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Originally published online as doi:10.1189/jlb.1105656 on July 24, 2006

Published online before print July 24, 2006
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(Journal of Leukocyte Biology. 2006;80:705-713.)
© 2006 by Society for Leukocyte Biology

Dual role of macrophages in tumor growth and angiogenesis

Chrystelle Lamagna*,{dagger}, Michel Aurrand-Lions* and Beat A. Imhof*,1

* Department of Pathology and Immunology, Centre Médical Universitaire, Geneva, Switzerland; and
{dagger} Department of Neurological Surgery and Brain Tumor Research Center, University of California San Francisco, San Fransico, California

1 Correspondence: Department of Pathology and Immunology, Centre Médical Universitaire, 1204, Geneva, Switzerland. E-mail: Beat.Imhof{at}medecine.unige.ch

ABSTRACT

During the neoplastic progression, macrophages as well as dendritic and NK cells are attracted into the tumor site and initiate the immune response against transformed cells. They activate and present tumor antigens to T cells, which are then activated to kill tumor cells. However, tumor cells are often capable of escaping the immune machinery. As the immune surveillance is not sufficient anymore, tumor-associated macrophages contribute to tumor progression. It is notable that tumor-associated macrophages promote the proliferation of tumor cells directly by secreting growth factors. They also participate in tumor progression by acting on endothelial cells and thus promoting the neovascularization of the tumor. Tumor-associated macrophages are indeed key protagonists during angiogenesis and promote each step of the angiogenesis cascade.

Key Words: chemokines • neovascularization • monocytes

INTRODUCTION

The causal relationship between inflammation and the neoplastic progression is a concept widely accepted. During the last century, the question of whether the immune system positively or negatively controls the neoplastic progression has been a matter of debate [1 ]. Accumulating data now validate the concept of cancer immunosurveillance, stating that one of the physiologic functions of the immune system is to recognize and destroy transformed cells. However, some tumor cells are capable of evading recognition and destruction by the immune system. Once tumor cells have escaped, the immune system participates in their growth, notably by promoting the vascularization of tumors. When it comes to this stage, the number of tumor-infiltrated hemopoietic cells correlates with poor prognosis [2 , 3 ].

Adaptive and innate immune cells participate in the surveillance and the elimination of tumor cells. Monocytes/macrophages may be the first line of defense in tumors, as they colonize rapidly and secrete cytokines that attract and activate dendritic cells (DC) and NK cells. In turn, activated DC and NK cells initiate the immune response against transformed cells [4 ]. Ideally, NK cells impair the progression of the tumor, and DC alert T lymphocytes of the presence of a danger in the tumor-draining lymph nodes [5 ]. Studies of gene-targeted mice lacking RAG-2 have led to elucidation of the cancer immunosurveillance concept. These mice cannot rearrange lymphocyte antigen receptors and thus cannot produce mature lymphocytes [6 ]. It is interesting that RAG-2–/– mice develop spontaneous epithelial tumors and chemically induced sarcomas more frequently than wild-type control mice [7 ], indicating that lymphocytes can protect against tumor formation. Further studies have defined the critical tasks responsible for the eradication of developing tumors. Among them, IFN-{gamma} is incontestably necessary for tumor immunity, as it prepares presentation of tumor antigens by macrophages and DC to T lymphocytes. Indeed, IFN-{gamma} is capable of protecting the host against the growth of spontaneous tumors, as well as transplanted or chemically induced tumors [8 9 10 11 ]. Activated by macrophages, NK cells, B cells, DC, as well as the macrophages themselves secrete IFN-{gamma} [12 13 14 15 ]. Gao et al. [16] have also shown that {gamma}{delta} T cells are a source of IFN-{gamma}. However, it remains unclear whether other RAG-2-dependent cellular sources of IFN-{gamma} exist. The critical function of cancer immunosurveillance is the ability of activated NK cells or T lymphocytes to kill the tumor. One excellent example of tumor eradication by immune cells is illustrated by manipulating perforin, which has been identified as one of the key cytolytic molecules of killer cells implicated in this process. Indeed, perforin-deficient mice develop more tumors than wild-type mice after chemical induction [11 , 17 , 18 ]. In summary, an antitumor response can be launched by a series of events, starting with inflammation mediated by monocyte/macrophages, which stimulates NK and DC and finally activates the cytotoxic lymphoid system.

