Lewis

Introduction As mentioned in previous chapters, tumours consist not only of malignant cells but also of various stromal cell types including tumour-associated macrophages (TAMs) (Sica et al. 2008). One early sign that TAMs might influence tumour angiogenesis was the finding that TAM numbers positively correlate with tumour angiogenesis in breast carcinomas (Leek et al. 1996). Several subsequent studies have confirmed such a link in a wide array of tumour types (Aharinejad et al. 2004; Bailey et al. 2007; Koide et al. 2004; Ohta et al. 2003; Saji et al. 2001) and showed that high-TAM numbers are also often linked to poor prognosis (Bingle et al. 2002; Lewis and Pollard 2006). However, definitive evidence for the pro-angiogenic effect of TAMs in tumours was provided using various murine tumour models. First, we demonstrated that macrophage infiltration into small breast tumour nodules (tumour spheroids) markedly enhanced their ability to induce neovascularisation following implantation into window chambers on the flanks of mice (Bingle et al. 2006). Then Lin and colleagues showed that, when MMTV-PyMT mice (which develop mammary tumours) were crossed with transgenic mice carrying a colony stimulating factor-1 (Csf1) null mutation (Csf1op/op), the absence of CSF-1 markedly decreased macrophage infiltration in pre-malignant tumours, which, in turn, resulted in the inhibition of tumour angiogenesis and delayed tumour progression (Lin et al. 2006; Lin et al. 2001). Indeed, they showed that TAMs control the ‘angiogenic switch’ associated with the malignant transition of the spontaneous mammary tumours that form in MMTV-PyMT mice (Lin et al. 2006). Other strategies employing an antibody to CSF-1 (Paulus et al. 2006) or clodronate liposomes (Zeisberger et al. 2006) to deplete TAMs have also resulted in a marked reduction in tumour angiogenesis.

2(UGHESs( 9&ANGs--UTHANAs#%,EWIS*) Academic Unit of Inflammation and Tumour Targeting, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK e-mail: claire.lewis@sheffield.ac.uk T. Lawrence and T. Hagemann (eds.), Tumour-Associated Macrophages, DOI 10.1007/978-1-4614-0662-4_2, © Springer Science+Business Media, LLC 2012

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R. Hughes et al.

%LEVATEDEXPRESSIONOFSUCHMACROPHAGECHEMOATTRACTANTSASTHE## CHEMOKINE CCL2 (MCP-1) in solid tumours correlates both with increased TAM recruitment (Hoshino et al. 1995; Leung et al. 1997) and with increased tumour neovascularisation (Barleon et al. 1996; Zhu et al. 2004). Additionally, the accumulation of TAMs INSOLIDTUMOURSISASSOCIATEDWITHTHEINCREASEDEXPRESSIONOFSUCHPRO ANGIOGENIC MOLECULESAS6%'&! ANDB&'& ANDCORRELATESWITHINCREASEDFORMATIONOFANGIOgenic blood vessels (Bolat et al. 2006; Tsutsui et al. 2005). Functional plasticity is a hallmark feature of macrophages and they can respond to diverse environmental signals with a wide array of functional phenotypes. Consistent with this, a variety of overlapping macrophage activation states have been reported with classically activated, ‘M1’ and alternatively activated, ‘M2’ MACROPHAGESREPRESENTINGOPPOSINGEXTREMESOFTHISCONTINUUM%XPOSURETOPRO inflammatory cytokines and microbial products such as interferon gamma (IFNJ) and lipopolysaccharide (LPS) polarizes macrophages into the pro-inflammatory, -PHENOTYPE WHICHISCHARACTERIZEDBYTHEINCREASEDEXPRESSIONOFTHEIROWNPRO INmAMMATORYMEDIATORSINCLUDINGINDUCIBLENITRICOXIDESYNTHASEI./3 REACTIVE NITROGEN INTERMEDIATES 2.) REACTIVE OXYGEN INTERMEDIATES 2/) AND THEIR increased microbicidal/tumoricidal activity. Anti-inflammatory molecules such as the Th2 cytokines, IL-4, IL-13 and IL-10, as well as apoptotic cells and immune COMPLEXESINCOMBINATIONWITH4OLL LIKERECEPTOR4,2 STIMULATIONINDUCEMACROPHAGES TO EXPRESS AN - ACTIVATION PHENOTYPE 3UCH @- MACROPHAGES ARE thought to be involved in promoting angiogenesis and tissue remodelling/repair, dampening inflammation by producing high levels of anti-inflammatory cytokines (e.g. IL-10 and transforming growth factor-E 4'&E) and up-regulating products of the arginase pathway (arginase II, ornithine and polyamines). They also display a HIGHLEVELOFPHAGOCYTICACTIVITYANDEXPRESSHIGHLEVELSOFBOTHMANNOSEANDSCAVENGERRECEPTORS'ORDON2003; Mantovani et al. 2004; Mantovani et al. 2002; Mills et al. 2000; Siciliano et al. 2006). However, it should be noted that variations within the M2 phenotype of macrophages have recently been described leading to the subdivisions, M2a, -b and -c. Interleukins 4 and -13 stimulate the onset of the M2a phenotype in macrophages (Mantovani et al. 2002; Martinez et al. 2009), whereas EXPOSURETOIMMUNECOMPLEXESINCOMBINATIONWITH4,2ACTIVATIONINDUCES-B TYPEMACROPHAGES&INALLY EXPOSURETOGLUCOCORTICOIDSOR), RESULTSINTHEPRODUCtion of M2c-type macrophages, with greater immunosuppressive, pro-angiogenic ANDTISSUEREMODELLINGFUNCTIONS'OERDTAND/RFANOS1999). Murine TAMs show many characteristics of an M2-polarized state (Biswas et al. 2008; Mantovani et al. 2002 &OREXAMPLE 4!-SOFTENUP REGULATEPRO ANGIOGENIC MEDIATORS SUCH AS 6%'&! "ALKWILL AND -ANTOVANI 2001 CYCLO OXYGENASE (COX)-2 (Nakao et al. 2005) and MMP9 (Coussens et al. 2000). They also have impaired EXPRESSION OF SUCH PRO INmAMMAOTORY MEDIATORS AS EG ), AND 4.&D), RNI AND MAJOR HISTOCOMPATIBILITY COMPLEX -(# )) WHICH FUNCTIONALLY CORRELATE TO REDUCEDCYTOTOXICITYANDANTIGENPRESENTINGCAPACITY#ONVERSELY 4!-SUP REGULATE ANTI INmAMMATORY MOLECULES SUCH AS ), AND 4'&E. Other hallmark markers of M2 activation, such as up-regulated levels of the M2-specific genes, arginase,

2

Role of Tumour-Associated Macrophages in the Regulation of Angiogenesis

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macrophage galactose-type C-type lectin-2 (Mgl2), found in inflammatory zone 1 &IZZ AND 9M -ANTOVANI ET AL 2003 6AN 'INDERACHTER ET AL 2006) are also EXPRESSEDBYMURINE4!-S(OWEVER ITSHOULDBENOTEDTHATSOME4!-SINMOUSE tumour models, as well as human tumours (e.g. breast, ovarian and renal cell carciNOMA EXHIBIT TYPICAL - CYTOKINES SUCH AS 4.&D, IL-1E, IL-6, IL-23p19 and CXCL8, depending on tumour type and stage (Biswas et al. 2006; Dinapoli et al. 1996; Langowski et al. 2006). This has lead to the suggestion that the phenotype of TAMs may vary between different stages of tumour development and possibly different regions of the same tumours (Murdoch et al. 2008; Ohno et al. 2004).

Pro-angiogenic Functions of Macrophages in Tumours 4!-SEXPRESSABROADNUMBEROFCYTOKINESTHATARECAPABLEOFDIRECTLYINDUCINGTHE FORMATIONOFBLOODVESSELSWITHINTUMOURSINCLUDING6%'&!ANDB&'&"OLATETAL 2006; Tsutsui et al. 2005). However, TAMs also produce a wide range of pleiotrophic factors including IL1E (Voronov et al. 2003 4'& E1, and TNFD (Luo et al. 2006; 9OSHIDA ET AL 1997), which promote new blood vessel formation by promoting THERELEASEOFSUCHFACTORSAS6%'&!FROMOTHERCELLTYPESCOMMONLYFOUNDWITHIN the tumour micro-environment (Pertovaara et al. 1994). TAMs also indirectly promote the formation of blood vessels in tumours using a variety of TAM-derived enzymes SUCHASTHEMATRIXMETALLOPROTEINASES--0S AND 'IRAUDOETAL2004; Hildenbrand et al. 1995; Houghton et al. 2006; Huang et al. 2002; Nozawa et al. 2006 4HESE ARE EXTRACELLULAR MATRIX %#- REMODELLING ENZYMES THAT ALTER THE STRUCTUREOFTHE%#-TOSUPPORTTHEATTACHMENT MIGRATIONANDINVASIONOFACTIVATED%#S)NADDITION THEINTERACTIONOF--0SWITHTHE%#-CANALSOINCREASETHE BIOAVAILABILITYOFSUCHPRO ANGIOGENICFACTORSAS6%'&!ANDB&'&!CCORDINGLY THERECRUITMENTOFMYELOIDCELLSEXPRESSING--0ISIMPORTANTFORTHELIBERATIONOF 6%'&! FROM THE %#- )T WAS THEREFORE HARDLY SURPRISING TO lND THAT --0 EXPRESSINGMYELOIDCELLSWEREESSENTIALFORTUMOURANGIOGENESISINAMURINEMODEL of glioblastoma (Du et al. 2008). In addition, mice lacking MMPs 2 and 7 show a reduced rate of tumour progression when implanted with B16 melanoma and Lewis Lung carcinoma cells or in a murine colorectal carcinoma model (Itoh et al. 1998; Wilson et al. 1997 4!-S ALSO EXPRESS THE IMPORTANT PRO ANGIOGENIC ENZYME thymidine phosphorylase in tumours (TP) and this correlates with tumour neovascularisation and poor prognostic outcome in patients with gastric cancer (Kawahara et al. 2010). This enzyme is capable of modifying DNA released by dead or dying cells by converting the nucleoside, thymidine, into thymine and the potent proANGIOGENIC MOLECULE DEOXYRIBOSE PHOSPHATE 4AKEN TOGETHER THESE lNDINGS highlight the importance of TAM-derived enzymes in the regulation of tumour neovascularisation. TAMs might also promote the formation of a tumour vasculature in a number of additional ways. These can include the recruitment of CD34+ AC133+ endothelial

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NORMOXIA

RECRUITMENT

HYPOXIA ACTIVATION

MONO E/MPC TEM ANGPT2 CXCL12

ANGPT2 CCL2 CSF1 EMAPII

TAM

MIF IL1B TP PDGFB VEGFA CXCL8 bFGF

BIOAVAILABILITY

ECM MODULATION

ECM

bFGF VEGFA MMP-2 MMP-7 MMP-9 MMP-12

ECM

Fig. 2.1 Mechanisms by which TAM promote the formation of the tumour vasculature. TAMs promote the formation of the tumour vasculature by a variety of mechanisms including the recruit OFSUCHPRO ANGIOGENICCELLTYPESASMONOCYTES-/./ AND4)% EXPRESSINGMONOCYTES4%- 4!-S ALSO ACTIVELY RECRUIT ENDOTHELIAL AND MYELOID PROGENITORS %-0#S CAPABLE OF DIRECTLY INCORPORATINGINTOTHETUMOURVASCULATURE&URTHERMORE 4!-SRELEASEAVARIETYOF%#- MODIFYING ENZYMES 4HESE ENHANCE THE ABILITY OF ACTIVATED %# TO MIGRATE AND INVADE THE TUMOUR MICRO ENVIRONMENTBYRESTRUCTURINGTHE%#-!DDITIONALLY THESESAMEMATRIX REMODELLINGENZYMESALSO INCREASETHEBIOAVAILABILITYOFSUCHPRO ANGIOGENICCYTOKINESANDGROWTHFACTORSAS6%'&!AND B&'&VIATHEIRLIBERATIONFROMTHE%#-&INALLY 4!-SALSODIRECTLYPROMOTEANGIOGENESISTHROUGH THE RELEASE OF SUCH PRO ANGIOGENIC CYTOKINES AND GROWTH FACTORS THAT ACT DIRECTLY ON %# THIS induces their activation, migration, proliferation and differentiation into tumour neo-vessels

PROGENITORSOR#$#$HUMANPERIPHERALBLOODMONOCYTES'ILLETAL2001; Peichev et al. 2000). Indeed, CD14+ CD34− peripheral blood mononuclear cells HAVE BEEN FOUND EXPRESSING THE CLASSIC %# MARKERS 6%'&2 AND V7& (Von Willebrand Factor) (Schmeisser et al. 2001 4HEPRESENCEOF6% CADHERIN6% '&!2'2#$B--0CELLSHASBEENFOUNDINSOLIDTUMOURSWHERETHEY contributed to tumour neovascularisation by becoming incorporated into the tumour ENDOTHELIUM9ANGETAL2004) (Fig. 2.1).

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Re-education of TAMs by Tumour-Derived Signals The tumour micro-environment is now thought to stimulate many of the proANGIOGENIC FUNCTIONS OF 4!-S &OR EXAMPLE 4.&D secreted by ovarian tumour CELLS ENHANCES 4!- EXPRESSION OF 6%'&! --0 AND OTHER IMPORTANT PRO angiogenic factors (Hagemann et al. 2006). Antibodies released from tumourinfiltrating B cells can also stimulate macrophages to promote tumour angiogenesis (Andreu et al. 2010). As mentioned in the chapter by Cramer and colleagues in this book, tumour HYPOXIA VERY LOW OXYGEN LEVELS ALSO PLAYS AN IMPORTANT PART IN RE EDUCATING MACROPHAGESINTUMOURS4HEOXYGENTENSIONINNORMALTISSUESRANGESFROMTO MM(G(YPOXIA ORLOWOXYGENTENSION DElNEDASANOXYGENTENSIONLESSTHAN 10 mmHg, arises when an imbalance occurs between the supply of O2 and its consumption by local cells. This usually results from the abnormally high proliferation of tumour cells or the presence of a defective vasculature (Shannon et al. 2003; Thews et al. 1998; Vaupel et al. 1989). This leads to cellular dysfunction and ultimately CELLDEATH-OSTTUMOURSWITHADIAMETERGREATERTHANMMCONTAINAREASOFHYPOXIA (Brown, 1979; Hill et al. 1996; Shannon et al. 2003 4HEOXYGENTENSIONSINHEAD and neck tumour metastases were found to be less than 30 mmHg with the median BEINGMM(GINDICATINGTHEPRESENCEOFSEVEREHYPOXIA"ROWN1999). Additional studies have reported similar findings in a variety of other tumour types including prostate (Movsas et al. 1999), cervical (Hockel et al. 1991), breast, brain, head/neck and soft tissue sarcoma (Vaupel et al. 1989; Vaupel et al. 1991). )NTERESTINGLY 4!-S HAVE BEEN SHOWN TO ACCUMULATE IN SUCH HYPOXIC ANDOR necrotic areas of breast, endometrial, ovarian, colorectal and buccal tumours (Lewis and Pollard 2006; Murdoch et al. 2008). Indeed, a positive correlation between the ACCUMULATIONOF4!-SANDTHEPRESENCEOFHYPOXIAHASALSOBEENREPORTEDINTHE liver metastases of breast and colorectal tumours (Stessels et al. 2004). It is thought THATTHESEAREASATTRACT4!-SBYRELEASINGSUCHHYPOXIA INDUCEDCHEMOATTRACTANTS AS6%'&! ENDOTHELINS %-!0))ALSOKNOWNAS3#9% AND#8#,3$& (Kioi et al. 2010; Murdoch et al. 2004; Murdoch et al. 2008). It might also involve THERELEASEOFFACTORSSUCHASHIGH MOBILITYGROUPBOX(-'" BYNECROTICCELLS INSITESOFCHRONICTUMOURHYPOXIAASTHISHASBEENSHOWNTOALTERTHEPHENOTYPE of macrophages (Andersson et al. 2000; Stros 2010). However, it remains to be seen whether necrotic debris can act as a chemoattractant to recruit monocytes into tumours. )T HAS BEEN SUGGESTED THAT ONCE DRAWN INTO SUCH HYPOXIC SITES 4!-S BECOME TRAPPEDANDUNABLETOLEAVEDUETOTHEIRREDUCEDEXPRESSIONOFCHEMOKINERECEPTORSANDTHEREDUCEDEXPRESSIONOFCHEMOKINESBYTUMOURCELLS.EGUSETAL1998; Sica et al. 2000 %XPOSURETOHYPOXIACANSTIMULATETHEEXPRESSIONOFMACROPHAGE migration inhibitory factor (MIF) by both macrophages and tumour cells (Koong et al. 2000; White et al. 2004). This may inhibit the ability of TAMs to migrate out of HYPOXICSITESINTOOTHERAREASOFTHETUMOUR2ECENTSTUDIESHAVEALSOSHOWNTHE UP REGULATEDEXPRESSIONOF#8#2 THERECEPTORFOR#8#,3$& BYHYPOXIC

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macrophages (Fang et al. 2009; Schioppa et al. 2003 ANDTHATHYPOXIA INDUCIBLE transcription factor-1 (HIF-1) influences the positioning and function of TAMs by REGULATINGTHEIREXPRESSIONOF#8#23CHIOPPAETAL2003). Moreover, HIF-1 actiVATIONALSOUP REGULATES#8#,INHYPOXICENDOTHELIALCELLS#ERADINIETAL2004).

