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The Breast 16 (2007) S27–S33

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Original article

Inflammation and cancer: Breast cancer as a prototype

Alberto Mantovania,b,�, Federica Marchesia, Chiara Portaa, Antonio Sicaa, Paola Allavenaa

aIstituto Clinico Humanitas IRCCS, Via Manzoni 56, 20089 Rozzano, Milan, ItalybCentro di Eccellenza per l’Innovazione Diagnostica e Terapeutica, Institute of Pathology, University of Milan, Italy

Abstract

Tumor-associated macrophages (TAM) represent the major inflammatory component of the stroma of many tumors, able to affect

different aspects of the neoplastic tissue. Many observations indicate that TAM express several M2-associated protumoral functions,

including promotion of angiogenesis, matrix remodeling and suppression of adaptive immunity. The protumoral role of TAM in cancer

is further supported by clinical studies that found a correlation between the high macrophage content of tumors and poor patient

prognosis and by evidence showing that long-term use of non-steroidal anti-inflammatory drugs reduces the risk of several cancers. Here,

we discuss evidence supporting the view that TAM represent a unique and distinct M2-skewed myeloid population and a potential target

of anti-cancer therapy.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Tumor-associated macrophages; Tumor; Inflammation; NF-kB; Metastasis; Hypoxia

Introduction

Epidemiological studies have revealed that chronicinflammation predisposes to different forms of cancerand that usage of non-steroidal anti-inflammatory agents isassociated with protection against various tumors. Aninflammatory component is present in the microenviron-ment of most neoplastic tissues, including those notcausally related to an obvious inflammatory process.Hallmarks of cancer-associated inflammation include theinfiltration of white blood cells, the presence of polypeptidemessengers of inflammation (cytokines and chemokines),and the occurrence of tissue remodeling and angiogenesis.

Already in the late 1970s it was found that a majorleukocyte population present in tumors, the so-calledtumor-associated macrophages (TAM), promote tumorgrowth.1,2 Accordingly, in many but not all human tumors,a high frequency of infiltrating TAM is associated to poorprognosis. Interestingly, this pathological finding has re-emerged in the post-genomic era: genes associated to

e front matter r 2007 Elsevier Ltd. All rights reserved.

east.2007.07.013

ing author. Istituto Clinico Humanitas IRCCS, Via

089 Rozzano, Milan, Italy. Tel.: +39 02 8224 2445;

4 5101.

ess: alberto.mantovani@humanitas.it (A. Mantovani).

leukocyte or macrophage infiltration (e.g. CD68) are partof molecular signatures which herald poor prognosis inlymphomas and breast carcinoma.3

Gene-modified mice, including some with cell-specifictargeted gene inactivation, allowed dissection of molecularpathways of inflammation leading to tumor promotion, aswell as the initial analysis of the role of distinct elements ofthe inflammatory process in different steps of tumorprogression. TNF, IL-1, the macrophage growth andattractant factor colony stimulating factor-1 (CSF-1),CCL2, a chemokine originally described as a tumor-derivedmacrophage attractant, the prostaglandin producing en-zyme cyclooxygenase 2, the master inflammatory transcrip-tion factor NF-kB, enzymes involved in tissue remodeling,all are essential elements for carcinogenesis and/or foracquisition of a metastatic phenotype in diverse organsincluding the skin, liver, mammary gland, intestine.4–9

Here we will review the available information on the roleof myelomonocytic cells, TAM in particular, in tumorinvasion and metastasis.

Angiogenesis and remodeling

Vascular and lymphatic endothelium are the majorroutes of metastatic spread of tumor cells. Tumor angiogenesis

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is often activated during the early, preneoplastic stages oftumor development10,11 and is controlled by a number ofpositive or negative regulators produced by cancer cellsand tumor-associated leukocytes.