Tumors that escape from the immune machinery can be a consequence of alterations occurring during the immunosurveillance phase. As an example, some tumor cells develop deficiencies in antigen processing and presentation pathways, which facilitate evasion from an adaptive immune response, such as the absence or abnormal functions of components of the IFN-{gamma} receptor signaling pathway [9 , 19 , 20 ]. Other tumors suppress the induction of proinflammatory danger signals, leading, for example, to impaired DC maturation. These defects result in the incapacity of DC to stimulate T cells and provide the developing tumor with a potential mechanism to escape immune detection [21 , 22 ]. Finally, the inhibition of the protective functions of the immune system may also facilitate tumor escape, such as the overproduction of the anti-inflammatory cytokines IL-10 and TGF-ß, which can be produced by many tumor cells themselves but also by macrophages or T regulatory cells [23 , 24 ].

As cancer immunosurveillance is not efficient anymore, tumor-infiltrated, inflammatory cells make peace with the enemy and help the neoplastic progression. This switch concerns mainly macrophages, which change their phenotype from proinflammatory to a proangiogenic one, contributing to the malignancy by releasing proteases, angiogenic factors, and cytokines [3 , 25 26 27 28 ]. In turn, tumor cells secrete cytokines, and the balance of proinflammatory versus anti-inflammatory cytokines controls the tumor outcome by regulating the number of inflammatory cells in the tumor mass and the development of a novel vascular network, allowing tumor cells to proliferate. It has been shown that reducing the number or function of macrophages has a blocking effect on angiogenesis, and elevated numbers of macrophages increased tumor angiogenesis and tumor growth [2 , 29 ]. Thus, the switch from antitumor immunity to tumor growth and angiogenesis seems to be controlled, at least in part, by monocytes/macrophages.

THE RECRUITMENT OF MACROPHAGES INTO THE TUMOR MASS

Macrophages are released from the bone marrow as immature monocytes. After circulating in the blood, they are recruited by chemokines into the tissue and undergo differentiation into macrophages [30 ] (Fig. 1 ). They can exhibit a variety of phenotypes and functions, depending on the physiologic or pathologic situation to which they are recruited. These various functions, essential for tissue remodeling, inflammation, and immunity, include endocytosis of foreign and necrotic debris, cytotoxicity, and secretion of more than 100 different substances [31 ]. Indeed, depending on their activation, macrophages are able to secrete growth factors, cytokines, proteases, or complement components. Moreover, specialization and activation of these cells are largely influenced by local stimuli. These can be delivered by cytokines, by the engagement of adhesion molecules, or by macrophage interaction with pathogens [32 ].


Figure 1
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  		Figure 1. Differential function of macrophages, which differentiate from tumor homing and blood monocytes and can acquire angiogenic (M2) or inflammatory (M1) phenotypes, depending on the environment of the tumor. The M1 macrophages induce antitumor reponses as a result of secretion of IFN-{gamma}, IL-12, or TNF-{alpha}; M2 macrophages suppress immune responses as a result of secretion of TGF-ß or IL-10 and stimulate angiogenesis and tumor growth as a result of secretion of IL-17, IL-23, vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), or endothelin.

 
On the basis of their activation state, macrophages can be referred to as Type I or Type II cells. The differences displayed by macrophages in term of receptor expression, cytokine production, and functions define these two populations. Numerous studies have permitted classifying Type I macrophages (M1) as cells capable of producing large amounts of proinflammatory cytokines, expressing high levels of MHC molecules, and implicated in the killing of pathogens and tumor cells. In contrast, Type II macrophages (M2) moderate the inflammatory response, eliminate cell wastes, and promote angiogenesis and tissue remodeling [26 , 33 , 34 ] (Fig. 1) . The macrophages present in neoplastic tissues are referred to as tumor-associated macrophages (TAMs) and mainly belong to the M2 population [27 ]. Both macrophage types or their monocyte progenitors are recruited to the tumor mostly from the blood circulation, although macrophages may also immigrate to tumors from the surrounding tissue [33 ].