Hypoxic Signalling and Regulation of Transcription in TAMs !NUMBEROFRECENTSTUDIESHAVESUGGESTEDTHAT INHYPOXICAREAS 4!-SEXPRESSA highly pro-angiogenic phenotype mainly due to their up-regulation of HIFs-1 and -2 (Burke et al. 2003; Fang et al. 2009). This is known to trigger increased tranSCRIPTION OF MANY ()& TARGET GENES THAT PROMOTE ANGIOGENESIS SUCH AS 6%'&! -)& !.'04AND#8#,&ANGETAL2009). Moreover, we showed that TAMs UP REGULATE THE PRO ANGIOGENIC CHEMOKINE @6%'&! IN AVASCULAR PERI NECROTIC tumours (Lewis et al. 2000). However, there is considerable debate regarding the relative contributions of ()&S AND TOHYPOXIA INDUCEDPRO ANGIOGENICGENEEXPRESSIONINMACROPHAGES Some studies have suggested that HIF-2 is the dominant form of HIF up-regulated BYHYPOXIAINSUCHCELLS'RIFlTHSETAL2000; Talks et al. 2000). Additionally, White ANDCOLLEAGUESUSEDADENOVIRALLYTRANSFECTEDHUMANMACROPHAGESTOOVER EXPRESS HIFs-1 and -2 and showed that HIF-2 rather than HIF-1 appeared to mediate the HYPOXICINDUCTIONOFVARIOUSPRO ANGIOGENICGENESSUCHAS6%'&! ), #/8AND B&'&7HITEETAL2004). However, their data is difficult to interpret as transfected MACROPHAGESEXPRESSEDSUPER PHYSIOLOGICALLEVELSOFTHESETWOTRANSCRIPTIONFACTORS ANDNORMOXICRATHERTHANHYPOXICCULTURESWEREUSEDTHROUGHOUT Other studies have shown that human macrophages accumulate high levels of BOTH()& THAN()& WHENEXPOSEDTOTUMOUR SPECIlCLEVELSOFHYPOXIAINVITRO (Burke et al. 2002). When siRNA was used to knock down the alpha subunit of each ()& THEMAJORITYOFHYPOXIA INDUCEDPRO ANGIOGENICGENESWERESEENTOHAVEEQUAL dependence on both HIFs, suggesting that each HIF was capable of compensating for the loss of the other (Fang et al. 2009). An intriguing recent study by Johnson and colleagues has shown that IL-4 up-regulates HIF-2 but not HIF-1 levels in macrophages and in doing so, skews them towards a more M2-like phenotype. This SUGGESTSTHAT()& MAYPLAYAPARTINMEDIATINGTHEINDUCTIONBYHYPOXIAANDOR ), OFTHE-PHENOTYPEEXPRESSEDBYSOME4!-S4AKEDAETAL2010) and accords WELLWITHTHElNDINGTHATHIGHNUMBERSOF()& EXPRESSING4!-SCORRELATEWITH increased tumour vascularity in breast carcinomas (Leek et al. 2002). Furthermore, THE MACROPHAGE EXPRESSION OF ()& HAS RECENTLY BEEN FOUND TO IMPACT ON THE progression of murine hepatocelullar carcinomas (HCC) – as evidenced by their reduced growth in myeloid-specific HIF-2D conditional knock-out mice. Moreover, TUMOURCELLSEXHIBITEDALOWERMITOTICINDEXINTHESETUMOURS)MTIYAZETAL2010). &INALLY ITSHOULDBENOTEDTHAT%LBARGHATIANDCOLLEAGUESSHOWEDTHAT()&S AND MAYBEDIFFERENTIALLYREGULATEDDURINGRE OXYGENATIONOFMACROPHAGESAFTERTHECESSATIONOFHYPOXIAFOREXAMPLE INHYPOXICTUMOURAREASUNDERGOINGREVASCULARIZATION

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()& UP REGULATION WAS RAPIDLY REVERSED DURING RE OXYGENATION WHEREAS ()& LEVELSTOOKLONGERTODROPTONORMOXICLEVELS%LBARGHATIETAL2008). Recently, the activation of another transcription factor, NF-NB, has also been IMPLICATED IN THE TRANSCRIPTIONAL REGULATION OF HYPOXIC MACROPHAGES 3HORT TERM EXPOSUREnH OFMURINEMACROPHAGESTOHYPOXIAACTIVATES.& NB, which in turn up-regulates HIF-1D levels (Rius et al. 2008). This study demonstrated the impaired ability of IKKE−/− macrophages to up-regulate HIF-1DINTHEPRESENCEOFHYPOXIA 2ECENTDATAFROMOURGROUPHASNOWSHOWNTHATALTHOUGHMACROPHAGESEXPOSEDTO MOREPROLONGEDPERIODSOFHYPOXIAEGH UP REGULATETHEEXPRESSIONOFSUCH NF-NB members as p65, p50 and IKKE, inhibition of canonical NF-NB signalling HADNOEFFECTONTHEIRUP REGULATIONOFVARIOUSGENESINHYPOXIA&ANGETAL2009). Together these findings suggest that NF-NB may be more important in the initial, EARLYRESPONSESOFMACROPHAGESTOTUMOURHYPOXIATHANWHENTHEYEXPERIENCEITFOR longer periods in tumours.

TIE2-Expressing Monocyte/Macrophages A subset of human and murine monocyte/macrophages have been identified that DISPLAY HIGHLY PRO ANGIOGENIC FUNCTIONS n THOSE THAT EXPRESS THE TYROSINE KINASE WITH)GAND%'&HOMOLOGYDOMAIN 4)% RECEPTOR WHICHISTYPICALLYEXPRESSED on endothelial cells, (De Palma et al. 2007; De Palma et al. 2005; De Palma et al. 2003; Murdoch et al. 2007; Nowak et al. 2004 ANDAREKNOWNAS4)% EXPRESSING MONOCYTEMACROPHAGES 4%-S 4%-S HAVE BEEN FOUND IN A VARIETY OF HUMAN tumours including kidney, colon, pancreas, lung and in soft tissue sarcomas (Venneri et al. 2007). 4HERECRUITMENTOF4%-SINTOSOLIDTUMOURSISTHOUGHTTOBEVIAMECHANISMS THATAREDISTINCTFROMTHOSEUSEDBY4)%n4!-S!SMENTIONEDPREVIOUSLY -#0 (CCL2) plays a prominent role in the recruitment of monocytes from the peripheral BLOODINTOSOLIDTUMOURS APROCESSMEDIATEDBYTHEEXPRESSIONOF##2 THERECEPTOR FOR##, ONTHEMAJORITYOFCIRCULATINGMONOCYTES4%-SDONOTEXPRESS##2 (Venneri et al. 2007) and therefore CCL2 appears not to play a significant role in THEIR RECRUITMENT )NTERESTINGLY WE AND OTHERS HAVE SHOWN THAT 4%-S COULD BE RECRUITED INTO TUMOURS BY THE 4)% LIGAND ANGIOPOIETIN !.'04 'U ET AL 2006; Murdoch et al. 2007; Stratmann et al. 1998; Venneri et al. 2007), a cytokine UP REGULATED BY ENDOTHELIAL CELLS IN TUMOUR BLOOD VESSELS !.'04 IS TYPICALLY stored in Weibel-Palade bodies in these cells, secretory granules that rapidly fuse with the cell membrane upon endothelial cell activation, and release stored cytokines and GROWTHFACTORS INCLUDING!.'04 INTOTHEEXTRACELLULARSPACE&IEDLERETAL2004). 4%-SAPPEARTOBEINHERENTLYBETTERATPROMOTINGTHEFORMATIONOFTUMOURBLOOD VESSELSTHAN4!-S!CCORDINGLY CIRCULATING4%-SWEREFOUNDTOEXPRESSHIGHER LEVELS OF IMPORTANT PRO ANGIOGENIC MEDIATORS SUCH AS 6%'&! --0 #/8 40 AND #ATHEPSIN " #ONSISTENT WITH THE ELEVATED EXPRESSION OF PRO ANGIOGENIC EFFECTORMOLECULES 4%-SPROMOTEDANGIOGENESISTOAGREATEREXTENTINBOTHINVITRO endothelial cell and murine in vivo tumour assays (Coffelt et al. 2010).

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When human U87 glioma cells were co-injected subcutaneously into nude mice WITH4%-S ASIGNIlCANTLYHIGHERLEVELOFTUMOURVASCULARISATIONWASSEENCOMPARED WITHMICERECEIVINGNON 4%-S$E0ALMAETAL2005). Moreover, De Palma and COLLEAGUESUSEDACONDITIONALGENETIC4%-DEPLETIONMODELTOSHOWTHEESSENTIAL pro-angiogenic effect of these cells in murine tumours. To do this, sub-lethally irradiated mice were implanted with orthotopic human glioma cells and their bone MARROWRECONSTITUTEDFROMAMURINETRANSGENICSTRAININWHICHTHEEXPRESSIONOFTHE thymidine kinase gene was under the control of the Tie2 promoter. When the pro-drug, ganciclovir, was then administered to tumour-bearing mice to specifically ablate the 4%-POPULATION ITRESULTEDINAMARKEDREDUCTIONINBOTHTUMOURANGIOGENESISAND growth (De Palma et al. 2005). Taken together, these data clearly demonstrate the ROLETHAT4%-SPLAYINREGULATINGTUMOURANGIOGENESIS

Concluding Remarks The findings discussed in this chapter highlight the multifaceted way in which TAMs stimulate tumour angiogenesis. These highly versatile cells up-regulate a plethora of cytokines and growth factors in response to such micro-environmental SIGNALSASCYTOKINESSECRETEDBYTUMOURCELLSANDHYPOXIA4HESEFACTORSEITHERACTIvate endothelial cells directly in the tumour vasculature or indirectly via a variety OF%#- REMODELLINGENZYMES SUCHASVARIOUS--0STHATRE SHAPETHE%#-TO FACILITATE%#INVASIONANDMIGRATIONANDINCREASETHEBIOAVAILABILITYOFVARIOUSPRO angiogenic mediators. Taken together, these findings indicate that the development of therapeutic strategies aimed at re-directing the phenotype of macrophages back towards more M1-like, anti-tumour functions in vivo might prove efficacious in the treatment of cancer. Acknowledgements The authors gratefully acknowledge the support of Cancer research UK, the "REAST#ANCER#AMPAIGNAND9ORKSHIRE#ANCER2ESEARCHFORTHEIRWORKINTHISAREAOFRESEARCH

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Koide N, Nishio A, Sato T, Sugiyama A, Miyagawa S (2004) Significance of macrophage CHEMOATTRACTANTPROTEIN EXPRESSIONANDMACROPHAGEINlLTRATIONINSQUAMOUSCELLCARCINOMA OFTHEESOPHAGUS!M*'ASTROENTEROLn +OONG!# $ENKO.# (UDSON+- 3CHINDLER# 3WIERSZ, +OCH# %VANS3 )BRAHIM( ,E14 4ERRIS $* ET AL #ANDIDATE GENES FOR THE HYPOXIC TUMOR PHENOTYPE #ANCER 2ES 883–887 Langowski JL, Zhang X, Wu L, Mattson JD, Chen T, Smith K, Basham B, McClanahan T, Kastelein RA, Oft M (2006) IL-23 promotes tumour incidence and growth. Nature 442: 461–465 ,EEK 2$ ,EWIS #% 7HITEHOUSE 2 'REENALL - #LARKE * (ARRIS !, !SSOCIATION OF macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 56:4625–4629 ,EEK2$ 4ALKS+, 0EZZELLA& 4URLEY( #AMPO, "ROWN.3 "ICKNELL2 4AYLOR- 'ATTER+# (ARRIS !, 2ELATION OF HYPOXIA INDUCIBLE FACTOR ALPHA ()& ALPHA EXPRESSION IN TUMOR INlLTRATIVEMACROPHAGESTOTUMORANGIOGENESISANDTHEOXIDATIVETHYMIDINEPHOSPHOrylase pathway in Human breast cancer. Cancer Res 62:1326–1329 ,EUNG39 7ONG-0 #HUNG,0 #HAN!3 9UEN34 -ONOCYTECHEMOATTRACTANTPROTEIN EXPRESSIONANDMACROPHAGEINlLTRATIONINGLIOMAS!CTA.EUROPATHOLn ,EWIS#% 0OLLARD*7 $ISTINCTROLEOFMACROPHAGESINDIFFERENTTUMORMICROENVIRONMENTS Cancer Res 66:605–612 ,EWIS *3 ,ANDERS 2* 5NDERWOOD *# (ARRIS !, ,EWIS #% %XPRESSION OF VASCULAR endothelial growth factor by macrophages is up-regulated in poorly vascularized areas of breast carcinomas. J Pathol 192:150–158 ,IN%9 .GUYEN!6 2USSELL2' 0OLLARD*7 #OLONY STIMULATINGFACTORPROMOTESPROGRESSIONOFMAMMARYTUMORSTOMALIGNANCY*%XP-EDn ,IN%9 ,I*& 'NATOVSKIY, $ENG9 :HU, 'RZESIK$! 1IAN( 8UE8. 0OLLARD*7 Macrophages regulate the angiogenic switch in a mouse model of breast cancer. Cancer Res 66: 11238–11246 ,UO9 :HOU( +RUEGER* +APLAN# ,EE3( $OLMAN# -ARKOWITZ$ 7U7 ,IU# 2EISFELD RA et al (2006) Targeting tumor-associated macrophages as a novel strategy against breast cancer. J Clin Invest 116:2132–2141 Mantovani A, Sozzani S, Locati M, Allavena P, Sica A (2002) Macrophage polarization: tumorassociated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23:549–555 Mantovani A, Schioppa T, Biswas SK, Marchesi F, Allavena P, Sica A (2003) Tumor-associated macrophages and dendritic cells as prototypic type II polarized myeloid populations. Tumori 89:459–468 Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M (2004) The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25:677–686 -ARTINEZ&/ (ELMING, 'ORDON3 !LTERNATIVEACTIVATIONOFMACROPHAGESANIMMUNOLOGIC functional perspective. Annu Rev Immunol 27:451–483 Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM (2000) M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol 164:6166–6173 -OVSAS" #HAPMAN*$ (ORWITZ%- 0INOVER7( 'REENBERG2% (ANLON!, )YER2 (ANKS'% (YPOXICREGIONSEXISTINHUMANPROSTATECARCINOMA5ROLOGYn -URDOCH# 'IANNOUDIS! ,EWIS#% -ECHANISMSREGULATINGTHERECRUITMENTOFMACROPHAGES INTOHYPOXICAREASOFTUMORSANDOTHERISCHEMICTISSUES"LOODn -URDOCH# 4AZZYMAN3 7EBSTER3 ,EWIS#% %XPRESSIONOF4IE BYHUMANMONOCYTES and their responses to angiopoietin-2. J Immunol 178:7405–7411 -URDOCH# -UTHANA- #OFFELT3" ,EWIS#% 4HEROLEOFMYELOIDCELLSINTHEPROMOTION of tumour angiogenesis. Nat Rev Cancer 8:618–631 .AKAO3 +UWANO4 4SUTSUMI -IYAHARA# 5EDA3 +IMURA9. (AMANO3 3ONODA+( 3AIJO9 .UKIWA 4 3TRIETER 2- ET AL )NlLTRATION OF #/8 EXPRESSING MACROPHAGES IS A

