Mononuclear phagocytes are essential cells for wound healing and tumors can be described as wounds that never heal [1]. Macrophages are numerous in the stroma of experimental and human tumors and mediate important biological functions that profoundly affect tumor cell behaviour. By secreting a number of diverse chemoattractants, primary tumors recruit blood circulating monocytes; here they differentiate to Tumor-Associated Macrophages (TAM) primarily because of the presence of M-CSF produced by neoplastic cells. Conditioned by the local milieu (rich in IL-10, TGFβ and prostaglandins), they acquire immune-suppressive and pro-tumoral effector properties. TAM are key players in cancer-related inflammation; with their continual deposition and degradation of the extracellular matrix (ECM) TAM actively contribute to the build-up the typical reactive micro-environment of tumors.

The interstitial matrix is an intricate and highly dynamic network of fibres composed of glycosaminoglycan (GAG)-containing glycoproteins. Different types of fibrous collagen together with fibronectin, hyaluronan and proteoglycans confer mechanical strength, elasticity and a precise spatial organization to tissues. In addition, the extracellular basement membrane, a specialized form of sheetlike ECM mainly composed by collagen IV and laminins, is very important to sustain the epithelial cell layer and maintain orientation of apicobasal polarity [2]. Besides structural support, the interstitial matrix and basement membranes are important to integrate complex signalling and to regulate cellular movement, proliferation and differentiation [3,4]. Furthermore, the ECM contains a wide range of growth factors that are bound in an inactive form to matricellular proteins, but can be rapidly released and activated in case of need, for example during tissue repair [5-7].

Neoplastic cells modify their stroma and vasculature through production and secretion of different growth factors and cytokines. The locally changed host microenvironment, in turn, regulates the proliferation and invasive behaviours of tumor cells. The tumoral ECM presents several different features compared to normal tissue ECM, not only for the presence of aberrantly expressed or modified structural proteins but also, and most importantly, because of the incessant remodelling operated by several proteolytic enzymes. ECM degradation has several important consequences: altered stiffness and composition of the ECM; fragmentation of basement membranes, which facilitates the motility and invasive ability of tumor cells; deregulated organization of the vessel network. All these processes, initially guided by tumor cells and gradually involving the contribution of host cells, lead to the construction of a reactive stroma where the cross-talk and signalling between the diverse cell types and ECM proteins is outside the normal control. Here we will review the role of tumor macrophages in neoplastic tissues, in particular their important contribution to the continuous remodelling of the tumor stroma.

Macrophages are versatile cells that are capable of displaying different functional activities, some of which are antagonistic; for instance they can be immuno-stimulatory or immuno-suppressive, and either promote or restrain inflammation [8,9]. This functional plasticity is regulated by local cues to which the macrophages respond. For instance during bacterial infections macrophages first orchestrate the acute inflammatory response and eliminate the invading pathogens; at later time points they transform into scavengers of tissue debris, and finally trigger the proliferative phase of healing by releasing a variety of growth factors and cytokines which recruit and activate fibroblasts and new vessels [10-15].

Macrophage heterogeneity has been simplified in the macrophage polarization concept where the two extreme phenotypes, the M1 and M2 macrophages, have distinct features. M1 or classically–activated macrophages are stimulated by bacterial products and Th1 cytokines (e.g., IFNγ); they are potent effectors that cope bacterial infections and may have cytotoxic activity towards transformed cells [16,17]. M2 or alternatively activated macrophages differentiate in micro-environments rich in Th2 cytokines (e.g., IL-4, IL-13); they have high scavenging activity, produce several growth factors that activate the process of tissue repair and suppress adaptive immune responses [18-20].

In established tumors, TAM resemble M2-like macrophages [21-23]. While M2-related activities are of extreme importance during wound healing to return to the homeostatic state, in the context of a growing tumor they may favour disease progression [16,17,21,24-27]. Indeed, neoplastic tissues show similarities to sites of tissue repair (Figure 1).

TAM are poorly cytotoxic against neoplastic cells and, instead, have been shown to influence fundamental aspects of tumor biology. Among the well documented pro-tumor functions of TAM is the production of many growth factors for tumor cells and for the nascent blood and lymphatic vessels, which are essential for the neo-angiogenesis switch and tumor proliferation. These include for instance epidermal, fibroblast and vascular growth factors (EGF, FGF, VEGF) [25,28-30]. TAM are also a major source of proteolytic enzymes that degrade the extra-cellular matrix thus favouring the invasion of neoplastic cells [25,31]. They contribute to the evasion of tumors from immune control by producing immune-suppressive cytokines such as IL-10 and TGF-beta [25,32].