Chemokines implicated in the recruitment of macrophages into the tumor
Most of monocytes, which become TAMs in the tumor, are attracted from the circulation into the tumor mass by the chemokines CCL2 (MCP-1) and CCL5 (RANTES) [35 , 36 ]. CCL2 has been described first as a tumor-derived chemotactic factor for monocytes and isolated from culture supernatants of human and murine tumor cell lines [37 ]. It can be detected in several tumors, such as sarcomas, gliomas, breast, and ovary carcinomas and melanomas. CCL2-deficient mice display abnormalities in monocyte recruitment in an inflammatory model, as well as delayed wound angiogenesis [38 , 39 ]. However, the number of macrophages within the wounded site is not affected by the absence of CCL2, suggesting that the monocyte recruitment into wounds is independent of the chemokine CCL2 [38 ]. Nevertheless, one could not exclude that CCL2 plays a critical role in healing wounds by activating macrophages or by acting on distinct cell types other than macrophages. This is in contrast to the important finding that tumor angiogenesis is reduced in CCL2–/– mice. It also correlates with reduced colonization of the tumor by macrophages and tumor growth [29 ]. In addition, in the model of choroidal neovascularization, inactivation of the CCR2, the unique receptor of CCL2, leads to the reduction of the number of infiltrating macrophages and decreases angiogenesis [40 ]. Accordingly, Nesbit et al. [41] have shown that low-level expression of CCL2 by tumor cells correlates with a modest monocyte infiltration and tumor development. High secretion of CCL2 is associated with bulk infiltration of macrophages into the tumor mass, leading to destruction of the tumor. This occurs probably by immigrating M1 macrophages exerting cytotoxic mechanisms such as induction of apoptosis or the secretion of highly reactive oxygen species or NO, both usually suppressed in M2 TAMs by the tumor. Thus, bulk overflow of the tumor with macrophages may allow partial persistence of M1-type macrophages in tumors. These results indicate that the biological effect of CCL2 is biphasic, depending on its level of secretion by tumor cells [41 ]. The abundant studies about CCL2 and its receptor CCR2 clearly demonstrate the role of CCL2 in recruiting monocyte/macrophages to the site of the tumor. These experiments show that the number and probably the type (M1 and/or M2) of macrophages in tumors are decisive for tumor destruction or tumor growth and angiogenesis.

CCL5 is not only produced by naive T cells but also by breast tumor cells, contributing to monocyte migration into tumor sites [42 ]. It is interesting that CCL5 stimulates human monocytes to express CCL2, CCL3 (MIP-1{alpha}), CCL4 (MIP-1ß), and CXCL8 (IL-8), all of them chemoattractants for myeloid cells [43 ]. CCL5 also stimulates the expression of the receptor CCR1 on monocytes, which is recognized by numerous chemokines. Hence, activation of monocytes by chemokines leads to further recruitment of more monocytes into the tumor mass, as well as that of other leukocyte populations [43 , 44 ].

At this time, studies about the role of chemokines in TAM recruitment have focused mainly on CCL2 and CCL5. Other chemokines, including CCL3, CCL4, CCL8 (MCP-2), and CCL22 (macrophage-derived chemokine), have been detected in ovarian tumors [45 46 47 ]. High levels of CXCL8 and CCL18 (MIP-4) have also been found in ascitic fluids from patients with ovarian carcinoma [47 ]. The presence of these chemokines in the tumor mass has been correlated with the presence of macrophages. It could be of interest to determine more precisely whether they play a role in the recruitment or in the maintenance of the TAM population in the neoplastic tissues.