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3TROS- (-'"PROTEINSINTERACTIONSWITH$.!ANDCHROMATIN"IOCHIM"IOPHYS!CTA 1799:101–113 4AKEDA. /$EA%, $OEDENS! +IM*7 7EIDEMANN! 3TOCKMANN# !SAGIRI- 3IMON-# Hoffmann A, Johnson RS (2010) Differential activation and antagonistic function of HIF-{alpha} ISOFORMSINMACROPHAGESAREESSENTIALFOR./HOMEOSTASIS'ENES$EVn 4ALKS +, 4URLEY ( 'ATTER +# -AXWELL 0( 0UGH #7 2ATCLIFFE 0* (ARRIS !, 4HE EXPRESSION AND DISTRIBUTION OF THE HYPOXIA INDUCIBLE FACTORS ()& ALPHA AND ()& ALPHA IN normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 157: 411–421 4HEWS / +ELLEHER $+ ,ECHER " 6AUPEL 0 "LOOD mOW OXYGENATION METABOLIC AND energetic status in different clonal subpopulations of a rat rhabdomyosarcoma. Int J Oncol 13: 205–211 4SUTSUI3 9ASUDA+ 3UZUKI+ 4AHARA+ (IGASHI( %RA3 -ACROPHAGEINlLTRATIONANDITS PROGNOSTICIMPLICATIONSINBREASTCANCERTHERELATIONSHIPWITH6%'&EXPRESSIONANDMICROVESSEL density. Oncol Rep 14:425–431 6AN'INDERACHTER*! -OVAHEDI+ (ASSANZADEH'HASSABEH' -EERSCHAUT3 "ESCHIN! 2AES' De Baetselier P (2006) Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion. Immunobiology 211:487–501 6AUPEL0 +ALLINOWSKI& /KUNIEFF0 "LOODmOW OXYGENANDNUTRIENTSUPPLY ANDMETABOLIC microenvironment of human tumors: a review. Cancer Res 49:6449–6465 6AUPEL0 3CHLENGER+ +NOOP# (OCKEL- /XYGENATIONOFHUMANTUMORSEVALUATIONOF TISSUEOXYGENDISTRIBUTIONINBREASTCANCERSBYCOMPUTERIZED/TENSIONMEASUREMENTS#ANCER Res 51:3316–3322 6ENNERI-! $E0ALMA- 0ONZONI- 0UCCI& 3CIELZO# :ONARI% -AZZIERI2 $OGLIONI# .ALDINI , )DENTIlCATION OF PROANGIOGENIC 4)% EXPRESSING MONOCYTES 4%-S IN human peripheral blood and cancer. Blood 109:5276–5285 6ORONOV% 3HOUVAL$3 +RELIN9 #AGNANO% "ENHARROCH$ )WAKURA9 $INARELLO#! !PTE2. (2003) IL-1 is required for tumor invasiveness and angiogenesis. Proc Natl Acad Sci USA 100:2645–2650 7HITE*2 (ARRIS2! ,EE32 #RAIGON-( "INLEY+ 0RICE4 "EARD', -UNDY#2 .AYLOR3 'ENETICAMPLIlCATIONOFTHETRANSCRIPTIONALRESPONSETOHYPOXIAASANOVELMEANSOF IDENTIFYINGREGULATORSOFANGIOGENESIS'ENOMICSn Wilson CL, Heppner KJ, Labosky PA, Hogan BL, Matrisian LM (1997) Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin. Proc Natl Acad Sci USA 94: 1402–1407 9ANG, $E"USK,- &UKUDA+ &INGLETON" 'REEN *ARVIS" 3HYR9 -ATRISIAN,- #ARBONE$0 ,IN0# %XPANSIONOFMYELOIDIMMUNESUPPRESSOR'R#$BCELLSINTUMOR BEARING host directly promotes tumor angiogenesis. Cancer Cell 6:409–421 9OSHIDA3 /NO- 3HONO4 )ZUMI( )SHIBASHI4 3UZUKI( +UWANO- )NVOLVEMENTOF interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol 17:4015–4023 Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA (2006) Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach. Br J Cancer 95:272–281 :HU9- 7EBSTER3* &LOWER$ 7OLL0* )NTERLEUKIN #8#,ISAGROWTHFACTORFORHUMAN lung cancer cells. Br J Cancer 91:1970–1976

Chapter 3

The Role of Tumour-Associated Macrophages in Malignant Invasion Claudia Binder

Introduction Infiltrates of leukocytes in various cancers were first observed more than 100 years ago and were interpreted as a defense mechanism of the host’s immune system. Attempts of clinicians such as Sir William Coley to eliminate malignant tumours by stimulating inflammation, however, were not reproducibly successful, not least because of uncontrollable side effects (Nauts et al. 1946). That there is also another side of the coin has long been known among pathologists, who observed high amounts of infiltrating leukocytes, particularly, in clinically unfavorable entities, such as inflammatory breast cancer. Nevertheless, it has only gradually become realized that these cells, in contrast to their expected cytotoxic function, can foster tumour promotion and progression (Steele et al. 1984; Leek et al. 1996). During the last two decades, evidence has accumulated that especially the tumour-associated macrophages (TAM), representing the main population of these infiltrates (Mantovani et al. 1992), are key actors in this dubious play. Among the various ways, TAM can promote malignant progression, we will here focus on their effect on tumour cell invasion. Movement of cells through 3D environments during invasion usually requires enhanced motile capacity as well as reorganization of the invaded structures through pericellular proteolysis. To acquire single cell motility, cells run through a program that involves cell polarization, loosening of cell–cell contacts, establishment of focal adhesions to the substrate as well as exertion of contractile force via actin-binding proteins, such as myosin, at the trailing edge (Friedl 2004; Kedrin et al. 2007) This is associated with extensive actin remodeling and development of various kinds of cellular protrusions at the leading edge, such as finger-like filopodia with

C. Binder (*) Department of Hematology/Oncology, University of Göttingen, Göttingen 37099, Germany e-mail: [email protected] T. Lawrence and T. Hagemann (eds.), Tumour-Associated Macrophages, DOI 10.1007/978-1-4614-0662-4_3, © Springer Science+Business Media, LLC 2012

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thin actin bundles, important for chemotaxis, and sheet-like lamellipodia, containing broad actin meshworks. A further type of protrusions is found in malignant cells, which use invadopodia to penetrate the surrounding extracellular matrix (ECM). Invadopodia extend vertically into the basement membrane and are capable of locally restricted proteolysis via the expression of the membrane-bound matrix metalloproteinase (MMP)-14, which activates the major ECM-degrading MMPs 2 and -9 (Yamaguchi et al. 2005). It has been reported that malignant cells can also move as amoebas and squeeze through the ECM without proteolysis (Friedl 2004). However, this applies predominantly to hematopoetic tumours and seems to play a rather secondary role in vivo (Sabeh et al. 2009). The above described type of single cell movement corresponds to a well-known process in development, called epithelial-mesenchymal transition (EMT), characterized by the downregulation of epithelial markers, such as E-cadherin, and the upregulation of mesenchymal ones (Yang and Weinberg 2008). Poorly differentiated cancer cells are often constitutively highly motile and strongly express the mesenchymal markers Vimentin, Snail, and Twist. Well-differentiated cells, in contrast, use a different kind of locomotion and frequently move in cohorts with largely preserved epithelial characteristics (Friedl and Gilmour 2009). Only the leading cells show certain features of EMT, and, to acquire these, need strong stimuli from the surrounding tissue. Macrophages express and secrete a huge arsenal of factors with well-described activity in this context. Thus, they are attractive candidates to stimulate tumour cell motility either directly through the activation of migrationassociated signaling cascades or indirectly through the supply of downstream effectors which enable tumour cells to penetrate tissue structures.

Experimental Evidence for Macrophage-Induced Invasion Tumour Cell–Macrophage Interactions Already in 1987, it was reported that the invasive capacity of AH 130 rat ascites hepatoma cells was strongly increased upon cultivation on a macrophage feeder layer (Mukai et al. 1987). Subsequently, an elegant proof that macrophages in fact are crucial for cancer cell invasion was provided by Lin et al. (2001) in the transgenic MMTV-PyMT mouse model. Due to targeted overexpression of the oncogenic polyoma middle T-antigen, these mice spontaneously develop hormone receptornegative, her2-positive breast cancer which rapidly metastasizes to the lungs (Lin et al. 2003). When crossed with mice deficient of colony-stimulating factor-1 (CSF-1), thus lacking significant amounts of macrophages, metastasis formation was greatly reduced, while the oncogene-driven development of the primary tumours remained unaffected. Upon reconstitution of CSF-1, either exogenously or through the transgene, macrophage counts were restored, and metastatic dissemination was reestablished. This indicates that macrophages in this model are not involved in tumour initiation, but

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The Role of Tumour-Associated Macrophages in Malignant Invasion

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are critical for tumour progression. The fact that they foster the escape from the primary tumour strongly suggests that they can modulate tumour cell invasiveness. To better understand how TAM influence tumour cell motility, Hagemann et al. (2004) co-cultivated peripheral blood-derived human macrophages with various breast cancer cell lines in a modified Boyden chamber assay. Coculture enhanced invasiveness of the otherwise only weakly invasive cancer cell lines, while a benign mammary epithelial cell line remained noninvasive. Induced invasion relied on the induction of MMPs as well as on the upregulation of the cytokine TNFD, both in the macrophages. Interestingly, treatment with the Toll-like receptor (TLR) 4-agonist bacterial lipopolysacharide (LPS) abolished this effect, presumably by shifting the tumour-educated macrophages into a classical inflammatory phenotype. These in vitro findings were reproduced also for other cancer entities (Lin et al. 2006; Zins et al. 2007; Wu et al. 2009), however, they cannot be generalized. Invasion of MDA-MB 231, a highly motile breast cancer cell line with mesenchymal features, is much less dependent on external macrophage signals, since motility is already constitutively upregulated through an autocrine CSF-1 loop (Patsialou et al. 2009). Direct evidence of what happens between macrophages and tumour cells in vivo came from Wyckoff et al. (2004). In the above-mentioned PyMT mouse model as well as in SCID mice orthotopically injected with the rat mammary adenocarcinoma cell line MTLn3, microneedles were placed into the tumours for collection and further analysis of the invading cells. Needles contained either epidermal growth factor (EGF) or CSF-1 to provide chemoattraction. Two cell types were found in the collected population. Macrophages comigrated with cancer cells in an approximate ratio of 1:4, which was much higher than expected from the primary tumour and indicated enrichment of macrophages in the motile population. That macrophages were indeed critical for malignant invasion was shown in CSF-1-deficient PyMT variants, where collection yielded not only fewer macrophages but also much lower amounts of tumour cells. The same group (Wyckoff et al. 2007) showed later that macrophages not only contribute to the first of the four steps of tumour progression, namely invasion from the primary tumour into the immediate surroundings (DeNardo et al. 2008). Obviously, they are equally involved in the second step, the intravasation of tumour cells into blood vessels as a prerequisite for further dissemination. Using intravital multiphoton imaging and the PyMT mouse model, Wyckoff and colleagues (2007) visualized the direct interaction of breast cancer cells with macrophages. Motile tumour cells were predominantly found at sites of macrophage accumulation at the tumour margin and in the perivascular space. There, tumour cells entered the vasculature together with the macrophages, again, in a CSF-1 and EGF-dependent way.

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Fig. 3.1 Tumour cells on their way into the brain. (a) MCF-7 breast cancer cells (black asterisks) are contacted by a resident brain macrophage (white arrow) protruding from living brain tissue (S). They are then pulled to the brain slice border (b) and use the microglial cell as a guiding rail for directional locomotion (c). Time lapse sequence of an organotypic brain slice coculture (obtained in collaboration with Chuang, Pukrop, and Hanisch, University of Göttingen)

The Role of Resident Macrophages To successfully accomplish colonization of distant tissues, circulating tumour cells have to extravasate from blood vessels and become invasive again. Since breast cancers in the PyMT mouse metastasize only on condition that the animals are not macrophage-depleted (Lin et al. 2001), it stands to reason that macrophages play a role also in this last step of tumour progression. However, apart from the peripheral blood-derived macrophages, most of the major target organs of metastasis harbor large populations of specialized macrophages with structural functions, also termed “trophic macrophages” (Qian and Pollard 2010). Until now, only few researchers have tried to answer the question of whether these resident macrophages are equally relevant for cancer cell motility and invasion. Liver-specific macrophages, the Kupffer cells, have been reported to exert tumoricidal as well as prometastatic effects (Gjoen et al. 1989; Heuff et al. 1995; Sturm et al. 2003; Gorden et al. 2007). In the lung, cells of the monocytic lineage establish the niche for successful colonization (Hiratsuka et al. 2002; Kaplan et al. 2005; Hiratsuka et al. 2008; Kim et al. 2009). Microglia, the resident macrophages of the central nervous system (CNS), representing a particularly secluded population, have been demonstrated to trigger invasion and secrete a variety of cytokines and growth factors which may contribute to successful immune evasion of CNSderived tumours (Markovic et al. 2005; Wesolowska et al. 2008). Recently, it has been shown that microglia are also essential for the invasion of solid tumours (Pukrop et al. 2010). Apart from enhancing the invasive capacity of breast cancer cells in Boyden chamber assays, microglia, as visualized in an organotypic slice coculture model, served as active transporters and guiding rails for the colonization of living brain tissues (Fig. 3.1). As a proof of principle, the bisphosphonate

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clodronate, a macrophage inactivator, abolished these effects. Unlike TAM, microglia did not undergo the expected cytokine profile switch to M2-like patterns. However, they were still able to react to exposure to LPS with an M1-like profile and the upregulation of TNFD, IL-6, CCL3 (MIP-1D), CCL5 (RANTES), CXCL1 (KC), and CXCL2 (MIP-2), which coincided with the loss of their pro-invasive function.

Signaling in Macrophage-Induced Invasion The EGF/CSF-1 Loop Establishment of cellular protrusions during invasion is based on enhanced actin turnover and extensive remodeling of the cytoskeleton. One of the key players in these changes is cofilin which was found upregulated in the invasive cancer cell population in a rat model (Wang et al. 2004). Local activation of cofilin defines the site of actin polymerization, protrusion and cell direction. Signaling through the EGF receptor was identified as the critical event (Yamaguchi et al. 2005). In particular, stimulation of the EGF receptor pathway was responsible for the formation of invadopodia, which was dependent on neural Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein (Arp) 2/3 complex, while cofilin was required for the stabilization and maturation of these protrusions. The relevance of EGF signaling for invasion was further emphasized by the observation that the overexpression of the EGF receptor in the rat mammary adenocarcinoma cell line MTLn3 fosters in vitro motility and enhances in vivo invasion and metastasis (Xue et al. 2006). Although constitutive overexpression of the EGF receptor as well as production of the respective ligand has been shown in many cancer cells, Wyckoff and colleagues (2004) were the first to identify TAM as the major source of EGF in this context. As blockade of the EGF receptor as well as of CSF-1 signaling inhibited comigration of macrophages and tumour cells, the authors postulated the existence of a paracrine loop, where CSF-1 was secreted by the tumour cells to attract macrophages which, in turn, responded by the release of EGF and stimulated invasion. Consistently, cocultivation of macrophages with MTLn3 cells resulted in the formation of protrusions and enhanced in vitro invasion dependent on EGF and CSF-1 (Goswami et al. 2005). EGF-induced actin remodeling was associated with the activation of phosphoinositide 3 kinase and phospholipase J, followed by the generation of phosphoinositide 2 and 3 phosphate. These signaling molecules can interact with a variety of actin-binding proteins, and also with other regulators of actin polymerization, such as the GTPases Rho and Rac. Interestingly, the latter two can additionally be activated by Wnt-signaling, which will be discussed in detail below.

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The impact of EGF for TAM-induced invasion was confirmed also in other cancer entities. Comparable effects were shown when hepG2 cells were cocultivated with conditioned media of various types of macrophages (Lin et al. 2006). This resulted in the reduction of E-cadherin in adherens junctions, acquisition of an EMT phenotype as well as enhanced invasive capacity. Again, the effect was dependent on EGF receptor signaling. The second partner in the pro-invasive loop, CSF-1 or M-CSF in humans, represents the major growth and differentiation factor for macrophages (Lin et al. 2002). It has long been known that CSF-1 promotes proliferation and, in particular, invasiveness of cancer cells expressing the cognate receptor CSF-1R, encoded by the c-fms protooncogene (Filderman et al. 1992). As mentioned above, MDA-MB 231 cells can upregulate invasion through their autocrine CSF-1 loop alone without any contribution of EGF (Patsialou et al. 2009). However, c-fms negative cells such as MCF-7, which have been shown to be unresponsive to the pro-invasive action of exogenous CSF-1 (Filderman et al. 1992), can still react with invasion to macrophage exposure (Hagemann et al. 2004). An elegant explanation to reconcile these divergent observations was recently provided by Abraham et al. (2010). They demonstrated that CSF-1 is also secreted by stromal cells, in particular, TAM and tumour-associated fibroblasts. In the same way as tumour-derived CSF-1, stromal CSF-1 serves as a chemoattractant to recruit additional macrophages, which then enhance the progression of c-fms positive as well as negative tumours, in the latter case via the upregulation of stromal target genes with pro-invasive function, such as MMP-2, VEG-F, and TNFD (Zins et al. 2007). Obviously, CSF-1 acts at the junction of several major tumour-promoting pathways, influencing not only cancer cell motility but also proteolytic tissue remodeling and neoangiogenesis.