Macrophages can exert a dual influence on blood vesselformation and function. On the one hand, macrophagesproduce molecules that are pro-angiogenic and on theother they can express anti-angiogenic molecules anddamage the integrity of blood vessels. On the anti-angiogenic side, in a murine model, CSF-induced, TAM-derived metalloelastase generates angiostatin. In general, asfor interaction with neoplastic cells, the pro-angiogenicfunctions of TAM prevail. In several studies, in humancancer TAM accumulation has been associated withangiogenesis and with the production of angiogenic factorssuch as VEGF and platelet-derived endothelial cell growthfactor.1 TAM accumulate in hypoxic regions of tumors andhypoxia triggers a pro-angiogenic program in these cells.A number of molecules with possible impact on angiogen-esis have been shown to be expressed by macrophage in lowoxygen conditions, such as VEGF, TNF-a, bFGF andCXCL8.12 Therefore, macrophages recruited in situ repre-sent an indirect pathway of amplification of angiogenesis,in concert with angiogenic molecules directly produced bytumor cells. Strikingly, it was recently reported thathypoxia-inducible factor-1 (HIF-1)-dependent chemokineCXCL1213 acts as a potent chemoattractant for endothelialcells of different origins bearing CXCR4 and is aparticipant in angiogenesis that is regulated at the receptorlevel by VEGF and bFGF. In agreement with theseobservations, our data suggest that the angiogenic programestablished by hypoxia relays also on the increasedexpression of CXCR4 by endothelial cells.14

Lymphoangiogenesis is mediated by the action of VEGF-C and VEGF-D acting on the receptor VEGFR-3. Morerecently VEGF-A, a chemotactic factor for monocytes, wasshown to increase lymphoangiogenesis, via the recruitmentof circulating monocytes.15 In human cervical cancer,VEGF-C production by TAMs was proposed to play a rolein peritumoral lymphoangiogenesis and subsequent dissemi-nation of cancer cells with formation of lymphatic metas-tasis.16 Additionally, TAM participate to the pro-angiogenicprocess by producing the angiogenic factor thymidinephosphorylase (TP), which promotes endothelial cell migra-tion in vitro and whose levels of expression are associatedwith tumor neovascularization.17

The contribution of chemokines toward angiogenesishas been the object of intensive investigation. A varietyof chemokines, including CCL2, CXCL12, CXCL8,CXCL1, CXCL13, CCL557 CCL17 and CCL22, havebeen detected in neoplastic tissues as products of eithertumor cells or stromal elements. CXCL1 and relatedmolecules (CXCL2, CXCL3, CXCL8 or IL-8) have animportant role in melanoma progression by stimulatingneoplastic growth, promoting inflammation and inducingangiogenesis.18 Strong evidence demonstrates that levels ofCCL2 are associated with TAM accumulation2 and that

CCL2 may play an important role in the regulation ofangiogenesis.19

Master genes

The capability to express distinct functional programs inresponse to different microenvironmental signals is abiological feature of macrophages, which is typicallymanifested in pathological conditions such as infectionsand cancer.20,21 Chronic infections can tightly regulate theimmune responses, being able to trigger highly polarizedtype I or type II inflammation and immunity. Central tothe development of type I or type II polarization is thespecificity of the host–pathogen interaction. While intra-cellular protozoa induce a type I polarized inflammation,with strong neutrophils, macrophage infiltrate, typical ingranulomas, parasites such helmints trigger strong type IIinflammation, characterized by extensive eosinophilia,mastocytosis and tissue remodeling.22

Classical or M1 macrophage activation in response tomicrobial products or interferon-g are characterized by:high capacity to present antigen; high interleukin-12(IL-12) and IL-23 production20 and consequent activationof a polarized type I response; and high production of toxicintermediates (nitric oxide (NO), reactive oxygen inter-mediates (ROI)). Thus, M1 macrophages are generallyconsidered as potent effector cells that kill microorganismsand tumor cells and produce copious amounts of proin-flammatory cytokines.Various signals elicit different M2 forms, able to tune

inflammatory responses and adaptive Th2 immunity,scavenge debris, promote angiogenesis, tissue remodel-ing and repair,2,20,23–25 which share selected functionalproperties (e.g. low IL-12).Microenvironmental signals expressed at the tumor

microenvironment have the capacity to pilot recruitment,maturation and differentiation of infiltrating leukocytes, andplay a central role in the activation of specific transcriptionalprograms expressed by tumor-associated leukocytes, mediat-ing either pro- or anti-tumoral functions.2,26