The recruitment of monocytes into the tumor is also under the influence of cytokines. Among them, the role of CSF-1 has been clearly demonstrated. CSF-1 stimulates the proliferation, differentiation, and survival of cells from the mononuclear phagocytic lineage, to which monocytes belong [48 ]. In addition, CSF-1 is produced by many cell types, such as monocytes and macrophages themselves, and is also implicated in the recruitment of macrophages into neoplastic tissues [49 ]. By crossing a transgenic mouse susceptible to mammary cancer with mice containing a recessive null mutation in the CSF-1 gene (op/op mice), it was shown that infiltration of monocytes into tumors was reduced markedly in the absence of CSF-1, and this correlated with delayed angiogenesis and tumor progression [2 ].

The angiogenic growth factor VEGF has also been correlated with the presence of macrophages within tumors. In addition, it has been shown to be a chemoattractant for monocytes and macrophages in vitro via activation of the receptor tyrosine kinase VEGF receptor-1 (VEGF-R1; flt-1) [50 51 52 ]. Indeed, a neutralizing mAb directed against VEGF-R1 reduces migration of the monocytes upon VEGF stimulation [52 ]. Furthermore, VEGF-induced migration of monocytes is suppressed strongly in transgenic mice with a nonfunctional tyrosine kinase domain of VEGF-R1, whereas embryonic development and normal angiogenesis occur [53 ]. In agreement, Grunewald et al. [54] recently demonstrated that VEGF by itself triggers the homing of circulating myeloid cells at a site of neovascularization in the adult. It is interesting that VEGF induces the expression of the chemokine CXCL12 stromal cell-derived factor-1, which retains the recruited cells around blood vessels. In addition to its role in attracting macrophages into the tumor mass, VEGF produced by breast tumor cells in response to local hypoxia also directs the movement of TAMs within the tumor [50 , 55 ].

Similar to chemokines, endothelins (ET-1, ET-2, and ET-3) are small vasoconstrictor peptides, which bind to G-protein-linked transmembrane receptors, ET-RA and ET-RB [56 ]. Endothelin expression has been correlated with the presence of macrophages within hypoxic areas of tumors. Studies show that ET-1 plays a role in tumor development by acting on mitogenesis, apoptosis, angiogenesis, tumor invasion, and metastasis [57 ] but also exhibits chemotactic activities toward human neutrophils and monocytes [58 ]. ET-2 rather mediates the chemoattraction and the activation of macrophages but not for freshly isolated monocytes [59 ]. Altogether, these findings suggest that ET-1 may be implicated in the recruitment of monocytes into tumors, whereas ET-2 directs their subsequent localization within hypoxic areas. Recent reports indicate that rather than favoring the migration of macrophages, ET-2 stimulates TAMs to produce molecules implicated in tumor progression and development, such as matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) [60 ]. The recently suggested role of endothelins in the attraction and migration of macrophages into the tumor mass represents a further leap in the understanding of the molecular mechanism. However, to be exploited for therapy, it needs further investigations.

ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN TUMOR GROWTH

The functions of TAMs within the tumor site are various and sometimes paradoxal. Initially, it was considered that the main function of TAMs was to exert direct, cytotoxic effects on tumor cells and to phagocyte apoptotic cells and waste products. Now it became clear that monocytes can differentiate into "friendly" M1 macrophages, which initiate tumor rejection or "foe" M2 TAMs, which stimulate tumor growth, metastasis, and angiogenesis [61 ].