Tumour Necrosis Factor a As the name implies, the inflammatory cytokine TNFD was originally discovered for its ability to elicit hemorrhagic necrosis in tumours. As a putative antitumour agent, it was even administered clinically in phase I/II studies. During these trials, however, it became increasingly clear that most of the TNFD effects point into the opposite direction (Balkwill 2009). In a murine model of chemically induced skin cancer, TNFD, while not interfering with tumour initiation, clearly promoted tumour progression (Moore et al. 1999). Although TNFD was produced by the tumour cells themselves, the fact that it was detected in whole epidermal homogenates of the tumour region (Scott et al. 2003) implies that stromal production may also have contributed to this effect. Consistently, macrophages were later identified as significant suppliers of TNFD, while enhancing invasion of breast cancer cells (Hagemann et al. 2004). Neutralization of TNFD inhibited the induction of downstream targets, such as MMPs, together with macrophageinduced invasion, indicating that TNFD is a critical modulator of the pro-invasive macrophage–tumour cell interactions. This was further underlined by the observation

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that TNFD was crucial for the macrophage switch to an alternatively activated M2-like phenotype (Hagemann et al. 2006). Of the two receptors for TNFD, the ubiquitously expressed 55 kDa TNFR I represents the key mediator of TNFD effects at the macrophage–tumour interface (Balkwill 2009). TNFR I governs several downstream signaling cascades which result either in the induction of cell death or in (chronic) inflammation and tumour promotion. Signaling via the latter branches involves the adaptor proteins TRADD and TRAF2 and is further propagated by the activation of Jun-N-terminal kinase (JNK) and the transcription factor AP-1, mitogen-activated protein kinases (MAPK) and the nuclear factor (NF)NB. The functional outcome of TNFD-signaling is determined not only by differential channeling of the signals, but also by the amount of available TNFD. High concentrations in classical M1-macrophages, especially in combination with other cytotoxic cytokines, clearly induce cell death, whereas lower levels in alternative activation do not. Instead, they induce mechanisms of enhanced survival and stimulate pro-invasive pathways (Sica et al. 2006). Among these pathways are the JNK as well as the NFNB cascade which have been described to mediate invasion in ovarian and breast cancer cell lines triggered by macrophage-derived TNFD (Hagemann et al. 2005). One of the downstream targets was the MMP-inducer EMMPRIN, whose inhibition by siRNA led to significant reduction of MMP secretion and invasion. Apart from increased proteolysis, TNFD can also directly influence cell motility. In pancreatic and colon cancer cell lines, macrophage-derived TNFD induced typical EMT features and enhanced invasion, acting cooperatively with another well-known EMT inducer, transforming growth factor (TGF)E (Bates and Mercurio 2003; Baran et al. 2009). Accumulation of the mesenchymal marker Snail due to TNFD-triggered inhibition of its degradation was responsible for this phenomenon (Wu et al. 2009). TNFD and the NFNB pathway also modulate cancer cell invasion via the activation of additional pro-migratory factors, such as VEGF and various chemokines. This has been demonstrated in ovarian cancer cell lines, where TNFD leads to the upregulation of VEGF, CCL2, CXCL12, and its receptor CXCR4, with corresponding immunohistochemical findings in primary ovarian cancers (Kulbe et al. 2005; Kulbe et al. 2007).

The Chemokine Connection Chemokines are small proteins which govern directional cell movement along chemotactic gradients. Although originally identified during leukocyte recruitment in tissue damage, it is now known that many cells can express chemokines and their receptors, in particular, malignant ones (Balkwill 2004). In cancer tissues, these cytokines are involved in various functions, most of them more or less based on their ability to induce directional migration (Barbieri et al. 2010). Of the many pairs of chemokines and cognate receptors, the CXCR4/CXCL12 axis is paradigmatic for the interplay between tumour cells and microenvironment.

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The receptor is mostly, but not exclusively, expressed on cancer cells (Schioppa et al. 2003), while the ligand CXCL12, as implied by its alternative name stromal-derived factor (SDF)-1, is predominantly produced by cells of the stromal compartment, not least of which, by macrophages (Xu et al. 2009; Zlotnik 2006). Activation of CXCR4 stimulates directional movement of cancer cells and increases their invasion through artificial basement membranes as well as monolayers of various cell types (Scotton et al. 2002). Muller et al. (2001) demonstrated that CXCL12, either recombinant or in conditioned media from bone-marrow cells, induced actin polymerization, pseudopodia formation and directed migration of breast cancer cells. In SCID mice, these cells preferentially metastasized into lung, liver, and lymph nodes, containing high amounts of CXCL12. This is in line with the observation that collectively migrating cells, as often seen in the colonization of distant tissues, rely on the activation of CXCR4 in the leading cells by signals from the stroma (Friedl and Gilmour 2009). The recently identified second receptor for CXCL12, CXCR7, which is expressed at the rear end of migrating cohorts, can exert inhibitory functions and specifically interfere with CXCL12/CXCR4-induced transendothelial migration of cancer cells (Zabel et al. 2009). There are many cross-links to other tumour-promoting signaling cascades. CXCR4 as well as its ligand are modulated by hypoxia through hypoxia-inducible factor (HIF) 1D, which upregulates both molecules in tumour cells and macrophages (Schioppa et al. 2003). Hypoxic necrosis is frequently observed in growing tumours, which additionally attracts macrophages, thus, amplifying the pro-invasive feedback loop. Moreover, CXCL12 has been shown to upregulate TNFD in ovarian cancer cells (Scotton et al. 2002), which, in turn, can trigger invasion. In breast cancer cells, motility and CXCR4 expression are regulated via the NFNB pathway (Helbig et al. 2003). There is evidence also for cross-talk with the EGF/c-erbB receptor family (Hernandez et al. 2009). Overexpression of her-2/c-erbB-2 in breast cancer leads to enhanced invasiveness through the upregulation of CXCR4 and the inhibition of its ligand-induced degradation (Li et al. 2004). Similarly, transactivation of the EGF/c-erbB-1 receptor via CXCR4 and CXCL12 has been demonstrated in ovarian carcinoma cells (Porcile et al. 2005). Comparable to the CXCR4/CXCL12 axis, which elicits a complex to and fro of para- as well as autocrine actions and reactions between tumour and stromal cells to foster invasion, there are many other chemokines and receptors (Robinson and Coussens 2005) following more or less the same functional patterns. For example, expression of CCL2 or monocyte chemoattractant protein (MCP)-1, present in tumour cells as well as macrophages, is upregulated in the latter through tumour cell coculture and results in enhanced macrophage chemotaxis (Fujimoto et al. 2009). Blockade of CCL2 production in non-small cell lung cancers inhibits tumour growth and metastasis not only by altered recruitment of TAM, but also by shifting their phenotype to more M1-like characteristics which then stimulate antitumour activity of the innate immune system via recruitment of CD8-positive T-cells (Fridlender et al. 2011). In conclusion, the chemokine network, apart from direct modulation of tumour cell motility, can potentiate this effect by controlling the amount and com-

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position of the leukocyte infiltrate, its action at the tumour–host boundary being crucial for tumour progression.

Wnt Signaling Wnt ligands are secreted glycoproteins, involved in cell proliferation and directional migration in embryonic development as well as in the adult organism (Clevers 2006). For example, so-called canonical Wnt signaling via E-catenin regulates cohort movement of lateral line cells in the zebrafish embryo by spatial distribution of the two CXCL 12 receptors CXCR4 and seven to either the leading or the trailing end (Aman and Piotrowski 2008). Given these functions, it is not surprising that Wnt signals contribute to cancer initiation and progression when deregulated. Wnts and their receptors are overexpressed in a variety of malignancies, in particular, in colon cancer (Fodde and Brabletz 2007). Pathologic activation of the Wnt/E-catenin pathway is associated with aggressive tumour biology and has been found especially at the invasive frontier between tumour and surrounding tissue (Brabletz et al. 2005). Invasion can also be mediated by another group of Wnts, referred to as noncanonical, whose signals are channeled either into the Wnt/Ca2+ or the planar cell polarity (PCP) pathway (Wallingford and Habas 2005). PCP-signaling results in the activation of the small Rho-GTPases RhoA and Rac, leading to reorganization of the cytoskeleton during establishment of cellular protrusions as well as phosphorylation of JNK and AP-1 (Montcouquiol et al. 2006). While most of the investigations focused on Wnt signaling in tumour cells, the first notion that stroma-derived Wnts might play a role in tumour progression came from Smith et al. (1999) who observed the expression of the noncanonical ligand Wnt 5a in TAM in colon cancer. It was not until 2006 that macrophage-induced invasion of breast cancer cells was demonstrated to depend in fact on the upregulation of macrophage Wnt 5a (Pukrop et al. 2006). Although a functional canonical pathway in the tumour cells was indispensable, noncanonical signaling via JNK was the critical event. The biological significance of these findings was underlined by the detection of Wnt 5a in TAM in primary breast cancers and at the invasive front of lymph node metastases. Upregulation of Wnt 5a has been observed in various types of inflammatory reactions, thus linking Wnt signaling to other inflammatory cytokines, such as TNFD, and the TLR pathway (Pereira et al. 2009). It was expressed in macrophages in granulomatous lesions in tuberculosis (Blumenthal et al. 2006) as well as in septic conditions, where signaling was conferred via Ca2+-influx (Pereira et al. 2008). It stands to reason that these macrophages correspond to the classical M1 phenotype with potential antitumour activity. Obviously, Wnt 5a in macrophages exerts a dual role with opposite functional outcome (Pukrop and Binder 2008). When part of an M1-like reaction and signaling through the Wnt/Ca2+ cascade, it may act as a tumour suppressor, whereas Wnt 5a in tumour-educated macrophages facilitates malignant invasion.

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A recent study of the transcriptome of TAM underlines the significance of Wnt signaling in this setting. Gene expression of murine macrophages comigrating with tumour cells, thus representing the actual pro-invasive subpopulation, was compared with that of the remaining nonmotile TAM (Ojalvo et al. 2010). The Wnt pathway was found enriched in this fraction with the upregulation of Wnt 5b and Wnt 7b, among other Wnt-associated molecules. The positive correlation between Wnt 7b expression and lymph node metastasis, identified by comparative analysis of previously published gene sets of human breast cancer, emphasizes the in vivo relevance of these findings. Interestingly, the pro-invasive interaction of tumour cells with the resident macrophages of the brain was also shown to be Wnt-dependent (Pukrop et al. 2010). Microglial cells constitutively express a series of Wnt ligands, among them Wnt 5a and b, and microglia-induced invasion was inhibited by the physiological Wnt-antagonists Dickkopf-1 and -2.

The Role of Proteases and the Extracellular Matrix Mostly as downstream effectors, but also as modulators of signaling by proteolytic modification of both cell surface proteins and ECM components, proteases play a key role in invasion. MMPs are the most well-known candidates in this context. They belong to a large family of zinc-dependent proteases with ECM-degrading activity and specificity for either collagens or other matrix molecules. During the 1990s, there were many reports that MMPs, especially the collagenases MMP-2 and -9, are crucial for tumour cell invasion (Westermarck and Kahari 1999). They are required, in particular, for the penetration of the collagen-rich basement membrane (Coussens and Werb 1996), their expression correlating with advanced tumour stage and unfavorable clinical outcome. Although many cancer cells express MMPs and are capable of local proteolysis through their invadopodia (Buccione et al. 2009), it has gradually become clear that the stroma significantly contributes to progression-associated MMP production, and a multitude of MMPs are supplied in high concentrations by TAM (Wiesen and Werb 1996; Swallow et al. 1996). MMP inhibitors can completely abolish macrophageinduced invasion in various settings (Hagemann et al. 2004; Grimshaw et al. 2004). The fact that these inhibitors simultaneously block coculture-induced TNFD production, presumably by antagonizing TNFD-shedding through MMP-7 (Haro et al. 2000), suggests that MMPs do not only act as terminal downstream effectors of invasion but also as signaling modulators. At the same time, MMPs are targets of several important regulators of macrophage-induced invasion, such as TNFD itself, SDF-1, EGF, and Wnt 5a (Jodele et al. 2006; Pukrop et al. 2006; Prieve and Moon 2003), indicating that they are involved in para- as well as autocrine loops within the complex signaling network between TAM and tumour cells. Cathepsins, a family of lysosomal cysteine proteases, also contribute to the proinvasive functions of TAM. They have been found upregulated in various cancers,

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where they are mislocated to the cellular surface and secreted into the ECM (Gocheva et al. 2006). Several members of this family, in particular cathepsin B, enhance tumour cell invasiveness via direct cleavage of E-cadherin, leading to loosening of cell–cell contacts and acquisition of an EMT phenotype. Interestingly, apart from the tumour cells themselves, cathepsins can also be produced by TAM (Vasiljeva et al. 2006). In the PyMT mouse as well as in the RIP1-Tag2 model of pancreatic cancer, cathepsin B is increasingly upregulated in macrophages, while they are recruited to the tumour and switch to the full TAM phenotype. This is dependent on IL-4 secretion by the cancer cells (Gocheva et al. 2010). TAMderived cathepsin B then enhances malignant invasion through matrigel and collagen I in vitro, as well as tumour growth and lung colonization in vivo. Obviously, proteases are crucial for the extensive ECM remodeling during tumour cell invasion. However, the ECM, mainly composed of collagens, fibronectin, laminins, elastin, and glycosaminoglycans, is not merely an inert structural scaffold for the embedded epithelial cells, where tumour cells just hack their way through. It rather represents a dynamic signaling network, which can either give rise to or modulate the activity of signaling molecules (Schenk and Quaranta 2003). Wnt molecules, due to their lipid modification (Mikels and Nusse 2006), are typical candidates for such interactions. Much of the secreted Wnts bind to heparan sulfate proteoglycans (Ai et al. 2003; Kurayoshi et al. 2007). Chemical linkage interferes with their signaling activity as well as with the establishment of gradients for directional cell movement. Similarly, matrix components can interact with chemokines and modulate the secretion of cytokines as well as MMP-9 by macrophages (Chung and Kao 2009). Versican, another ECM proteoglycan, found in interstitial tissues at the invasive margins of various cancers, interacts with EGF receptor signaling and promotes migration (Wu et al. 2005; Zheng et al. 2004). When produced by Lewis lung carcinoma cells, it activates TLR 2 and its coreceptors TLR 6 and CD14 and induces IL-6 and TNFD in macrophages, resulting in strongly enhanced metastasis to the lungs (Kim et al. 2009).

Conclusion Taken together, whether a malignant cell invades is not only determined by its intrinsic invasive capacity, but also is regulated by TAM. Invasiveness of poorly differentiated cancer cells with mesenchymal features is constitutively high, and often only marginally influenced by TAM, in contrast to their more epithelial-like counterparts, found in the majority of solid cancers. Differentiated cells require support from TAM, which either directly induce EMT in the tumour cells, or serve as mesenchymal guides at the tip of invading tumour cell cohorts with largely preserved epithelial characteristics. Both settings are driven by the activation of pro-invasive signaling pathways which either end or start in tumour or stromal cells and vice versa, thus, modulating and amplifying the respective effects (Fig. 3.2).

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Fig. 3.2 Hypothetical model of macrophage-induced invasion. Tumour cells secrete cytokines and chemoattractants to recruit macrophages. This switches macrophages to an alternatively activated phenotype and leads to the induction of stimulators and effectors of motility and invasion, among them EGF, which is part of the pro-invasive EGF/CSF-1 loop, TNFD, a major inductor of the NFkB pathway, chemokines as well as Wnt ligands and matrix metalloproteinases. Several of these can also be provided by other stromal cells, in particular CXCL12/SDF-1, which fosters migration and invasion by interaction with the CXCR4 chemokine receptor on cancer cells. Their effects on malignant invasion are additionally regulated by interaction with components of the extracellular matrix as well as by autocrine loops. In some cases, these loops can substitute for the assistance of macrophages, e.g., the autocrine stimulation by CSF-1 without EGF in cells with constitutive mesenchymal features. Macrophages contribute to invasion by directly inducing EMT and enhanced invasive capacity in the tumour cells via the provided stimuli. Additionally and/or alternatively, they can act as mesenchymal assistants at the tip of invading tumour cell cohorts with largely preserved epithelial characteristics. (T = tumour cells, M = macrophages, F = fibroblasts, V = vasculature, L = lymphocytes, ECM = extracellular matrix, EMT = epithelial–mesenchymal transition)

Given the complexity of the underlying signaling and effector networks, it is clear that therapeutic intervention is not trivial. These networks are cross-linked at multiple levels, and activation patterns in the cytotoxic and the pro-invasive setting are at least partially overlapping. Thus, attempts to modulate activity of single factors may result in unexpected upregulation of substitute pathways and other escape mechanisms and have, until now, not proven satisfactorily successful. Targeting the TAM to shift them back from a tumour-promoting phenotype to their

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genuine defense functions, seems a more promising strategy, as this would interfere not only with their pro-invasive but also with their angiogenic and growth-supporting actions. Coming back to Coley, in principle, he seems to have been on the right track. However, there is still a lot more to know to reprogram inflammation and disrupt the involved cascades at the appropriate point to eventually achieve anti-invasive features without undesired side effects.