In line with the above evidence, high density of TAM has been significantly associated with poor prognosis in the majority of tumors [17,20,25,33]. Indeed, markers of macrophages or their products are present in the stroma-associated gene signature predicting clinical outcomes (see below).

Some studies in human colorectal cancer, however, indicated that macrophages may have anti-tumor activity [34-36]. TAM localization appears of primary importance: the number of peritumoral macrophages, but not of those within the cancer stroma, was associated with improved disease-free survival. Peritumoral macrophages had higher expression of costimulatory molecules (CD80 and CD86) and were able to induce apoptosis in cancer cells by a Fas ligand-dependent mechanism [37,38]. It may be possible that by being less exposed to tumor-derived immune-suppressive cytokines outer macrophages are able to differentiate into cytotoxic effectors.

In normal tissues resident stromal cells (fibroblasts, leukocytes, endothelial cells) are typically quiescent. In tumors, the contrast is macroscopically evident, as observed by pathologists over 100 years ago. The tumor stroma is highly inhomogeneous with more abundant ECM, activated fibroblasts, irregular vessels and numerous inflammatory leukocytes [39-41].

In the normal stroma each cell type displays surface receptors appropriate to its environment. ECM proteins function cooperatively to modulate the interaction between different cellular components, basement membranes and interstitial matrix proteins. These processes keep under strict control various cellular processes such as growth, death, adhesion, migration, gene expression and differentiation, and are of relevance either to maintain homeostasis and to cope tissue repair in case of injury [4,42].

The tumor stroma is characterized by a remarkable subversion of the tissue architecture, especially in poorly differentiated carcinoma, and by a different composition of some ECM components. Ultrastructural and immunohistochemical analyses revealed the up-regulation of several proteins such as tenascin, decorin, byglican, α-smooth muscle actin, osteopontin, fibulin-1, fibronectin, and the appearance of spliced protein isoforms that are not normally expressed [4,43-45]. While the vascular network in normal tissues is characterized by regular dichotomous branching, the tumor vasculature is disorganized, with numerous capillary branches, blind buds or dilated vessels, and a decreased number of pericytes. Indeed, neoplastic tissues appear to be in a constant state of tissue damage [43,46-48].

Probably the most remarkable characteristic of tumoral stroma is the high level of proteolytic degradation [49-51]. This phenomenon has several consequences: first, it alters stroma stiffness and removes the physical barriers between cells, facilitating the invasion of migrating cells (neoplastic and endothelial cells); second, cleaved ECM proteins may reveal cryptic sites and generate abnormal signalling; third, ECM-stored growth factors are released in active form and directly stimulate tumor cell survival, proliferation, motility and the neo-angiogenic switch.

Even in normal tissues the ECM is not a static structure and microscopic changes are determined by a careful balance between matrix synthesis, secretion, modification and enzymatic degradation. Such dynamic remodeling is amplified, in a deregulated manner, in tumor tissues. Matricellular proteins are degraded by specific proteases which can be grouped in large families and include matrix metalloproteases (MMPs), cathepsins, hyaluronidases, ADAM proteases, but also heparanase, elastase, urokinase-type plasminogen activator (uPA), plasmin and others [69,70].

Tumors have high turnover of ECM proteins and protease activity. Although neoplastic cells and fibroblasts are able to produce proteolytic enzymes, macrophages are considered the major cell type expressing protease activity in tumor tissues [50,71,72]. Immunohistochemical and enzymatic analyses in different tumors have shown that increased expression of proteases or changes in cell localization are important prognostic factors which correlate with tumor progression [51,73] Proteolysis of ECM proteins disrupts integrin-mediated anchorage and focal adhesion kinase (FAK) and is pivotal for cancer cell invasion into the adjacent space [31,74-76].

TAM and their released factors (e.g., IL-1 and TNF) have long been known to augment tumor metastasis [77,78]. In addition they are an important source of proteolytic enzymes, especially MMPs and uPA [49,50,79]. Of note, TAM produce several chemokines which beyond regulating cell motility–are able to activate MMPs [80,81]. The role of TAM in cancer cell invasion has been visualized in experimental tumors in vivo by multiphoton microscopy; by using fluorescently labelled cells Wyckoff and colleagues showed that tumor cell intravasation occurs next to perivascular macrophages in mammary tumors [82,83]. Further, it has been recently shown that the cathepsin protease activity of IL-4-stimulated TAM promotes tumor invasion [84]. IL-4 is produced by tumor-infiltrating CD4 T cells and there is mounting evidence of its relevance in the polarization of macrophages with pro-tumor functions [85,86].