Activation of an antitumor immune response by M1 tumor-associated macrophages
Macrophages have well-documented functions in the regulation of the antitumor immune response. As the presence of a growing tumor is associated to stromal remodeling and to the production of proinflammatory molecules that act as danger signals, M1 macrophages, as well as NK cells and DC, are thus attracted into the injured site, resulting in a massive secretion of IFN-{gamma} and IL-12 [62 , 63 ]. Indeed, the products generated during the stromal remodeling induce macrophages to produce IL-12, which stimulates NK cells to produce IFN-{gamma}, which in turn activates macrophages to produce more IL-12, leading to a positive feedback increasing the IFN-{gamma} production by NK cells [64 ]. In addition, a newly described subset of DC, called IKDC, contributes to IFN-{gamma} production [65 , 66 ]. Upon activation by IFN-{gamma}, M1 macrophages release tumoricidal products, such as reactive oxygen intermediates and NO, which kill tumor cells [31 , 67 ]. As a result, tumor antigens from dead tumor cells become available, and the adaptive immune system is recruited. Activated macrophages also produce TNF-{alpha}, which as its name implies, can kill tumor cells by direct effect on tumor cells or by inhibiting the developing vasculature [68 , 69 ]. Finally, M1 macrophages, which are APC, process tumor antigens and present them to lymphocytes after migration into the draining lymph nodes. As a result, the different subsets of T lymphocytes are activated, proliferate, and thus infiltrate the tumor, where they can exert their immune function [1 ].

Stimulation of tumor growth by M2 tumor-associated macrophages
By the means of their secretory products, M2 TAMs stimulate tumor growth. This can be directly by producing cytokines able to stimulate the proliferation of tumor cells or indirectly, by stimulating endothelial cell proliferation and angiogenesis [55 , 70 ]. As an example, the growth of subcutaneous Lewis lung tumors is impaired in the CSF-1-deficient, macrophage-deficient op/op mice, indicating a role for macrophages in tumor growth [71 ]. Moreover, treatment of tumor-bearing op/op mice with human recombinant CSF-1 corrects this impairment. M2 TAMs and tumor cells also produce immunosuppressive cytokine TGF-ß, which effectively blunts the antitumor response by cytotoxic T cells [27 , 72 ]. Finally, TAMs have also been implicated in the proteolytic remodeling of the extracellular matrix (ECM), which is important at several points during multistage progression of tumors [73 ].

Tumor-associated M2 macrophages and metastasis
The percentage of TAMs within a tumor has been positively correlated with the metastatic potential of the tumor, suggesting a role for TAMs in the distant dispersion of tumor cells [74 ]. Indeed, monocytes secrete enzymes capable of degrading the ECM, such as the MMP-19 and the urokinase-type plasminogen activator receptor (uPAR) [43 , 75 ]. By relaxing the connective tissue surrounding the tumor, these molecules allow tumor cells to detach from the tumor mass and to disseminate, leading to the formation of distant metastases. Moreover, by favoring angiogenesis and lymphangiogenesis, macrophages increase the availability for tumor cells to enter blood or lymphatic vessels and invade distant tissues [76 77 78 ]. This is supported by the fact that tumor vessels exhibit an anarchic organization compared with normal blood vessels with immature, interendothelial junctions and fenestrations, thus exposing tumor cells to the blood circulation [79 , 80 ].

THE ANGIOGENIC PHENOTYPE OF TUMOR-ASSOCIATED MACROPHAGES

The angiogenic phenotype of macrophages is in part defined by their ability to secrete molecules that promote or inhibit angiogenesis. Depending on their activation state, M2 TAMs produce a variety of proangiogenic and lymphangiogenic growth factors, cytokines, and proteases [29 , 77 , 81 ]. However, it has been demonstrated that endothelial cell stimulatory and inhibitory fractions can be purified from conditioned medium of monocytes [82 , 83 ]. More recently, Pakala et al. [84] have shown that conditioned medium from resting monocytes inhibits endothelial cell proliferation, and conditioned medium from activated monocytes (macrophages) stimulates endothelial cell proliferation. These studies show that the activation of monocytes induces their pro- or antiangiogenic activities. In addition, elevated levels of TAMs correlate with increased tumor angiogenesis in breast cancer and endometrial carcinoma [85 ], pulmonary adenocarcinoma [86 ], and malignant melanoma [78 ].