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derived cathepsin B promotes progression and lung metastasis of mammary cancer. Cancer Res 66:5242–5250 Wallingford JB, Habas R (2005) The developmental biology of Dishevelled: an enigmatic protein governing cell fate and cell polarity. Development 132:4421–4436 Wang W, Goswami S, Lapidus K, Wells AL, Wyckoff JB, Sahai E, Singer RH, Segall JE, Condeelis JS (2004) Identification and testing of a gene expression signature of invasive carcinoma cells within primary mammary tumors. Cancer Res 64:8585–8594 Wesolowska A, Kwiatkowska A, Slomnicki L, Dembinski M, Master A, Sliwa M, Franciszkiewicz K, Chouaib S, Kaminska B (2008) Microglia-derived TGF-beta as an important regulator of glioblastoma invasion–an inhibition of TGF-beta-dependent effects by shRNA against human TGF-beta type II receptor. Oncogene 27:918–930 Westermarck J, Kahari VM (1999) Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J 13:781–792 Wiesen JF, Werb Z (1996) The role of stromelysin-1 in stromal-epithelial interactions and cancer. Enzyme Protein 49:174–181 Wu YJ, La Pierre DP, Wu J, Yee AJ, Yang BB (2005) The interaction of versican with its binding partners. Cell Res 15:483–494 Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP (2009) Stabilization of snail by NF-kappaB is required for inflammation-induced cell migration and invasion. Cancer Cell 15:416–428 Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, Graf T, Pollard JW, Segall J, Condeelis J (2004) A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 64:7022–7029 Wyckoff JB, Wang Y, Lin EY, Li JF, Goswami S, Stanley ER, Segall JE, Pollard JW, Condeelis J (2007) Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors. Cancer Res 67:2649–2656 Xu L, Duda DG, di Tomaso E, Ancukiewicz M, Chung DC, Lauwers GY, Samuel R, Shellito P, Czito BG, Lin PC, Poleski M, Bentley R, Clark JW, Willett CG, Jain RK (2009) Direct evidence that bevacizumab, an anti-VEGF antibody, up-regulates SDF1alpha, CXCR4, CXCL6, and neuropilin 1 in tumors from patients with rectal cancer. Cancer Res 69:7905–7910 Xue C, Wyckoff J, Liang F, Sidani M, Violini S, Tsai KL, Zhang ZY, Sahai E, Condeelis J, Segall JE (2006) Epidermal growth factor receptor overexpression results in increased tumor cell motility in vivo coordinately with enhanced intravasation and metastasis. Cancer Res 66:192–197 Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, Symons M, Segall J, Eddy R, Miki H, Takenawa T, Condeelis J (2005) Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J Cell Biol 168:441–452 Yang J, Weinberg RA (2008) Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 14:818–829 Zabel BA, Wang Y, Lewen S, Berahovich RD, Penfold ME, Zhang P, Powers J, Summers BC, Miao Z, Zhao B, Jalili A, Janowska-Wieczorek A, Jaen JC, Schall TJ (2009) Elucidation of CXCR7-mediated signaling events and inhibition of CXCR4-mediated tumor cell transendothelial migration by CXCR7 ligands. J Immunol 183:3204–3211 Zheng B, Lavoie C, Tang TD, Ma P, Meerloo T, Beas A, Farquhar MG (2004) Regulation of epidermal growth factor receptor degradation by heterotrimeric Galphas protein. Mol Biol Cell 15:5538–5550 Zins K, Abraham D, Sioud M, Aharinejad S (2007) Colon cancer cell-derived tumor necrosis factor-alpha mediates the tumor growth-promoting response in macrophages by up-regulating the colony-stimulating factor-1 pathway. Cancer Res 67:1038–1045 Zlotnik A (2006) Chemokines and cancer. Int J Cancer 119:2026–2029

Chapter 4

Tumour-Induced Immune Suppression by Myeloid Cells Serena Zilio, Giacomo Desantis, Mariacristina Chioda, and Vincenzo Bronte

Tumour-Induced Immunosuppression Since the description of the immunosurveillance theory, tumour immunologists have faced one critical paradox: the host maintains a general good responsiveness to exogenous antigens in spite of the profound dysfunction of immune reactions against tumour-antigens, except under some chemotherapeutic and radiotherapy treatments or in advanced stages of the disease. Even with the identification of tumour-associated antigens, which could be used as selective targets for cancer immunotherapy, the inability of the immune system to provide a strong response against the tumour, and the consequent low efficiency in eliminating tumour cells represents one of the major limitations to clinical translation. Indeed, the process of immune selection can be exploited by tumours for evolving in cells escaping the host immune response. This allows the generation of clinically identifiable tumour variants growing in immunocompetent hosts. It is, therefore, clear that the immune system not only has the potential of protecting the host in early stages of tumourigenesis but can also select cancer cells to give rise to species with reduced immunogenicity (Dunn et al. 2004).

S. Zilio Department of Oncology and Surgical Sciences, University of Padova, Via Gattamelata 64, 35128 Padova, Italy Istituto Oncologico Veneto (IOV), IRCCS, Via Gattamelata 64, 35128 Padova, Italy G. Desantis Department of Oncology and Surgical Sciences, University of Padova, Via Gattamelata 64, 35128 Padova, Italy M. Chioda Istituto Oncologico Veneto (IOV), IRCCS, Via Gattamelata 64, 35128 Padova, Italy V. Bronte (*) Verona University Hospital and Department of Pathology, Immunology Section, Piazzale L.A. Scuro 10, 37134 Verona, Italy e-mail: [email protected] T. Lawrence and T. Hagemann (eds.), Tumour-Associated Macrophages, DOI 10.1007/978-1-4614-0662-4_4, © Springer Science+Business Media, LLC 2012

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Tumours can elude immune response by three main mechanisms: (1) cancer cells can mask their identity to escape recognition by the immune effectors, (2) they can directly modulate immunity, or (3) recruit other immune regulatory cells whose normal function is to dampen immune reactions to avoid the detrimental effects of an excessive immune stimulation. Probably, the most robust and efficient mechanism of “tumour escape” relies on tumour ability to create a tolerant microenvironment through the production of tumour-derived soluble factors (TDSFs). TDSFs modify the differentiation of myeloid precursors into either dendritic cells (DCs) or myeloid-derived suppressor cells (MDSCs) (Smyth et al. 2001; Drake et al. 2006) in the bone marrow and other hematopoietic organs. The imbalance in the number and type of myeloid cells has also profound influences on myeloid cell recruitment and function at the tumour site and in secondary lymphoid organs. The first part of this chapter considers cancer-induced modification in myelopoiesis, whereas the second one analyzes how tumour microenvironment can alter the function of recruited cells to enforce a tolerogenic mileu.

Tumour-Dependent Alteration in Myelopoiesis Results in Defective Accumulation of DCs and Enhanced MDSC Generation The presence of tumours alters the distribution and function of cells of the innate immune system. One of the main cell population targeted by this mechanism is represented by DCs, potent antigen-presenting cells (APCs) able to initiate the activation of naïve T cells (Guermonprez et al. 2002). In the bone marrow, hematopoietic progenitors cells (HPCs) give rise to immature DCs (iDCs). This first step of lineage commitment of the HPC is due to both exposure to soluble growth factors such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and fms-related tyrosine kinase 3 ligand (FLT3L) (Gabrilovich 2004) and to cell–cell contacts with bone marrow stroma cells. Differentiation of HPCs in iDCs is tightly regulated by the cooperation between the signaling pathways of Notch-Dll1 and the canonical Wnt cascade (Cheng et al. 2010). To achieve complete maturation, iDCs require further stimuli because, although able to uptake, process, and present antigens, they express little or no costimulatory molecules such CD80, CD86, and CD40 necessary to exert their full set of functions (Rabinovich et al. 2007). Maturation occurs physiologically thanks to microbial stimuli, proinflammatory cytokines, or CD40L-expressing T cells. During inflammation iDCs are activated by the contact with both exogenous antigens such as bacterial and viral products (for example LPS, flagellin, and viral particles) and endogenous antigens released from dying cells, as double-stranded RNA and other small molecules, such as urea and ATP (Macagno et al. 2007). In theory, stimuli for promoting iDC terminal differentiation are not induced only by microbial antigens, given that an inflammatory context is also often associated to several pathologies including cancer.

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When iDCs become activated DCs, they upregulate costimulatory and MHC class II molecules, increase IL-12 production and lose the ability to process antigens (Mellman and Steinman 2001; Banchereau et al. 2000). At the tumour site, the large number of tumour proteins released by dying or necrotic cancer cells represent, in theory, potent activators of DCs (Sauter et al. 2000). Activated DCs engulf these cells, process and present their antigens. Once they have reached the draining lymph nodes, DCs loaded with tumour antigens have the potential to initiate an immune response against tumour. However, despite this potential, DCs do not contribute to trigger an efficient immune response aimed at eliminating tumours, as often these manage to progress until the death of the host. One of the first explanations of this DC “failure” could derive from the fact that some tumours can affect the generation of functionally active DCs. This hypothesis is substantiated by the observation that tumour-bearing hosts have fewer activated DCs and that a physiological number of DCs can be restored upon resection of the primary tumour (Ishida et al. 1998; Gabrilovich et al. 1996a). Reduction of DCs was also measured in lymph nodes, skin, and spleen (Ishida et al. 1998). Growing tumours affect the number of activated DCs by inhibiting the transition from iDCs to DCs. In fact, DCs found at tumour site had a phenotype similar to the iDCs that leave the bone marrow after the initial phase of their differentiation. Furthermore, the higher number of iDCs stems out from defects in myelopoiesis rather than simply from the lack of appropriate activation signals at the tumour site. In vitro treatment of tumour-infiltrating DCs with appropriate stimuli (GM-CSF and TNF-D or CD40L) was in fact not sufficient to induce the expression of markers proper of activated DCs (Chaux et al. 1997). Taken together, this evidence supports the concept that the reduced functionality of DCs against some tumours is most probably due to defects in differentiation from their iDC progenitors. iDC are not the only myeloid cell population altered in tumour pathologies. In the bone marrow, hematopoietic stem cells give rise to the lymphoid (LP) and the myeloid (MP) multipotent precursors cells. From the MP originate other pluripotent cell types: the common DC (CDP) and the immature myeloid cell precursors (IMC); the first gives rise to iDCs and plasmacytoid dendritic cells (PDC), the second is the common progenitor of macrophages, granulocytes, and monocyte-derived DCs such as the Tip-DCs (TNF and inducible iNOS-producing DCs). In healthy organisms, IMCs rapidly differentiate into their descendant lineages; consequently they represent a relative low percentage of myeloid cells. However, under pathological conditions, including cancer, there is a partial block of differentiation of the IMCs leading to the accumulation of MDSCs and a consequent reduction of mature myeloidderived cells (Geissmann et al. 2010; Gabrilovich and Nagaraj 2009). Key features of MDSCs, besides their myeloid origin, are their immature phenotype and the exceptional ability to suppress T-cell-mediated immune response. (Gabrilovich et al. 2007). MDSCs are not a uniform cell population rather they include several subgroups distinct on their morphology and expression of surface markers. MDSCs have been described in several mouse tumour models and in human cancer patients. In mice, these cells are defined by the co-expression of the myeloid-cell lineage differentiation

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marker Gr-1 and of CD11b (Serafini et al. 2004; Serafini et al. 2006). Based on the expression levels of Gr-1 (low, intermediate, and high), MDSCs cells were more precisely defined as a macropopulation composed by at least three different groups. (Dolcetti et al. 2010). MDSCs were also distinct depending on their phenotype and morphology into monocytic MDSCs (CD11b+Ly6C+F4/80+Gr-1low/int) and granulocytic MDSCs (CD11b+ Ly6G+F4/80-Gr-1high). MDSCs also express markers associated at the early stage of myeloid differentiation (CD31 and ER-MP58), low levels of MHC class II and co-stimulatory molecules (e.g., CD80), according to their origin from immature myelo-monocytic precursors (Bronte et al. 2000; Rossner et al. 2005). Although the reciprocal interplay among these subpopulations requires further studies, the immunosuppressive role of the monocytic MDSC subset has been confirmed by several groups (Dolcetti et al. 2010; Movahedi et al. 2008; Huang et al. 2006). Fundamental for the suppressive activity of MDSCs are their expansion and activation. Their activation is regulated by factors released by activated T-cells and tumour stromal cells, while their expansion is due mainly to factors secreted by the tumour itself (TDSFs). TDSFs induce both stimulation of myelopoiesis and the block of differentiation of myeloid cells with the consequent accumulation of immature myeloid precursors. It appears, therefore, that such a cancer-induced massive imbalance of myelopoiesis has profound consequences on the ability of the immune system to react to tumours: a lower number of mature DCs (DC maturation is blocked from both the iDC and the IMC branches) of macrophages and granulocytes results in a reduced potential of tumour antigen presentation, T-cell activation, and elimination of cancer cells. The immune system is then further compromised by the suppressive activity of the MDSC population (see next section). Many are the factors known to play a role in tumour-mediated MDSC or iDC expansion and alteration of myelopoiesis: they include cytokines, chemokines, and other factors such as prostaglandins, stem-cell factor (SCF), M-CSF, IL-6, GM-CSF, and the vascular endothelial growth factor (VEGF) (Gabrilovich and Nagaraj 2009). We will illustrate some examples of how TDSFs can regulate the number of mature and immature myeloid cells. VEGF is responsible of tumour-dependent angiogenesis and contributes to both tumour growth and metastasis. Tumour-produced VEGF (tVEGF) also impairs the recruitment and differentiation of DCs and expands MDSCs by suppressing the nuclear factor-kB (NF-kB) in hematopoietic progenitor cells. In facts, NF-kB is a key transcription factor in myelopoiesis regulating differentiation and survival of many precursors (Ouaaz et al. 2002) thus impacting on the composition of the mature myeloid population. Further, there is a feedback loop between tVEGF and MDSC expansion as treatment with tVEGF increases recruitment of MDSCs in the spleen of tumour-free mice; MDSCs, in turn, can regulate the bioavailability of the VEGF through the upregulation of matrix metallo-protease 9 (MMP9) (Yang et al. 2004). MMP9 acts as the molecular switch in tumour-dependent angiogenesis mediating release of VEGF (Oyama et al. 1998; Fricke et al. 2007). Through a VEGF receptor (R)2-mediated mechanism, VEGF was also shown to

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cause a profound alteration in hematopoiesis, by impairing the bone marrow hematopoiesis and, although, a parallel activation of extramedullary hematopoiesis in liver and spleen was seen, it was not sufficient to compensate for the defects of the first one (Xue et al. 2009). Another key cytokine is IL-10 as it can induce the downregulation of MHC II, co-stimulatory and adhesion molecules on DC surface, converting competent DCs in tolerogenic APCs (Steinbrink et al. 1997). IL-10 functions also in preventing differentiation of monocyte precursors to monocytes-derived DCs and rather forces them toward macrophagic lineage (Allavena et al. 1998; Buelens et al. 1997). Furthermore, DCs matured in the presence of IL-10 showed a reduced production of inflammatory cytokines and a lack of IL-12 synthesis, resulting in the induction of anergic CD8+ and CD4+ T regulatory lymphocytes, named CD8+/CD4+ Tr cells, with an antigen-specific suppressive activity. These cells present high expression of the CTLA-4, which is a inhibitor of T-cell activation (Koch et al. 1996; Steinbrink et al. 2002). However, the immunosuppressive activity of IL-10 is limited to iDCs since during terminal differentiation, DCs downregulate the IL-10 receptor, becoming non-responsive to IL-10 (Mahnke et al. 2002). IL-13 is a fundamental cytokine for the activation of the suppressive function of MDSCs. IL-13 interacts with the D chain of IL-4 receptor and cooperates with IFN-J required for the stable expression of IL-4RD on cell surface. IL-13 and IFN-J are essential for the induction of the two enzymes (ARG1 and NOS2) involved in l-arginine metabolism, whose upregulation has been linked to the immunosuppressive activation of MDSCs (see next section) (Gallina et al. 2006). Key roles in regulating myelopoiesis are played by M-CSF and IL-6, which also prevent myeloid precursor cells to become DCs and promote differentiation toward the monocytic/IMC lineage (Gabrilovich et al. 1996b; Menetrier-Caux et al. 1998). Treatment of iDC with IL-6 induces inability of these cells to respond to stimulation with LPS or TNF-D and peptidoglycan and therefore to achieve full maturation as DCs (Park et al. 2004). Another important cytokine involved in tumour tolerance is the granulocyte– monocyte colony stimulating factor (GM-CSF). GM-CSF is produced by a wide range of mouse and human tumour cell lines (Bronte et al. 1999). Chronic administration of GM-CSF results in the accumulation of MDSCs in the bone marrow, spleen, and blood of healthy mice mirroring the pattern induced by tumours (Bronte et al. 1999). Recent reports in both mice and humans show that bone marrow cells treated in vitro with GM-CSF and IL-6, become phenotipically similar to MDSCs induced by cancer. These in vitro-derived MDSCs also retain the ability to suppress in vivo the activity of T lymphocytes in an allogenic transplant models (Marigo et al. 2010). The molecules described above influence the ability of the HPCs in the bone marrow to commit to a given cell lineage by either favoring or preventing one of several differentiation routes. This is achieved molecularly by a restricted number of signal transduction pathways where many of these factors converge. Recent evidence suggests that at least one of these common pathways could be controlled by

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Janus-activated kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). The JAK and STAT families are involved in the transcriptional regulation of cellular survival, apoptosis, proliferation, and differentiation (Rane and Reddy 2000). JAK2 has been associated to the immune system because of its correlations to various cytokines and growth factor receptors including IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, and IFN-D,E,J (Ihle et al. 1997; Imada and Leonard 2000). Further, direct stimulation of STAT3 activity in HPCs was demonstrated for all the described factors involved in DC dysfunction (Nefedova et al. 2004). Cytokines activate the JAK2 pathway causing the recruitment and phosphorylation of STAT3. Phosporylated STAT3 omodimerizes and traslocates to the nucleus where it regulates the expression of target genes involved in the control of cell survival and proliferation. Indeed, the aberrant activation of JAK or its constitutive expression has been observed in many tumours (Nosaka and Kitamura 2000; Steelman et al. 2004). The JAK2– STAT3 pathway is physiologically required for the early steps of differentiation of myeloid cells (Duarte and Franf 2002; Smithgall et al. 2000). Studies carried on conditional STAT3 knockout mice described the abrogation of the signaling pathway of Flt3L responsible for the activation of many hematopoietic progenitors and the consequent impairment in the development of DCs (Laouar et al. 2003). However, full differentiation of DCs requires a time-depending downregulation of STAT3 activation (Nefedova et al. 2004), emphasizing that a tight control of this signaling route is required for regulating various stages of myeloid differentiation and survival. (Nefedova et al. 2004). Another molecular switch involved in the expansion of MDSC population is represented by the signaling pathway of CCAAT-enhancer-binding protein-beta (C/EBPE). Tumour-produced GM-CSF, IL-6, and G-CSF are potent inducer of C/ EBPE a known regulator of ‘emergency’ granulopoiesis (Hirai et al. 2006). This factor plays a fundamental role in MDSCs expansion since the ablation of C/EBPE in the myeloid lineage results in a reduced accumulation of MDSCs, preferentially of the monocytic subset, in the spleen of tumour bearing mice. The reduced expansion of this population of MDSCs correlates with the full restoration of CD8+ T lymphocyte function and in a less invasive tumour behavior measured by the number of secondary metastasis (Marigo et al. 2010). Arginase 1 (ARG1) (Gray et al. 2005) and nitric oxide synthase 2 (NOS2) (Gupta and Kone 1999) are also regulated by C/ EBPE; therefore, the induction of this transcriptional factor by tumour-derived GM-CSF, IL-6, or G-CSF is one of the direct links between tumour and myelopoiesis resulting in the expansion of a MDSC monocytic population permissive to tumour and metastasis (Marigo et al. 2010). We described TDSFs as the main mechanism by which tumour can alter homeostasis of myelopoiesis. It is important to mention that TDSFs normally act locally and they rapidly undergo degradation in the blood stream. Tumour can use an additional strategy, physiologically employed for trafficking molecules. Recent evidences point out that cancer cells could release microvescicles containing bioactive molecules, nucleic acid, and protein (Mytar et al. 2008; Ratajczak et al. 2006; Cocucci et al. 2009). Probably, by protecting TDSFs from degradation, this mechanism facilitates their delivery to distant tissues, such as the bone marrow.