To the extent that they have been investigated, differ-entiated mature TAM have phenotype and function similarto type II macrophages. In an attempt to clarify themolecular basis of the TAM phenotype, biochemicalstudies have identified the transcriptional factors NF-kBand HIF-1 as master regulators of their transcriptionalprograms and indicated these factors as central regulatorsof tumor progression and metastasis.Clinical evidence has long suggested that cancers arise at

sites of chronic inflammation and this hypothesis hasrecently received molecular confirmations in inflammation-associated cancer models,5,7 which provide in vivo evidencessupporting a causal relationship between NF-kB-mediatedinflammation and tumorigenesis. NF-kB induces severalcellular alterations associated with tumorigenesis and moreaggressive phenotypes, including self-sufficiency in growthsignals, insensitivity to growth inhibition, resistance to

ARTICLE IN PRESSA. Mantovani et al. / The Breast 16 (2007) S27–S33 S29

apoptotic signals, immortalization, angiogenesis, tissueinvasion and metastasis.27

Constitutive NF-kB activation often observed in cancercells may be promoted by either microenvironmentalsignals, including cytokines, hypoxia and ROI, or bygenetic alterations.28 In particular, proinflammatory cyto-kines (e.g. IL-1 and TNF), expressed by infiltratingleukocytes, can activate NF-kB in cancer cells andcontribute to their proliferation and survival.5,7,29

While several in vitro and in vivo evidence suggest thatNF-kB-induced proliferation and cell survival are twomajor mechanisms of NF-kB-mediated tumorigenesis, adirect role of NF-kB in metastasis formation has beenrecently confirmed in vivo by Luo et al., in a murine cancermetastasis model of colon adenocarcinoma, and theydemonstrated that the LPS-induced metastatic growth isdependent on both TNF-a production by hematopoieticcells and NF-kB activation by tumor cells.30 In addition,defective NF-kB signaling by retroviral delivery of adominant negative inhibitor of NF-kB resulted in thedownregulation of prometastatic metalloproteinase, aurokinase-like plasminogen activator, and heparanaseand reciprocal upregulation of antimetastatic tissue in-hibitor of metalloproteinases 1 and 2 and plasminogenactivator 2.31 These observations are in line with severalreports indicating that NF-kB regulates a myriad of genesplaying a role in invasion and metastasis, such as cytokinesand chemokines, adhesion molecules, matrix metallopro-teinases, stress response genes, growth and angiogenicfactors.32

Other evidence suggests that inhibition of NF-kBsuppressed angiogenesis along with vascular endothelialgrowth factor (VEGF) and IL-8,33 thus preventing initialsteps of tumor cells spread. NF-kB regulates expression ofadhesion molecules34,35 and cell-surface metalloproteases,such as MMP-9 and MMP-2.36 Consistent with theobserved association between inflammation and cancer, itwas shown that upregulation of NF-kB in head and necksquamous cell carcinoma promotes inflammatory cytokinesproduction and metastasis.37

These findings propose NF-kB as a possible target fordevelopment of anti-cancer treatments and clinical trialswith drugs that block NF-kB are currently in progress withpromising results.38,39

To the extent they have been investigated TAM displaydefective NF-kB activation in response to different pro-inflammatory signals.40 Detective NF-kB activation inTAM correlates with impaired expression of NF-kB-dependent inflammatory functions (e.g. expression ofcytotoxic mediators, such as NO, and cytokines, TNF-a,IL-1 and IL-12)2,40,41 observed in these cells. However,these observations were obtained in TAM isolated fromtumors characterized by advanced stages40 and are incontrast with a protumor function of inflammatoryreactions and TAM in particular.5,7 This apparent dis-crepancy may reflect a dynamic change of the tumormicroenvironment during the transition from early neoplastic