Monocytes and resting macrophages exhibit a nonangiogenic phenotype [82 , 87 ]. Microbial agents such as LPS trigger monocytes into M1 immunostimulatory macrophages, which are, however, able to partially initiate angiogenesis [26 ]. Stimulation of monocytes by tumor products such as IL-10 clearly drives monocytes into M2 angiogenic macrophages, secreting the highly angiogenic growth factor VEGF. Furthermore, activation signals delivered by metabolic conditions found in injured tissues, such as low oxygen tensions or elevated concentrations of lactate, pyruvate, or hydrogen ions, also result in the expression of angiogenic and lymphangiogenic factors by macrophages [88 89 90 91 92 ]. Most TAMs found during human cervical carcinogenesis are of M2 type and express VEGF-C, VEGF-D, as well as the VEGFR-3, all of which are implicated in the formation of lymphatic vessels and finally, lymphatic metastases [77 ]. CSF-1, in addition to its role in the recruitment of TAMs, also regulates their phenotype. Indeed, by inoculating tumor cell lines derived from the CSF-1-deficient op/op mice into SCID recipients, Dougherty et al. [93] have observed that infiltrating TAMs appear less mature. In addition, recombinant human CSF-1 induces normal human monocytes to produce and release the biologically active form of angiogenic VEGF [94 ]. Recently, IL-23 and IL-17 have been described as two proangiogenic cytokines released by TAMs [95 , 96 ]. Although the effect of IL-23 on tumor growth and angiogenesis in IL-23-deficient animals is clear, the molecular mechanism seems to be complex.

Tumor-associated macrophages preferentially accumulate in hypoxic and necrotic regions within the tumors and become M2-angiogenic [97 98 99 ]. Many studies have investigated the induction of genes encoding angiogenic factors in macrophages exposed to hypoxia. CXCL8, VEGF, FGF-2, (basic FGF), and platelet-derived growth factor B (PDGF-B) have been found to be hypoxia-regulated molecules in macrophages [100 101 102 ]. In addition, low oxygen concentration induces the expression of CXCR4, the chemokine receptor of CXCL12, by monocytes, monocyte-derived macrophages, tumor-associated macrophages, as well as endothelial and tumor cells [103 ]. It has also been suggested that the chemokine CXCL12 is regulated by hypoxia [104 ]. However, how M2 TAMs respond to the oxygen level within the tumor mass is still unclear. It is admitted that the response of cells to hypoxia is mediated by the hypoxia-inducible factor (HIF) system [105 ], notably the HIF-1 (composed of HIF-1{alpha} and HIF-1ß) and the HIF-2 system (composed of HIF-2{alpha} and HIF-2ß). It is interesting that the HIF-2{alpha} subunit is expressed by subsets of TAMs, suggesting that the response of TAMs to hypoxia is mediated by the HIF-2 system [99 , 106 ]. However, an additional study shows that human macrophages express higher levels of HIF-1 than HIF-2 when exposed to hypoxia in vitro, and a high level of HIF-1 is detected in macrophages in human breast and ovarian tumors [107 ]. As the response of tumor cells to hypoxia is dependent on their microenvironment, one could imagine that depending on the tumor localization and environment, M2 macrophages activate the HIF-1 or HIF-2 pathway.

TUMOR-ASSOCIATED MACROPHAGES SHAPE THE TUMOR MICROENVIRONMENT FOR ANGIOGENESIS

By secreting a wide range of chemokines, enzymes, and growth factors, M2 macrophages are able to promote each step of the angiogenesis cascade, leading to the formation of new, mature blood vessels (Fig. 2 ). It is important to state that the events leading to newly formed vessels are intricate, and macrophage secretory molecules can be implicated in different steps.


Figure 2
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  		Figure 2. Three steps in angiogenic blood vessel formation. Tumor-associated macrophages of the angiogenic type (M2) produce a series of factors such as the VEGFs, FGFs, chemokines, endothelin, IL-17, IL-23, or TGF-ß, which contribute to angiogenesis, occurring in three steps: 1) induction of endothelial cell proliferation; 2) production of metalloproteases, which degrade the vascular basement membrane, allowing sprouting and migration of endothelial cells into the tumor; and 3) tube formation and maturation of the new vessel, followed by its stabilization by attaching mural cells.