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Generation of Tumour Permissive Microenvironment The ongoing discovery of the fine regulation of the adaptive immunity by myeloid cells (a sort of “historic revaluation” of the innate immunity importance in antigenic responses) is providing us some answers to the puzzle regarding the apparent loss of immunocompetence at tumour sites. To simplify the complexity of this regulation, we can say that tumour-established suppressive network aims at two main goals: the direct inhibition of CTL antitumoural responses and the creation and maintenance of an immunosuppressive environment where this is needed, with the consequent induction of regulatory phenotypes in cells of the immune system that reside in those sites. To achieve these two goals, tumour-induced myeloid populations must first migrate to strategic key points throughout the organism, i.e. secondary lymphoid organs and the tumour site. The spleen in tumour-bearing hosts is an evident example of the abnormal tumour-driven accumulation of myeloid cells with immunosuppressive activity, most of which are CD11b+ Gr-1+ (in case of the mouse host) MDSCs (Gabrilovich and Nagaraj 2009; Diaz-Montero et al. 2009; Almand et al. 2001). These cells are very powerful suppressors of CD8+ T-cell response; their ability requires direct cell-to-cell contact, antigen presentation through MHC class I (Gabrilovich et al. 2001) and acts through the generation of free radicals and metabolism of l-arginine in the microenvironment by ARG1 and NOS2 enzymes (Kusmartsev et al. 2004; Bronte and Zanovello 2005). Although matter of debate, it seems that MDSC immunosuppression may be both antigen- or non-antigen-specific, depending on the tissue where these cells reside. In peripheral lymphoid organs, MDSCs seem to suppress in an antigen-specific manner. Among the possible mechanisms, the post-trasductional modifications of the TCR through the generation of peroxynitrites at the site of the immunologic synapse has been advanced (Willimsky et al. 2008; Nagaraj et al. 2007). Peroxynitrites (ONOO−) are generated by the combination of reactive oxygen species (ROS) with nitric oxide (NO), mainly produced by NOS2 with minor contribution of other NOS isoforms. During presentation of antigens, MHC-I molecules expressed by MDSCs engage with the TCR of CD8+ T cells, and this strict interaction favors the nitration and nitrosylation of TCR amino acids by MDSC-released peroxynitrites, finally resulting in the modification of the TCR specificity against those antigens presented in the context of MHC-I on MDSC. Reduced TCR specificity causes an impairment of TCR ability to respond to antigenic stimulation (Nagaraj et al. 2007). MDSC generation has another important effect on lymphoid tissues. As we discussed earlier, an increased production of these cells will result in a lower number of mature competent professional APCs (i.e., DCs) in lymphoid tissues (Ishida et al. 1998; Almand et al. 2000; Della Bella et al. 2003); moreover, the few DCs present in lymph nodes and tumour site display an immature phenotype. The lack of stimuli due to the absence of mature DCs has an active detrimental effect on T-cell function, causing anergy, which is a state of non-responsiveness to antigenic stimulation (Bonifaz et al. 2002). In this way, all tumour antigens presented in draining lymph nodes by iDCs will induce tolerance instead of immune reaction. Interestingly,

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a class of lymphoid DCs called plasmacytoid DCs is not affected by tumour-induced alteration in myelopoiesis (Della Bella et al. 2003; Hoffmann et al. 2002), and this particular DCs can induce IL-10+ CCR7+ suppressive CD8+ T cells, with inhibitory effects on priming of tumour-specific T-cell in draining lymph nodes (Wei et al. 2005). All these phenomena may explain how the tumour manages to suppress the immune response against its own antigens, while leaving substantially unaltered the immunocompetence against general non-self-antigens. At the tumour site, the aim of dampening the immune response is achieved by a quite different strategy. In this case, MDSC suppression is believed to be non-antigenspecific and to be mediated by the concomitant upregulation of ARG1 and NOS2 induced by local stimuli (Gabrilovich and Nagaraj 2009; Gallina et al. 2006). The effects of the combined activity of these two enzymes (Bronte and Zanovello 2005) bring to a general inability of T cells to respond to proliferative stimuli, leading them to apoptosis, eventually. ARG1 and NOS2 have as common substrate l-arginine. Upregulation of these two enymatic activities implies a greater consumption of this essential aminoacid. l-Arginine starvation limits protein synthesis and reduces the re-expression of the ]-chain subunit of the TCR, which is usually internalized after TCR-mediated stimulation (Baniyash 2004). This mechanism could be mediated by GCN2, a sensor of amino acid deprivation, whose activation leads to an impairment of protein synthesis (Zhang et al. 2002; Lee et al. 2003). A second similar strategy operated to reduce immunocompetence at tumour sites, consists in controlling the availability of other essential nutrients to limit the proliferation of particular cell types, as in the case of indoleamine 2,3 dioxygenase (IDO) enzyme, which catalyze tryptophane degradation, expressed in a particular subset of DCs (Munn et al. 2002; Uyttenhove et al. 2003). An alternative way through which MDSCs achieve non-antigen-specific suppression is the production of NO by NOS2. NO levels impair IL-2 signaling by destabilizing IL-2 mRNA (Fischer et al. 2001; Macphail et al. 2003) and by blocking downstream effectors of the IL-2 receptor signaling pathway such as JAK1, JAK3, STAT5, ERK, and AKT (Bingisser et al. 1998; Mazzoni et al. 2002). The expression of ARG1 and NOS2 in MDSCs is a peculiar feature of these cells, which makes them quite different from tumour-associated macrophages (TAMs). In fact, the mutually exclusive expression of these enzymes has been historically used to define the polarization of mouse macrophages driven by antithetical cytokines. NOS2 is generally considered a marker of M1 macrophage orientation, while ARG1 defines the M2 state. Induction of both NOS2 and ARG1 is not only a way through which MDSCs can exert suppression in tumour environment, but these enzymes are also necessary for the MDSCs in lymphoid compartment to acquire full suppressive potential. At least in some tumour models, splenic MDSCs need to be armed by antithetic cytokines (IFN-J and IL-4/IL-13) to sustain a long-lasting upregulation of both NOS2 and ARG1 (Gallina et al. 2006). In particular, IFN-J released by antigen-activated CD8+ T cells and a membrane-coupled signal are sensed by MDSCs, which react to this stimulus by autocrine secretion of both IFN-J and IL-13. The combination of the signal transduction pathways activated by these two

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cytokines (which requires the expression of the marker CD124, the D chain of the IL-4/IL-13 receptor), leads MDSCs to upregulate ARG1 and NOS2 (Gallina et al. 2006). Interestingly, CD124 is one of the first markers to be upregulated in bone marrow cells under cytokine regimes that generate human and mouse MDSCs (Marigo et al. 2010). The peculiarity of MDSCs to respond with an M2-oriented program to Th1-cytokines is common to TAMs (Sica and Bronte 2007), but in both cell types the molecular ground for this phenotypic behavior needs further investigation. Actually, the relationship between MDSCs and TAMs may be an “ontologic” one. Some data suggest that, once they reach the tumour site, circulating MDSCs are capable of differentiating into TAMs, as indicated by the downregulation of Gr-1 and upregulation of F4/80 surface markers (Kusmartsev and Gabrilovich 2005); given the strict connection in their molecular pathways, just discussed, this could indeed be the case (Sica and Bronte 2007). Although TAMs are capable of expressing either ARG1 or NOS2, their suppressive capacity has been mainly associated with their ability to secrete large amounts of IL-10, TGF-J, and prostaglandins (Mantovani et al. 2002). Due to their extraordinary ability to release a wide range of cytokines, TAMs are some of the main actors of the immunosoppressive environment established at the tumour site. For example, they attract Tregs through the release of CCL22 (Curiel et al. 2004), and Treg regulatory activity is enhanced by the secretion of IL-10 and TGF-E. The role of TAMs as “masters of puppets” in the induction of other regulatory cells is strictly connected with the relationship between cancer and inflammation illustrated by the model of the “balance needle.” For several years, a relationship between chronic inflammation and incidence of cancer has been observed, and tumour is often considered an example of smolder inflammation (Mantovani et al. 2008). Although many issues need to be clarified, neoplastic lesions are thought to begin with an initial phase of inflammation, which promotes the neoplastic transformation and evolve into a state of persistent mild inflammation. This latter favors proliferation and survival of tumour cells, promotes angiogenesis, metastasis and prevents an opportune immune reaction to be initiated. As a confirmation of this, the transcriptome of human tumour cells in which the oncogene RET has been activated (which is sufficient for the pathogenesis of papillary thyroid carcinoma), includes among others upregulation of mRNAs encoding for various inflammation-linked molecules, such as colony stimulating factors (CSFs), IL1-E, COX2 (which synthetize prostaglandins), chemokines attracting monocytes, and DCs (CCL2 and CCL20) (Borrello et al. 2005). TAMs could represent the most probable candidates for explaining the link between cancer and inflammation as they inflict tissue damage during chronic inflammation and favor tumourigenesis (through an M1 phenotype), while sustain smolder inflammation during tumour progression (through an M2 phenotype) (Sica and Bronte 2007). A molecular explanation for the polyedric behavior of macrophages during various steps of tumourigenesis can be related to the transcription factor NF-kB. Given the broad implications that NF-kB has on cell

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survival, proliferation, and differentiation, its link to TAMs is described in depth in a separate chapter of this book. According to the model of the “balance needle,” the immunologic fate of an antigen (induction of response or tolerance) depends on the kind of signals provided in the context of its expression and/or presentation. Each signal (cytokines, co-stimulatory signals, activity of immunoregulatory enzymes, etc.) supports the preservation or the elimination of that antigen, and their combination determines its fate. At the tumour site, there is a clear imbalance toward immunological tolerance to or disregard of tumour antigens, due to the secretion of molecules that suppress DC maturation, as above described (Gabrilovich 2004; Zou 2005); these molecules are secreted by tumour cells, stromal cells, and TAMs. Further, low levels of cytokines necessary for DC differentiation (e.g., IL-4, IL-12, and IFN-J) are produced at the tumours site, where the presence of a smolder inflammation phenotype also implies that insufficient danger/activating signals are delivered. These conditions lead to the accumulation of iDCs responsible for CD8+ T cell anergy. Furthermore, the imbalance of cytokines induces a regulatory behavior of DCs together with the expression of co-inhibitory molecules such as B7-H1 and B7-H4 (Zou and Chen 2008), which determines both T-cell unresponsiveness and generation of Tregs (Krupnick et al. 2005). TAMs contribute to the establishment of a tolerogenic microenvironment also by the secretion of chemokines (in particular CCL2, CCL22, and CCL18) that attract other regulatory cells. CCL2 is the principal chemoattractant for TAMs (Mantovani et al. 2002), CCL22 for Tregs, while CCL18 acts on naïve T cells (Schutyser et al. 2002), which are caught in a signaling web where they are rendered either anergic, tolerant, or regulatory. MDSCs seem to contribute to the induction of Tregs, but this aspect of their biology has been poorly studied so far. Several works have shown, both in vitro and in vivo, that MDSCs are able to expand Tregs through stimulation of natural occurring Tregs, instead of converting CD4+ T effector cells into regulatory ones (Huang et al. 2006; Ghiringhelli et al. 2005; Serafini et al. 2008; Yang et al. 2006; Liu et al. 2008; MacDonald et al. 2005). Many mechanisms for the MDSC-mediated Treg expansion have been proposed: these involve the secretion of IFN-J, IL-10, and TGF-E, the expression of arginase and the co-stimulatory molecules CD80 and B7-H1. Although these data propose different models and require further validation, the majority of them clearly shows the need for MHC class II-restricted antigen presentation in MDSC-Treg direct interaction. Moreover, it has been shown that APCs acquire the ability of expanding Tregs after direct or indirect interaction with tumour cells, both in vivo and in vitro (Ghiringhelli et al. 2005; Serafini et al. 2008; Yang et al. 2006). However, this concept is still a matter of debate as other lines of evidence have shown no correlation between MDSC expansion and induction of Tregs during tumour progression (Movahedi et al. 2008; Dugast et al. 2008). In summary, the induction of immunoregulatory phenotypes at tumour site is a complex network where signal transduction, transcriptional regulation, cytokines secretion, surface molecules, nutrients metabolism, and reciprocal cell–cell interaction are tightly bound. Tumour-induced tolerance is not simply hierarchical or unidirectional and it requires fine modulation in the function of lymphoid cells involved, as they have the potential of either inducing tolerance or initiating an immune reaction:

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for example, Tregs can trigger alternative activation of macrophages or suppress the expression of IL-12 in DCs, by reverse signaling of B7-H1 (Tiemessen et al. 2007; Curiel et al. 2003).

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Chapter 5

TAM: A Moving Clinical Target Simon Hallam and Thorsten Hagemann

TAM and Potential Therapeutic Strategies As previously detailed, TAM are an important component of a tumour-supporting microenvironment and consist of newly recruited and resident tissue macrophages. TAM have roles in supporting angiogenesis, tumour growth, invasion and metastasis. TAM phenotype is variously described as “wound healing” and it has been postulated that this may indeed describe how they have evolved this phenotypic response to the presence of tumours. It is possible that TAM are, maladaptively, responding to tumour-inflicted tissue damage in a similar way that they would to sterile wounds, and so display a pro-angiogenic and immune-suppressive phenotype. Rational therapeutic approaches can be considered in terms of whether they involve macrophage ablation, inhibition of monocyte recruitment, re-education or adoptive transfer.