Cleavage of matrix molecules also reveals available binding sites that were previously masked to cell surface receptors, and fragments with new functional effects. For instance, MMP-2 degradation of collagen unveils integrin-binding sites that rescue melanoma cells from apoptosis [47] while the trimeric NC1 domain of collagen XVIII induces angiogenesis [87]. Cryptic epitopes of fibronectin trigger angiogenesis and tumor growth [88,89].

Over the last decade there has been recognition that proteins of the ECM can modulate multiple functions of innate immune cells. A cryptic peptide of laminin-10, a prominent component of basement membranes, is chemotactic for neutrophils and macrophages and induces the up-regulation of TNF, chemokines and MMP-9 [90]. Particular attention has been given to proteolytic ECM fragments and the activation of Toll-like receptors: versican activates TLR2 and TLR6 on TAM and stimulates the production of IL-6 and TNF, two prototypic cytokines of cancer-related inflammation [91]. Hyaluronan fragments induce the expression of inflammatory genes in immune cells through activation of TLR4 and TLR2 as well as the CD44 receptor [92]. Thus ECM glycoproteins and glycosaminoglycans can directly stimulate inflammatory cells and contribute to fuel inflammation at tumor sites.

We recently performed an Affymetrix gene profiling of TAM isolated from human ovarian carcinoma and found that among the most up-regulated genes were several genes coding for ECM proteins or related to its remodelling (Figure 2). Among proteolytic enzymes, the most expressed were MMPs (12, 9, 1 and 14), Cathepsins (L,C,Z and B), uPA, lysosomal enzymes and ADAM proteases (Figure 2).

The matrix is a valuable repository of growth factors: members of the EGF and FGF families, TGF-beta and related members, as well as PDGF and VEGF, bind to the various components of ECM and are stored, in an inactive form, until released and activated by matrix proteases. In the tumor context, increased proteolytic activity releases active growth factors which stimulate tumor and stromal cells [47,50,93,94].

For example, MMPs, plasmin and heparanase degrade the angiogenic factor FGF-beta [95]. MMP-3 has been shown to cleave the matrix molecule decorin, thereby delivering active TGF-beta [96] and MMP13 appears to be essential for the release of VEGF from the ECM in squamous cell carcinoma [97]. Of note, during ECM proteolysis fragments with angiostatic activity can also be generated. Thus the ultimate biological response really depends on the balance between pro-and anti-angiogenic factors.

Degraded matrix proteins need to be eliminated. A major function of macrophages, dictated by their name, is the phagocytosis of apoptotic cells and cellular debris, and their final disposal in specialized lysosomal compartments. As mentioned above, TAM expresses high levels of lysosomal-enriched cathepsins, which facilitate the elimination of ingested proteins.

Fibroblasts are master regulators of matrix deposition in the stroma and are influenced by stimuli coming from both inflammatory cells (macrophages) and neoplastic cells. Several growth factors produced by TAM are able to activate fibroblasts: EGF, FGF, PDGF and, above all, TGF-beta. In turn, activated fibroblasts (or myofibroblasts, as they start producing a-smooth muscle actin) release growth factors for epithelial cells (IGF, EGF, HGF), chemokines for macrophages (CCL2, CXCL12) and activated MMPs [28,29,106]. Thus, tumor-associated fibroblasts are key cells of the reactive tumor micro-environment. In addition, also TAM are very active producers of matricellular proteins. TAM contribute to matrix architecture by producing for instance osteopontin, fibronectin, proteoglycans, SPARC and different collagen types [14,15,107].

As summarized above, TAM functional activities importantly contribute to the construction of the reactive tumor micro-environment and are, therefore, amenable targets of biological therapies. Macrophage depletion in experimental settings has been successful in decreasing tumor growth and metastatic spread [11,137,138]; furthermore their depletion may contribute to a better response to conventional chemotherapy and anti-angiogenic therapy [62,63,65-67]. Several approaches have been followed to target TAM in tumors such as inhibition of their recruitment at tumor sites or the use of cytotoxic drugs, for example, biphosponates.

A number of studies have shown that the bisphosphonate clodronate-encapsulated in liposomes is an efficient reagent for the depletion of macrophages. Clodronate-depletion of TAM in tumor-bearing mice resulted in reduced angiogenesis and decreased tumor growth and metastasis [139]. Moreover, the combination of clodronate with sorafenib significantly increased the efficacy of sorafenib alone in a xenograft model of hepatocellular carcinoma. In clinical practice, bisphosphonates are employed to treat osteoporosis; current applications in cancer treatment include their use to treat skeletal metastases in multiple myeloma, prostate and breast cancer. Treatment with zoledronic acid was associated with a significant reduction of skeletal-related events and, possibly, direct apoptotic effects in tumor cells [140-142].