events toward advanced tumor stages, which would resultin progressive modulation of NF-kB activity expressedby infiltrating inflammatory cells. While full activation ofNF-kB in inflammatory leukocytes resident in preneoplasticsites may exacerbate local inflammation, thus favoringtumorigenesis, tumor growth may result in the progressiveinhibition of NF-kB in infiltrating leukocytes, as observedin both myeloid40,41 and lymphoid42 cells associated withsolid tumors.Hypoxia is a common feature of solid tumors that has

been associated with decreased therapeutic response,malignant progression, local invasion and distant metas-tasis. The transcription factor HIF-1 is a major regulator ofcell adaptation to hypoxic stress43 and therefore a potentialtarget for anticancer therapies.44 HIF-1 mediates switchfrom aerobic to anaerobic metabolism thus conferring aglycolitic phenotype to cancer cells and ensuring theirenergy requirements, thereby allowing their survival in ahostile environment. It was also proposed that theglycolitic phenotype of cancer cells is required for invasivetumor growth and observed that persistent increase inglycolysis results in chronic acidification of the localmicroenvironment, a condition which stimulates in vitroinvasion and in vivo metastasis.45 Increased glycolysis wasassociated with enhanced incidence of metastasis in cervicaland head neck cancers.46–48 HIF-1 activation in tumor cellsactivates several mechanisms leading to angiogenesis,glycolysis, inhibition of apoptosis, upregulation of growthfactors (e.g. PDGF, TGF-a, IGF-2, EGF, VEGF) andprotein involved in tumor invasion (e.g. urokinase-typeplasminogen activator). Moreover, hypoxia downregulateadhesion molecules thus contributing to cancer celldetachment.49,50

TAM accumulates preferentially in the poorly vascular-ized region of tumors which are characterized by lowoxygen tension. Such environment promotes TAM adapta-tion to hypoxia, which is achieved by the increasedexpression of hypoxia inducible and pro-angiogenic genes,such as VEGF, bFGF and CXCL8, as well as glycoliticenzymes, whose transcription is controlled by the tran-scription factors HIF-1 and HIF-2.51,52. The in vivorelevance of this metabolic adaptation to hypoxia bymacrophages was recently demonstrated by Cramer et al.53

Ablation of the hypoxia responsive transcription factorHIF-1a resulted in impaired macrophage motility andcitotoxicity, in low oxygen conditions. This evidencehighlights the relevance that the hypoxia-HIF-1 pathwaymay play in the recruitment and activation of TAM intosolid tumors and may be instrumental for TAM-mediatedangiogenesis and tumor metastasis. In support of this, wehave recently described that hypoxia can influence thepositioning and function of cancer and stromal cells,including TAM, by selectively upregulating expression ofthe chemokine receptor CXCR4.14 Moreover, a recentwork has shown that HIF-1 activation may play a role inthe induction of the CXCR4 ligand, CXCL12,54 achemokine involved in cancer metastasis.55

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The Met tyrosine kinase, a high-affinity receptor forhepatocyte growth factor (HGF), plays a crucial role incontrolling invasive growth and is often overexpressed incancer. It was shown that hypoxia activates transcriptionof the met protoncogene through HIF-1 activation andthat inhibition of Met expression prevents hypoxia-inducedinvasive growth. These data show that hypoxia promotestumor invasion by sensitizing cells to HGF stimulation,providing a molecular basis to explain Met overexpressionin cancer.56

Similar to NF-kB, inhibition of HIF-1a is considered apromising therapeutic approach against cancer57 and infact some of its inhibitors (e.g. farnesyl transferaseinhibitors, PI3K inhibitors) are now in clinical trials asantitumor drugs.58

Interplay with adaptive immunity

In established tumors, available information suggeststhat TAM have a skewed M2 phenotype as discussedabove. M2 mononuclear phagocytes are characterized byan IL-12low IL-10high phenotype and TAM also producetransforming growth factor-b (TGF-b). In addition,tumor-associated dendritic cells (DC) have an immaturephenotype, various cytokines (M-CSF, IL-6, IL-10) presentin the tumor microenvironment, contribute to blocking DCmaturation in tumors. Immature myeloid cells are ex-panded in chronic infections and cancer and act as potentsuppressors of T cell-dependent antitumor immunity viaunbalanced iNOS and arginase-1 activity.59 Thus, tumor-associated myelomonocytic cells favor progression bytaming and skewing anti-tumor T-cell responses.