Macrophage Ablation An attractive strategy would be to selectively ablate pro-tumoural TAM, while preserving physiological macrophages in other tissues. In the 1980s, a “macrophage suicide” technique was developed, whereby liposome-encapsulated hydrophilic bisphosphonates were delivered into cells, exploiting the preferential engulfment of liposomes by cells of the mononuclear–phagocyte system. Intracellular accumulation of bisphosphonate led to apoptosis, primarily of monocytes and tissue macrophages. The most effective bisphosphonate for this purpose has been clodronate

3(ALLAMs4(AGEMANN*) Centre for Cancer and Inflammation, Barts Cancer Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK e-mail: [email protected]; [email protected] T. Lawrence and T. Hagemann (eds.), Tumour-Associated Macrophages, DOI 10.1007/978-1-4614-0662-4_5, © Springer Science+Business Media, LLC 2012

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(dichloromethylene bisphosphonate) (Van Rooijen & Sanders 1996). Liposomal clodronate has been extensively used to try and demonstrate proof-of-concept in mouse models of cancer (Zeisberger et al. 2006; Gazzaniga et al. 2007; Hiraoka et al. 2008; Miselis et al. 2008), to establish a functional relationship between the presence of TAM and tumour progression. Liposomal clodronate was recently evaluated as therapy in dogs with a spontaneous histiocytic neoplasm (Hafeman et al. 2010). Two of five dogs demonstrated tumour regression with an intravenous schedule of 0.5 ml/kg over 60 min, repeated once or twice at 2-week intervals. The authors believe that direct tumour cell killing was at least partly responsible for the clinical response observed in this situation, rather than only that mediated by macrophage depletion. Nonetheless, this work showed that such a short course of liposomal clodronate was feasible and well tolerated. Encouraging results have recently been published with a murine model whereby macrophage ablation with liposomal clodronate given prior to radiotherapy inhibits tumour relapse (Meng et al. 2010). Identification of such critical therapeutic windows for macrophage ablation, perhaps to augment responses to chemotherapy or radiotherapy, offers the prospect of optimising anti-tumour effects while minimising detrimental effects to physiological processes. Consequently, there have been calls for urgent clinical trials of liposomal clodronate in humans (Hafeman et al. 2010; Meng et al. 2010). However, this relatively unselective macrophage ablation technique destroys many physiological tissue macrophages, and in its unrefined form is likely to be limited in its human application. Systemic delivery of un-encapsulated bisphosphonates has been common clinical practise for many years, intended to inhibit osteoclast-mediated bone resorption in the setting of osteoporosis, multiple myeloma, and cancer metastasising to bone. Intriguingly, recent data strongly suggests that the effects of systemic bisphosphonates go beyond simply reducing bone resorption or metastasis, also appearing to modulate overall cancer progression. In a clinical trial of 1,803 pre-menopausal women with oestrogen-responsive early breast cancer, the addition of the intravenous bisphosphonate zoledronic acid, improved disease-free survival (Gnant et al. 2009). There were reductions in loco-regional recurrence, distant recurrence, bone metastasis and disease in the contralateral breast, implying anti-tumour effects extending beyond the bone microenvironment. Similarly, the Medical Research Council Myeloma IX Study of 1,960 patients is reporting a survival benefit from zoledronic acid, distinct from that associated with the prevention of skeletal-related events, again implying anti-cancer properties by mechanisms that remain unclear (Oral presentation, Prof G. Morgan ASCO 2010: Evaluating the Effects of Zoledronic Acid (ZOL) on Overall Survival (OS) in Patients (Pts) With Multiple Myeloma (MM): Results of the Medical Research Council (MRC) Myeloma IX Study. Abstract 8021). The contribution of macrophages to this effect remains unclear, as there may well also be direct anti-cancer cell effects. In vivo work in a mouse model of mesothelioma has indicated that systemic zoledronic acid might impair myeloid cell differentiation to tumour-associated macrophages (Veltman et al. 2010), and further work is needed to clarify and confirm this mechanism of action.

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Inhibition of Monocyte Recruitment A recent seminal study has exploded the dogma of TAM simply arising from circulating monocytes recruited to sites of cancer-related inflammation by chemokines including CSF-1 (Macdonald et al. 2010). This work reveals that monocyte production and inflammatory function are independent of maturation and replacement of resident tissue macrophages. It is the latter population of resident macrophages, whose development depends upon intact CSF1-Receptor signalling, that constitute TAM in the context of malignancy. In a tumour model, administration of a novel CSF1R blocking antibody reduced the numbers of TAM, but had no effect on monocyte recruitment using non-cancer inflammation models induced by lipo-polysaccharide, wound healing, peritonitis or acute graft-versus-host disease. Identification that TAM arise along separate maturation pathways from inflammatory monocyte/macrophages raises the prospect of reducing TAM numbers without severely impairing physiological monocyte/macrophage functions. This is conceptually more appealing than the far less selective macrophage ablation achieved with liposomal clodronate. It will be exciting to follow the development of human trials of CSF1R blocking antibodies or small molecules. Further insights into leukocyte trafficking have been gained by the study of a humanised monoclonal antibody against CD11b and CD18 cell adhesion proteins, expressed on macrophages and neutrophils. In a rabbit model of ischaemic brain injury, Rovelizumab (LeukArrest, ICOS) reduced neutrophil infiltration by 90%. Rovelizumab is well tolerated in humans and entered Phase III clinical trials in the late 1990s for use in ischaemic stroke (Jones 2000). Development of this drug for reducing inflammatory insults after ischaemic injury was halted in the light of disappointing clinical outcomes in this and other inflammatory conditions. Interest in Rovelizumab has been re-ignited by work investigating post-radiotherapy use of a CD11b antibody in a xenograft model of human hypopharyngeal carcinoma in mice (Ahn et al. 2010). Radiotherapy is known to induce tissue hypoxia, which in turn drives not only macrophage infiltration but also a pro-tumoural phenotype (Vujaskovic et al. 2001). In this way, TAM may promote tumour re-growth following radiotherapy. Having observed recruitment of CD11b + myeloid cells to tumours growing in previously irradiated tissues (Ahn and Brown 2008), it was found that systemic administration of a neutralising CD11b monoclonal antibody following irradiation reduced infiltration of myeloid cells, which correlated with the inhibition of tumour re-growth. Additionally, murine tumours transplanted to CD18 hypomorphic mice were more sensitive to irradiation than wild-type controls. CD11b and CD18 are expressed on such a large population of circulating and resident macrophages and neutrophils that timing of administration of neutralising antibodies is likely to be crucial when trying to influence TAM infiltration. CX3CL1, also known as Fractalkine, mediates several properties of inflammatory cells, including chemotaxis, adhesion, maturation and survival, and has a highly specific receptor, CX3CR1. This variety of functions as both a chemokine and adhesion molecule results from CX3CL1 existing in both membrane-tethered and

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soluble isoforms (Bazan et al. 1997). Murine monocytes have been classified as CX3CR1hi or CX3CR1lo, correlating with their migratory properties (Geissmann et al. 2003) such that CX3CR1lo/CCR2+/GR1+ monocytes are actively recruited to inflammatory tissues but short lived, and CX3CR1hi/CCR2-/GR1− monocytes are recruited in a CX3CR1-dependent manner to non-inflamed tissues and are longer lived. A murine model of ovarian cancer found that tumour progression was associated with massive accumulation in the peritoneum of CX3CR1hi TAM (Hart et al. 2009). A wealth of research has identified important roles for the CX3CL1–CX3CR1 axis in the pathogenesis of many inflammatory as well as malignant disorders. CX3CL1 antagonists as well as blocking antibodies to CX3CR1 (Furuichi et al. 2006), depletion of CX3CR1 positive cells and CX3CR1 knockout animals (Landsman et al. 2009; Jung et al. 2000; Yu et al. 2007) have developed this proof-ofconcept, but as yet there are no associated clinical agents undergoing human trials in cancer.

TAM Re-education Ablation of macrophage populations must be extremely selective to not only disable tumours, but also to enable normal healthy physiology. Phenotypic plasticity is a hallmark of macrophages, and one that, we believe, is hijacked by tumours to facilitate their growth and spread, such that TAM adopt an alternatively activated phenotype. An attractive approach, therefore, is to exploit this phenotypic plasticity and re-educate TAM towards a classically activated phenotype. In principal, this might not only result in the withdrawal of pro-tumoural phenomena, but also incorporate anti-tumoural activities through recruitment of Th1 immune responses. Previous chapters have outlined in great detail the nature of macrophage plasticity and phenotype determination. Animal models have clearly demonstrated the potential therapeutic benefits of re-educating macrophages in a number of different settings including cancer (Hagemann et al. 2008; Movahedi et al. 2010; Fong et al. 2008; Guiducci et al. 2005). Realising this potential with clinically feasible compounds is a goal that is being actively pursued. Small molecule inhibitors of enzymes at critical points in pathways of alternative activation of macrophages, such as IKKE, hold promise. Designing such drugs to be macrophage-specific is a huge challenge, yet in the case of the IKKE/NF-NB pathway, there may be advantages to inhibition in both cancer cells and macrophages. Using IKKE deletion in epithelial cells and separately myeloid cells, Greten et al. demonstrated attenuation of colitis-associated tumours with both approaches (Greten et al. 2004). Similar benefits of IKKE inhibition in both tumour cells and macrophages have been demonstrated in a chemically induced hepatocellular cancer model (Maeda et al. 2005). Inhibition of the NF-NB pathway appeared to become a clinical reality, with the development of Bortezomib, a highly effective anti-myeloma agent. Recent work has exposed the complexity of its true mechanism of action, such that it cannot be considered simply a pure NF-NB inhibitor of myeloma cells and highlighted the need for specificity regarding target


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cell type, and inhibition of inducible versus constitutive NF-NB activation in rational drug design (Hideshima et al. 2009). In this regard, it was found that Bortezomib did not actually induce NF-NB inhibition in patient myeloma cells, nor in peripheral blood mononuclear cells, but did inhibit NF-NB in bone marrow stromal cells. Novel small molecule inhibitors of IKKE, such as MLN120B (Nagashima et al. 2006), were found to inhibit myeloma cell NF-NB activation. It is interesting to speculate on the future clinical benefits that may be derived from combined specific inhibition of tumour cell and micro-environmental cell NF-NB, including in TAM. As we improve our understanding of existing therapies, it is becoming evident that certain anti-cancer drugs and radiotherapy act also as immuno-modulatory agents, and re-educate the tumour microenvironment, some exploiting TAM in their mechanism of action. One example of this exploitation is Rituximab, a chimaeric monoclonal antibody directed against CD20, as found on malignant B-lymphocytes. Rituximab is highly effective therapy against B-lymphomas, such as Follicular Lymphoma (FL), and is thought to exert its effects partly through direct lymphoma killing and partly through immune-mediated mechanisms, in which activatory FcJ receptor-expressing macrophages play a prominent role (Beers et al. 2010; Glennie et al. 2007). Although there is not yet a consensus on this matter, there is a growing body of evidence that a large number of infiltrating TAM predict a poor prognosis in FL when treated with conventional chemotherapy (Farinha et al. 2005). Moreover, TAM display alternatively activated features in this disease. However, when treated with Rituximab alone or in conjunction with chemotherapy, a large number of TAM predict a good response to treatment (Taskinen et al. 2007; Canioni et al. 2008). A fascinating insight has been provided by in vitro work with malignant B-lymphocytes, demonstrating that alternatively activated macrophages phagocytose Rituximab-opsonised malignant B-cells more efficiently than classically activate macrophages, leading the authors to suggest that TAM actually switch from a tumour-promoting to a tumour-inhibiting function with the addition of Rituximab (Leidi et al. 2009). Radiotherapy, at sufficient doses, certainly seems to exert some of its beneficial effects through re-education of the tumour microenvironment. Radiation induced tumour cell damage and death releases “danger signals,” provoking in situ autoimmunisation, augmenting tumour-specific T-cell and macrophage responses. The direction and consequences of macrophage activation following radiotherapy appear to depend upon the dose, the cancer being treated and the genetic background of the individual host. A better understanding of these variables is required to optimise this phenomenon (Formenti and Demaria 2009). Nanoparticles have been extensively investigated as vehicles capable of delivering anti-cancer drugs directly to tumour cells, aiming to avoid drug resistance mechanisms and collateral damage to normal tissue. However, as previously discussed in the example of liposomal clodronate, macrophages avidly take up simple liposomal nanoparticles, and prior to liposome PEGylation, was one of the major obstacles encountered in their use against cancer cells (Illum et al. 1984). This phenomenon does, of course, present the opportunity to target macrophages themselves with

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nanoparticles in such a way that they are activated toward a tumouricidal phenotype. In vitro studies with lung cancer cell lines and murine alveolar macrophages suggested that empty endotoxin-free poly(isobutylcyanoacrylate) nanoparticles are able to induce tumouricidal activity in macrophages with an associated increase in Th1 cytokines in the co-culture supernatant (Al-Hallak et al. 2010). Silica nanoparticles, under investigation as gene delivery vectors to the central nervous system, induce phenotypic changes in rat brain resident macrophages (microglia) (Choi et al. 2010). Increasing the production of reactive oxygen and nitrogen species, and downregulating TNF-D gene expression, macrophages adopt a potentially tumouricidal classically activated phenotype. Nanoparticles with additional functionality can be engineered with surface ligands to enhance binding to target cells, be they cancer cells or TAM. An alternative approach has been to explore combining the, often hypoxiadriven, tumour-homing behaviour of macrophages, with the low-toxicity and other benefits of nanoparticle drug-encapsulation, by immobilising nanoparticles on the cell surface of macrophages (Holden et al. 2010).

Adoptive Transfer Having considered ablation or re-education of pro-tumoural TAM, it is now logical to discuss the merits of adoptive transfer of monocytes or macrophages as a therapeutic strategy in cancer. Given that tumours appear to recruit monocytes from the circulation by a variety of mechanisms, it is reasonable to suppose that supplementary populations of injected monocyte/macrophages might preferentially accumulate within tumours. Without manipulation, these cells might contribution to the TAM population and so encourage tumour growth. Manipulation of the monocyte/ macrophages either in vitro or subsequently in vivo might provide the potential to deliver to the tumour, classically activated macrophages with anti-cancer properties. Alternatively, or in addition, these macrophages might be used as vectors to express genes within the tumour, such as inflammatory cytokines, or pro-drug conversion genes to optimise local delivery of active chemotherapeutics (Griffiths et al. 2000). In vivo studies in mice have demonstrated the therapeutic potential of macrophage adoptive transfer. In 1974, Isaiah Fidler and colleagues studied the ability of syngeneic macrophages to inhibit pulmonary metastases in C57BL/6 mice bearing a progressively growing B16 melanoma (Fidler 1974). Their work demonstrated that peritoneal macrophages, harvested after thioglycollate-induced inflammation, reduced the number of pulmonary metastases. This activity was dependent upon the macrophages being specifically treated in vitro and then injected intravenously. Whilst unable to describe the macrophage phenotype in language we are now familiar with, it was clear that the tumour-cytotoxic activity of the macrophages was influenced by their prior in vitro exposure to sensitising agents during maturation. Those exposed to tumour supernatants as well as rat lymphocytes sensitised to the tumour in vivo were highly active in preventing pulmonary metastases, compared with those exposed


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only to tumour supernatants, which had no effect. Later work described that the subsequent tissue distribution in a normal host mouse of transferred macrophages was determined by the method used to elicit and activate them from the donor peritoneum (Wiltrout et al. 1983). It is much clearer to us now that macrophage subtype and phenotype is likely to be crucial in successfully populating and influencing growing tumours. Similarly successful adoptive transfer of macrophages with tumour regression has been repeated and refined in other mouse models of cancer (Hagemann et al. 2008; Ben-Efraim et al. 1994). Human studies in the late 1980s demonstrated that macrophage adoptive transfer is feasible and safe in patients with cancer (Lacerna et al. 1988; Stevenson et al. 1987). Greater than 3×109 autologous monocyte-derived macrophages, sensitised in vitro with interferon-J or lipopolysaccharide, or in vivo with GM-CSF, can be safely infused by intravenous (i.v), intraperitoneal (i.p) or intrapleural routes. Systemically delivered macrophages first are retained in the lungs before pooling in the liver and spleen, concentrating at sites of metastases (Andreesen et al. 1998). Macrophages delivered i.p remain within this cavity for more than 7 days and accumulate at sites of major tumour growth. In one study of patients with ovarian or gastric cancer, i.p macrophages significantly reduced the volume of malignant ascites in three of seven patients, but without impacting on tumour mass or activity (Andreesen et al. 1990). Unfortunately, these results proved to be exceptional, with the majority of early human in vivo studies showing little or no measurable benefit in a variety of cancers (Lacerna et al. 1988; Faradji et al. 1991a; Faradji et al. 1991b; Lopez et al. 1991; Eymard et al. 1996; Hennemann et al. 1995; Hennemann et al. 1997). One reason for this failure of effect, despite successful transfer, might be an overwhelming ability of the tumour to manipulate the phenotype of transferred macrophages, so down-regulating their tumour-cytotoxic and pro-inflammatory functions. This might be overcome by transfecting macrophages with critical pro-inflammatory cytokines. In vitro and in vivo studies in mice support the feasibility of this approach with respect to IFN, IL4, IL6, TNF, also showing increased tumouricidal activity (Nishihara et al. 1995).

Concluding Remarks As the field of macrophage biology continues to expand, so the prospect of effective and highly specific TAM therapies comes closer to a reality. Studying the macrophagerelated mechanisms of action of existing therapies will continue to provide valuable insights, but it is likely to be the development of novel molecules, specific to certain macrophage subtypes and almost certainly adjuvant to other treatment modalities, that will deliver the greatest rewards. The specific aims in TAM therapy are likely to differ between cancer diagnoses and situations. The objectives, however, will remain constant: removing pro-tumoural influences, introducing, maximising and maintaining tumouricidal effects, whilst preserving physiological macrophage functions in normal tissues.

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Part II

Mechanisms of Action

Chapter 6

Arginine Metabolism and Tumour-Associated Macrophages Melissa Phillips and Peter W. Szlosarek

Introduction The metabolism of the amino acid l-arginine is critical in several key biological processes, including maintenance of normal immune cell function. Recent findings indicate that dysregulated metabolism of l-arginine by macrophages in the tumour microenvironment may promote tumour growth and development by impairing the anti-tumour immune response. Two enzymes that compete for l-arginine as a substrate – arginase and nitric oxide synthase (NOS) – are critical components of this immune suppression pathway. In contrast, argininosuccinate synthetase (ASS1)negative tumours, which depend on exogenous l-arginine for growth (‘arginine auxotrophs’), may instead succumb to an l-arginine deficient tumour microenvironment secondary to arginase released by alternatively activated macrophages or via the delivery of pharmacological doses of pegylated arginine-degrading enzymes. Since the rate-limiting enzyme for l-arginine biosynthesis, ASS1, may be elevated in macrophages, understanding its role in the context of l-arginine auxotrophic tumours and in channelling l-arginine to the enzymes arginase and NOS, remains a priority for further research. This chapter reviews the role of arginine and macrophages in tumour growth and development and examines the therapeutic potential of strategies that modulate the metabolism of this amino acid in the treatment of cancer.