Another approach is to inhibit the recruitment of circulating monocytes in tumor tissues. Among the many chemokines expressed in the tumor micro-environment, CCL2 (or monocyte chemotactic protein-1) occupies a prominent role and has been selected for therapeutic purposes. Pre-clinical studies have shown that anti-CCL2 antibodies or antagonists to its receptor CCR2, given in combination with chemotherapy, were able to induce tumor regression and yielded to improved survival in prostate mouse cancer models [143-145].

A third and more recent approach is to ‘re-educate’ TAM to exert anti-tumor responses protective for the host, ideally by using factors able to switch the M2-phenotype of TAM into that of M1-macrophages with potential anti-tumor activity. This was achieved in experimental mouse tumors by injecting the TLR9 agonist CpG- oligodeoxynucleotide (CpG-ODN), coupled with anti-IL-10 receptor [146] or the chemokine CCL16 [147]. CpG-ODN synergized also with an agonist anti-CD40 mAb to revert TAM displaying anti-tumor activity [148]. A remarkable anti-tumor effect of re-directed macrophages has been recently reported in human pancreatic cancer with the use of agonist anti-CD40 mAb [149]. A recent report showed that the plasma protein histidine-rich glycoprotein (HRG), known for its inhibitory effects on angiogenesis [150,151] is able to skew TAM polarization into M1-like phenotype by down-regulation of placental growth factor (PlGF), a member of the VEGF family. In mice, HRG promoted anti-tumor immune responses and normalization of the vessel network [152].

The intense protease activity present within tumors has been the object, over several years, of pharmaceutical research, looking for specific MMPs inhibitors [49,50]. The first generation of developed compounds were competitive inhibitors (e.g., batimastat), and later were derivatives of tetracycline, which inhibited MMP gene transcription and enzymatic activity. Of note, also the biphosponates inhibit MMP activity. When tested in clinical trials several years ago, these compounds gave overall disappointing results. The field, however, is still active and considering the use of monoclonal antibodies specific for membrane-bound MMPs, such as MMP14 [51,153].

Few years ago, by studying a new anti-tumor agent of marine origin, trabectedin, we unexpectedly observed that this compound was highly cytotoxic to monocytes and macrophages, with a remarkable selectivity, as neutrophils and lymphocytes were not killed [154]. Trabectedin has now been registered in 2007 in Europe for the treatment of soft tissue sarcoma, and in 2009 for ovarian cancer [155-157]. Trabectedin also affects gene transcription with a peculiar selectivity; some inflammatory cytokines and chemokines are reduced by the drug, such as IL-6, CCL2, CXCL8, VEGF, while TNF is not inhibited [158]. Another affected gene is collagen [159]. We therefore tested whether other ECM-related genes produced by macrophages were reduced by the drug. In in vitro experiments with monocytes/macrophages we found that low non-cytotoxic concentrations of trabectedin significantly decreased the expression of both the full length fibronectin and MSF. Also osteopontin and MMP2 were inhibited, while osteoactivin was not (Liguori, unpublished data). These results indicate that trabectedin may reduce the high turnover of the tumor stroma. This effect of “normalization” of the micro-environment, combined with its cytotoxic effect on macrophages and tumor cells, makes trabectedin an interesting compound in oncology.

In the last decades the concept that the ECM is simply a supporting structure for the preservation of tissue architecture has dramatically changed [160]. Indeed, ECM components provide signals affecting cell adhesion, migration, proliferation and differentiation. In particular, degraded/proteolytic fragments of ECM molecules or their aberrant expression, as occurs during neoplastic transformation, can sustain the activation of inflammatory cells. TAM are key players of the cancer-related inflammation present at tumor sites. In addition to releasing cytokines and growth factors, a distinguished feature of TAM is their high rate of remodelling of the tumor stroma, in which they vigorously participate by expressing proteolytic enzymes and ECM proteins, some of which are specific to the neoplastic tissues. Such a reactive micro-environment eventually supports tumor cell proliferation and the full-blown development of neo-angiogenesis. There is increasing evidence that successful anti-cancer therapies are not only dependent on the cancer phenotype but also on the normalization of the tumor stroma. In this view, depletion of the unfaithful TAM, or their “re-education”, may contribute to the success of conventional anti-tumor therapies.