Transgenic mice carrying the early region genes ofHPV16 under the control of the human keratin 14promoter offer a useful model which recapitulates tumorprogression of squamous cell carcinoma from hyperplasiato displasia to overt malignancy. Innate immunity cells,most prominently mast cells and granulocytes, infiltrateHPV16 premalignant tissues, followed by macrophages incarcinoma. They drive a chronic inflammatory processwhich promotes epithelial hyperproliferation, tissue remo-deling and angiogenesis, followed by displasia and invasivecarcinoma.60 By crossing HPV transgenic mice withseverely immunodeficient mice (RAG-1�/�), it was foundthat genetic elimination of T and B lymphocytes blocksrecruitment of innate immunity cells, tissue remodeling andangiogenesis, with an arrest of the carcinogenesis process atthe stage of epithelial hyperplasia.61 Dissection of cells andmolecules involved revealed that B cells, which do notinfiltrate the lesions, act as remote orchestrators of theinnate immune cells in situ. Circumstantial evidencesuggests that this remote control mechanism of cancerpromoting inflammation operates via deposition of im-munoglobulins, but this was not formally proven.

Interestingly, immune complexes in concert with micro-bial molecules or inflammatory cytokines have been shownto elicit an M2 form of macrophage activation14 and TAM

are a prototypic M2 population.2,20,23 M2 cells tuneinflammation and adaptive immunity and promotecell proliferation, angiogenesis, tissue remodeling andrepair. B cells, by causing formation of immune complexes,may contribute from a distance to the M2 polarizationof phagocytes which are set in a tissue remodeling andrepair mode and orchestrate the smouldering and pola-rized chronic inflammation associated to establishedneoplasia.22,29

Invasion and metastasis

Several lines of evidence indicate that inflammatory cellsand cytokines found in tumors are more likely to contributeto tumor growth, progression, and immunosuppressionthan they are to mount an effective host anti-tumorresponse.1

Macrophages play an important role in this scenario asthese cells produce large quantity of pro- and anti-inflammatory cytokines, which can promote cancer dis-semination and metastasis. TNF-a is a proinflammatorycytokine generally produced by macrophages in responseto pro-inflammatory signals.62,63 Direct evidence forthe involvement of TNF in malignancy comes from theobservation that mice lacking the gene for TNF areresistant to skin carcinogenesis.64 A recent report showedthat co-cultivation of tumor cells with macrophages leadsto enhanced invasiveness of the malignant cells due toTNF-a-dependent MMP induction in the macrophages.65

Ablation of IL-1b in mice resulted in absence ofmetastasis development, either with melanoma cell modelsor with mammary and prostate cancer cells, suggesting theimportance of microenvironmental IL-1b. Both IL-1b andto a minor extent IL-1b were required for in vivoangiogenesis and invasiveness of tumors in vivo.66

CSF-1 is a potent chemoattractant of macrophages intosolid tumors.67 The intercross of transgenic mice suscep-tible to mammary cancer (PyMT) with mice containing arecessive null mutation in the (CSF-1) gene (Csf op)68

demonstrated that TAM recruitment is an absoluterequirement for productive metastatic growth. Hiratsukaet al.67 provided evidence suggesting that primary tumorsinduce endothelial cell MMP-9 through an interactionbetween endothelial cells and lung macrophages, via aVEGFR-1-dependent mechanism. MMP-9 expression inalveolar endothelial cells as well as in TAM, renders thepulmonary metastatic site fertile for secondary malignantcell growth, depending on the presence and activation ofthe VEGF-VEGFR signaling cascade.Macrophages produce endothelins (ETs), a family of

small related, vasoactive peptides that have a great numberof physiological roles in many tissues. TAM contribute toETs production in the breast tumor microenvironment,69

thus promote an invasive phenotype of breast cancer cells,which is the result of an interplay with other factors,including cytokines, matrix metalloproteases and activa-tion of TAM.70 Interestingly, the ET-1 was reported to

ARTICLE IN PRESS

TAM

EGF

Chemokines (CCL2; CCL5)

CSF1

Recruitment

M2 skewing Progression (adenoma → carcinoma),

Growth, Invasion, Metastasis

CARCINOMA

Fig. 1. The interplay between carcinoma cells and TAM in breast cancer.