-0HILLIPSs073ZLOSAREK*) Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and The London School of Medicine, Queen Mary College, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK e-mail: [email protected] T. Lawrence and T. Hagemann (eds.), Tumour-Associated Macrophages, DOI 10.1007/978-1-4614-0662-4_6, © Springer Science+Business Media, LLC 2012

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l-Arginine Metabolism: An Overview l-Arginine (l-2-amino-5-guanidinovaleric acid) is a dibasic D amino acid first discovered over 100 years ago from lupin seedlings (Boger and Bode-Boger 2001). The L-form is one of the 20 most common natural amino acids. In humans, it is classified as a semi-essential or conditionally essential amino acid, as the body’s ability to synthesise sufficient quantities depends on the developmental stage and health status of the individual. Newborns, for example, are unable to produce l-arginine efficiently, but healthy adults can synthesise enough so that it is not nutritionally essential. It does, however, need to be supplemented in cases of physical stress and illness. Free l-arginine in the body is derived from the diet, endogenous synthesis, and protein turnover. Thus, in healthy adults, homeostasis of this amino acid is mainly achieved by the modulation of intake and modulation of its catabolism (reviewed by Morris 2007). Endogenous l-arginine synthesis occurs predominantly within the kidney, where it is formed from l-citrulline and aspartate by successive actions of the rate-limiting enzyme ASS1 and argininosuccinate lyase (ASL). The liver is also capable of synthesising considerable amounts of l-arginine, but this is compartmentalised within the urea cycle, rather than contributing to plasma l-arginine levels (Boger and Bode-Boger 2001). l-Arginine is regarded as a highly versatile amino acid, playing an important role in the synthesis of proteins and nitric oxide (NO). In addition, l-arginine is a precursor for at least 6 other biologically important compounds in mammals: polyamines, nucleotides, proline, glutamate, creatine and urea (Fig. 6.1). These processes are differentially expressed according TOCELLTYPE AGEANDDEVELOPMENTALSTAGE DIETANDSTATEOFHEALTHORDISEASE7UAND Morris 1998). The metabolism of l-arginine is complex and tightly regulated by several enzymes, including ASS1, arginase, and NOS, which are expressed as a number of different isoforms (reviewed by Husson et al. 2003; Fukumura et al. 2006; Morris 2009; Fig. 6.1). Two different isoforms of arginase have been identified in humans with 58% sequence identity at the amino acid level, similar enzyme activity, but differential tissue expression. Arginase I (argI) is constitutively present in the liver as part of the urea cycle and is induced in different myeloid cells by exposure to pro-inflammatory cytokines, as discussed below. On the other hand, arginase II (argII) is located in the mitochondria of various cell types, including renal cells, neurons and enterocytes and macrophages. Three distinct isoforms of NOS have also been identified with ~50% identity, but differing in intracellular localisation, regulation, catalytic properties and inhibitor sensitivity. NOS1 (also known as nNOS) is prevalent in neuronal tissue, NOS 2 (or iNOS) is the inducible isoform, present in cells of the immune system and various cancer cells, and NOS3 (or eNOS) is found in endothelial cells. Although there are 10–14 copies of the argininosuccinate synthetase gene in the human genome, all but one, residing on 9q34 (ASS1), are pseudogenes. Most of the evidence to date suggests that activities of iNOS, argI/II and ASS1 play major roles in determining the metabolic fates of l-arginine in health and disease (Peranzoni et al. 20077UETAL2009).

6

Arginine Metabolism and Tumour-Associated Macrophages

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Fig. 6.1 Arginine utilisation in the tumour cell. Arginine is a substrate for multiple metabolic and inflammatory pathways in health and disease (see text). Arginine may be sourced via the cationic amino acid transporter, ASS1, or autophagy. The subcellular locations of key enzymes are shown and enumerated in the figure as follows: 1, arginase 1; 2, ornithine transcarbamylase (OTC); 3, argininosuccinate synthetase (ASS1); 4, argininosuccinate lyase; 5, nitric oxide synthase; 6, ornithine decarboxylase; 7, pyrroline-5-carboxylate reductase; 8, pyrroline-5-carboxylate dehydrogenase; 9, proline oxidase (dehydrogenase); 10, ornithine aminotransferase; 11, pyrroline-5carboxylate synthase; 12, arginine decarboxylase

l-Arginine and the Macrophage Interest in l-arginine metabolism has increased greatly in the last 20 years, triggered primarily by the discovery that in mammals l-arginine is the sole precursor for the multi-functional messenger molecule, NO. Initially NO production from l-arginine was identified in endothelial cells, followed by the key discovery that murine macrophages express iNOS upon stimulation by cytokines and other inflammatory stimuli (Hibbs et al. 1988). Macrophages are crucial to the regulation of an effective immune response, playing an important role in both innate and adaptive immunity. In addition to their role as resident tissue macrophages (i.e., Kupffer cells in the liver and microglial cells of the brain), activated macrophages are also recruited to sites of inflammation and infection, where they become involved in complex interactions with various cells present at the site. As a result, macrophages have a diverse range of biological functions essential for tissue remodelling, inflammation and immunity, depending

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on their mode of activation. These include phagocytosis, cytotoxicity, and secretion of a wide array of cytokines, growth factors, lysozymes, proteases, complement components, coagulation factors and prostaglandins (Lin and Pollard 2004). To perform their functions, macrophages must be activated either by Th1-type cytokines IL-1, TNF-D, IFN-J and IL-2 and bacterial lipopolysaccharide (often referred to as M1 or ‘classically’ activated macrophages) or by Th2-type cytokines IL-4, IL-10, IL-13 and TGF-E, as well as GM-CSF, prostaglandin and catecholamines (M2 or ‘alternatively’ activated macrophages). Importantly, both pathways of macrophage activation employ l-arginine as a key substrate: Th1-type cytokines transcriptionally upregulate the enzyme iNOS, and Th2-type cytokines activate arginase expression (reviewed by Bronte and Zanovello 2005; Classen et al. 2009). The activities of these enzymes are tightly co-ordinated with several counter-regulatory feedback loops. For example, N-hydroxy-l-arginine (NOHA), an intermediate product in the iNOS pathway, is known to strongly inhibit arginase function. Conversely, arginase can reduce iNOS activity by competitively utilising the common substrate, l-arginine. The activities of iNOS and arginase are also dependent on l-arginine availability. Macrophages utilise both exogenous and endogenous sources of l-arginine to meet their metabolic requirements. l-arginine can enter a cell through several transporters, although an Na+-independent transport system, named system y+ is postulated to be the main route for l-arginine entry in most cells (Closs et al. 2004). Transporters in the y+ system include those of the high-affinity cationic amino acid transporter (CAT) family. To date, four members in the CAT family have been identified, CAT-1 to CAT-4, encoded by Slc7A1–4GENES RESPECTIVELY7HILE#!4 AND are known to transport cationic amino acids – such as l-arginine, l-ornithine, and l-lysine – through a pathway that is independent of pH, sensitive to stimulation, and saturable, the function of CAT-4 is unclear. The most relevant CAT member involved in macrophage function is CAT-2, which can be expressed as two splice variants (Yeramian et al. 2006). The high-affinity transporter CAT-2B is inducible in many cell types including macrophages and is strongly upregulated by IFN-J (±LPS), IL-4, and GM-CSF, while CAT-2A is constitutively expressed in several cell types and is poorly sensitive to trans-stimulation. Several data implicate the CAT system in macrophage activation. First, genetic deletion of CAT-2 in mice revealed a reduction in l-arginine uptake by 95% and led to a 92% decrease in NO production, indicating that the uptake of l-arginine via this transporter is critical for iNOS activity in macrophages (Nicholson et al. 2001). Second, there is evidence that CAT-2 on macrophages supplies arginase with l-arginine in a rodent model of bleomycin-induced fibrosis, although inflammation per se was not modulated by the deletion of CAT-2 (Niese et al. 2010). Interestingly, high levels of extracellular l-arginine are catabolised preferentially by iNOS rather than arginase, generating proapoptotic levels of NO in the murine macrophage-like 2!7 CELL LINE 'OTOH AND -ORI 1999). Third, IFN-J-mediated growth enhancement of leishmania amazonensis amastigotes in murine macrophages is associated with l-arginine transport via CAT-2B, as parasite growth was found to be ABSENTINMACROPHAGESDERIVEDFROM#!4 "DElCIENTMICE7ANASENETAL2007). Since the activities of iNOS (i.e., classical pathway for parasite killing) and arginase

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(i.e., alternative pathway for parasite growth) were unchanged in these IFN-J-treated, infected macrophages, this implicates a novel direct pathway of arginine supply via CAT-2B for parasitic proliferation. Alternatively, l-arginine can be obtained by endogenous synthesis, as first described with the conversion of l-citrulline into l-arginine in rat peritoneal macROPHAGES7UAND"ROSNAN1992). Furthermore, LPS-activated macrophages were shown to be threefold more active in the synthesis of l-arginine from l-citrulline than unstimulated macrophages. LPS and certain cytokines are able to induce ASS1, but not ASL, both of which are normally only present at low levels in resting macrophages. ASS1 induction occurs in parallel with upregulation of iNOS such that a large fraction of the l-citrulline, produced as a by-product of the iNOS reaction, is recycled back to l-arginine, termed the ‘citrulline-NO’ cycle (Nussler et al. 1994). In summary, l-arginine from exogenous and endogenous sources can be utilised by macrophages in a variety of metabolic pathways, with the differential catabolism via iNOS and arginase being the best characterised to date. Arginine utilisation, therefore, may directly affect the role of macrophages and the type of host immune response within the tumour microenvironment.

TAMs, Cancer Cells and the Tumour Microenvironment An inflammatory component consisting of cells and their secreted products is present in the microenvironment of most neoplastic tissues, and it is increasingly recognised that inflammation plays an indispensable role in cancer progression (reviewed by (Mantovani et al. 2008). Indeed, tumour-associated macrophages (TAMs) are key regulators of the link between inflammation and cancer, and although most are derived from peripheral blood monocytes recruited into the tumour mass from the circulation, there is also evidence of local macrophage proliferation. Thus, the fate of a developing tumour is determined by the properties of the malignant cells and by the phenotype of the tumour infiltrating myeloid cells, with the ‘arginine metabolome’ of both tumour and TAMs playing a potentially critical modulatory role.

TAMs Recent work has identified varying proportions of M1 (argI low and iNOS high) and M2 (argI high and iNOS low) TAMs depending upon the type of chemically or genetically induced epithelial tumour murine model. For example, macrophagesexhibited argI(high)iNOS(low) polarisation in early stage chemically induced lung tumours, whereas a mixed population of M1 and M2 TAMs was observed with late stage lung adenocarcinomas (Redente et al. 2010). Moreover, lung tumour regression secondary to the inactivation of the Kras or FGF10-driven transgene was associated with a switch from an argI(high)iNOS(low) TAM polarisation to an argI(low) iNOS(low) pulmonary macrophage phenotype (no polarisation). In contrast, studies


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L-ARGININE

IFN-G IL-1 IL-2 TNF-A

CAT2B

TAM 1 ‘Classically’ activated macrophage

L-ARGININE

iNOS

NOHA ASS1 ASL L-CITRULLINE

extracellular

TAM 2 ‘Alternatively’ activated macrophage

IL4 IL10 IL13

Arginase

ORNITHINE L-Arginine Due to high ODC arginase activity PUTRESCINE

OAT

+ RNS ROS

PROLINE

NO SPERMIDINE + SPERMINE

L-Arginine

Pro-tumourigenic Tumour cytotoxicity (high NO) Cell proliferation T cell hypo-responsiveness effect (DNA damage) Pro-tumourigenic effect (low NO) Pro-tumourigenic Depletion by excess arginase effect may inhibit tumour growth

Fig. 6.2 Schematic representation of TAM 1 and TAM 2 metabolic pathways. The activities of the enzymes in both ‘classically’ activated (M1), and ‘alternatively’ activated (M2) macrophages in the tumour microenvironment are illustrated. Solid arrows indicate the main enzymatic activity, whereas dashed arrows indicate alternative metabolic activity. In particular, when l-arginine concentrations are low, iNOS activity changes from the prevalent production of NO to the generation of superoxide and highly reactive nitrogen species (RNS) (Ohshima et al. 2003, Bronte and Zanovello 2005). Low extracellular l-arginine concentration, overexpression of ARG 1, or reduction of the capacity for l-arginine uptake can all decrease intracellular l-arginine levels. The T helper 1 cytokines (IFN Ź) and T helper 2 cytokines (IL4, IL13) are the main inducers of iNOS and Arginase 1 (ARG 1), respectively. Pro-inflammatory signals (such as IL1 and TNF ŷ) and antiinflammatory signals (IL10) can contribute to regulate the final balance between iNOS and ARG 1 activity. Moreover, ARG 1 and iNOS directly activate several biochemical circuits that negatively regulate each other. The depletion of l-arginine by overexpression of ARG 1 reduces the activity of iNOS in the production of NO. Polyamines can also inhibit production of NO. Conversely, NOHA can inhibit ARG 1. l-arginine depletion causes T cell hyporesponsiveness, affecting the anti-tumour immune response. However, if enough ARG 1 is produced by increasing numbers of alternatively activated macrophages, the further l-arginine depletion that results can inhibit tumour growth in l-arginine auxotrophic tumours (Ellyard et al. 2010). For further details, see main text. NOHA, N hydroxy l-arginine; OAT, ornithine aminotransferase; ODC, ornithine decarboxylase; RNS, reactive nitrogen species; ROS, reactive oxygen species; ASS1 argininosuccinate synthetase; ASL, argininosuccinate lyase

of TAMs associated with different stages of melanoma progression, revealed dominant iNOS expression in in situ and thin melanomas which declined with the development of thicker melanomas (Massi et al. 2007). The shift in the iNOS/arginase balance may reflect a change in biology from tumour promotion secondary to iNOS activity (producing low prosurvival levels of NO) to proliferation secondary to comparatively greater argI activity (Fig. 6.2).

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Arginase Arginase generates urea and ornithine, the latter a precursor for various polyamines produced via ornithine decarboxylase (ODC), and proline via ornithine aminotransferase (OAT), thereby playing an important role in tumour cell proliferation and collagen production, respectively (Gerner and Meyskens 2004; Morris 2007). Co-culture experiments using murine macrophages transfected with rat Arg I and the human breast cancer cell line ZR-75-1, have revealed enhanced growth of the latter via the upregulation of ornithine and the polyamine putrescine (Chang et al. 2001). This was accompanied by a reduction in tumour cytotoxic NO levels, illustrating the opposite actions of iNOS and argI in this model. TAMs expressing argI and argII may also be directly involved in tumour angiogenesis, by providing a source of polyamines for increased tumour vascularisation. A marked reduction in vessel diameter was observed with arginase compared with NOS inhibition in the context of peritoneal TAMs in the LMM3 xenograft model (Davel et al. 2002). Particularly relevant to immunogenic tumours, such as melanoma and renal cell carcinoma, is the finding that M2-polarised macrophages impair T-cell anti-tumour activity by depleting l-arginine (references by Rodriguez and Ochoa 2008; and Fig. 6.2). Macrophages stimulated with Th2 cytokines (IL-4 and IL-13) produce ArgI, rapidly reducing l-arginine concentration in the microenvironment and inducing T-cell dysfunction. In the absence of l-arginine, T cells failed to upregulate cyclin D3 and cyclindependent kinase 4 and remained in the G0–G1 phase of the cell cycle. There was a concomitant downregulation of the principal signal transduction element of the T-cell receptor (TCR) or the ] chain (CD3]), which is rate-limiting in TCR assembly and memBRANE EXPRESSION 7HILST !RG) INHIBITION OR ARGININE SUPPLEMENTATION RESTORED 4 CELL function, no effect on T-cell function was observed with ArgII or iNOS, confirming the importance of arginine and ArgI in this host-dependent immunosuppression pathway. Similarly, myeloid-derived suppressor cells (MDSCs), a heterogenous population of inflammatory cells that includes precursors of the macrophage, upregulate argI and CAT2B resulting in efficient depletion of extracellular l-arginine and T-cell suppression using a murine lung carcinoma cell line. This mechanism appears to be relevant for tumour escape in vivo, because injection of the arginase inhibitor NOHA reduced lung tumour growth in a dose-dependent manner. Although ArgI is absent in human lung epithelial carcinoma cells, a large infiltrate of strongly ArgI-expressing MDSCs cells has been identified, consistent with the impaired T-cell immunity described in patients with non-small cell lung cancer. In contrast, a recent study revealed that ArgI depletion of l-arginine by alternatively activated (M2 polarised) macrophages directly inhibited B16-F1 melanoma cell proliferation in vitro, a process that was enhanced with exogenous IL-4 or IL-13 treatment (Ellyard et al. 2010). Furthermore, the ratio of alternatively activated macrophages (effector, E) to tumour (T) cells impacted tumour cell survival in vitro: low E:T ratios (

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