A. Mantovani et al. / The Breast 16 (2007) S27–S33 S31

activate inflammatory pathways in human monocyte,through the activation of NF-kB,71 while binding of theET-1 to ovarian tumor cell lines triggers activation andstabilization of HIF-1a, which then increases VEGFmRNA and protein levels in these cells.72

Local growth and invasion of solid tumors as well asmetastasis depend on the controlled degradation ofcomponents of the extracellular matrix (ECM). TAM arerecognized as important player in cancer metastasis andclinical evidence showed a strong correlation between thenumber of TAM and poor prognosis.73,74 In turn, geneticstudies in mice have shown decreased rates of metastasis tobe associated with decreased TAM number.68,75 A modelby which macrophages promote tumor invasion andmetastasis includes expression of their proteolitic activityand subsequent break down of the basement membranearound the pre-invasive tumors, thereby enhance theability of tumor cells to escape into the surroundingstroma.67

TAM express molecules that affect tumor cell prolifera-tion, angiogenesis and dissolution of connective tissues.These include epidermal growth factor (EGF), members ofthe FGF family, TGF-b, VEGF, chemokines. In lungcancer, TAMmay favor tumor progression by contributingto stroma formation and angiogenesis through their releaseof PDGF, in conjunction with TGF-b1 production bycancer cells.2 TAM produce several matrix-metallopro-teases (e.g. MMP-2, MMP-9) that degrade proteins of theextra-cellular matrix, and also produce activators ofMMPs, such as chemokines. TAM also produce factorssuch as TGF-b, platelet-derived growth factor, IL-6,urokinase plasminogen activator and Tissue-type Plasmi-nogen Activator (t-PA) that may cause degradation ofECM to facilitate the tumor cell invasion and migration.76

Ahmed et al. described a method to observe theorientation of individual tumor cells as they enter bloodvessels, in real time and in a living animal.77 They foundthat tumor cells seem to be attracted to macrophages,which line the outside of the vessels. Goswami et al.78

described a paracrine signaling loop between tumor cellsand macrophages, in which tumor cells secrete macrophagecolony stimulating factor (M-CSF, also known as Csf1).This, in turn, causes macrophages to secrete EGF, a

chemoattractant for the tumor cells. Interrupting either ofthese signals results in decreased tumor-cell motility. Directevidence have been presented that MMP-9 derived fromhematopoietic cells of host origin contributes to skincarcinogenesis.60 Chemokines have been shown to inducegene expression of various MMPs and, in particular,MMP-9 production, along with the uPA receptor.79

Evidence suggests that MMP-9 has complex effects beyondmatrix degradation including promotion of the angiogen-esis switch and release of growth factors.60

Conclusions

Macrophages are a key component of cancer-promotinginflammatory reactions. Several lines of evidence, rangingfrom adoptive transfer of cells to genetic manipulations,suggest that myelomonocytic cells can promote tumorinvasion and metastasis, although under certain conditionsthey can express antitumor reactivity. In particular,mammary carcinoma has served as a prototypic tumorfor the TAM-cancer cell interplay (Fig. 1).9,65,71–73 Thus,therapeutic targeting of macrophage-derived mediatorsmay provide innovative therapeutic strategies againstinvasion and metastasis.

Conflict of Interest Statement

None declared.

Acknowledgments

This work was supported by Associazione Italiana per laRicerca sul Cancro (AIRC), Italy; European Commission;Ministero Istruzione Universita e Ricerca (MIUR) andIstituto Superiore di Sanita (ISS).

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