 
Local degradation of the ECM by TAMs
As mentioned above, angiogenic M2 TAMs release enzymes that are able to change the composition of the ECM and thus, to modulate the mechanical stress applied by the ECM on endothelial cells [108 ]. Under the action of CCL2 and CCL5, monocytes and macrophages abundantly secrete MMP-9 and MMP-19 and the uPAR [43 , 75 ]. These enzymes degrade the sustaining basement membrane and the ECM, thus promoting the migration and proliferation of endothelial cells, as well as tumor cells. In addition, the uPA has been found to play a non-negligible role in tube formation of microvascular cells in vitro, as its inhibition subsequently leads to suppression of angiogenesis [109 ].

The degradation of the ECM also permits the release of angiogenic factors, normally sequestered by the ECM. This is the case for FGF-2, released by the degradation of heparan sulfate by uPA, TGF-ß, VEGF, and GM-CSF [110 111 112 113 114 ]. Similarly, angiostatin is the resulting molecule of the proteolytic cleavage of plasminogen by a serine protease, which is mainly produced by tumor-infiltrating macrophages [115 ]. Angiostatin inhibits endothelial cell proliferation in vitro and in vivo [116 ].

TAMs stimulate proliferation and migration of endothelial cells
M2 tumor-associated macrophages release endothelial cell growth stimulatory factors such as FGF-2 [117 ], VEGF [118 ], G-CSF, and GM-CSF [119 , 120 ]. The role of VEGF as a proliferating and survival signal for endothelial cells has been clearly demonstrated. Crowther et al. [121] have demonstrated in breast carcinomas that when tumor cells do not produce VEGF, TAMs represent the unique source of this growth factor. In addition, macrophages secrete the chemokine CXCL8 under activation by CCL2 and CSF-1 [122 ]. CXCL8 has been reported to mediate angiogenesis by stimulating endothelial cell proliferation in a dose-dependent manner [123 ].

Macrophage-released growth factors such as TGF-ß, TNF-{alpha}, and IFN-{gamma} [124 125 126 127 ] exhibit effects that often appear conflicting, such as stimulation or inhibition of endothelial cell growth. This occurs through differential activation of signal transduction pathways [128 ].

A further level of complexity is brought in by macrophages that produce several factors inducing migration of endothelial cells by chemotactic effects. Most of them also support other stages of the angiogenic process, such as mitosis or differentiation of endothelial cells. The poorly characterized factor human angiogenic factor (HAF) was initially found in conditioned medium of the human melanoma cell line A375, but it is also produced by macrophages. It supports endothelial cell migration and angiogenesis in vivo but is not mitogenic for endothelial cells [129 , 130 ]. In addition, the factor angiotropin, also known as monocyto-angiotropin, has been isolated from activated peripheral blood monocytes and macrophages [131 ]. Whereas angiotropin has vasodilatory activity in vivo, culture of confluent endothelial cells in the presence of angiotropin induces the generation of three-dimensional, capillary-like structures [132 , 133 ]. The migratory effects of HAF and angiotropin are sufficient for initial neovascularization, as migrating endothelial cells can form sprouts without proliferating. FGF-2 also stimulates migration of cultured endothelial cells and promotes formation of differentiated capillary tubes in vitro. These effects are associated with selective up-regulation of integrins on endothelial cells and induction of proteases such as plasminogen activator.

TAMs badly sustain blood vessel maturation
Inhibition of neovascularization is necessary to restrict the extent of the new vascular network and to facilitate differentiation of capillary sprouts into functionally mature capillaries. Although factors such as angiopoietins have been implicated in this maturation step, there is no evidence that macrophages secrete such factors. However, they participate in the maturation of newly formed vessels by releasing several factors that inhibit the proliferation of endothelial cells.

TAMs are the only blood cells except platelets that produce PDGF [134 ]. PDGF-B is mainly implicated in the recruitment of pericytes to newly formed vessels and is also expressed by endothelial cells during angiogenesis [135 136 137 138 ]. Mice that lack PDGF-B or PDGF receptor-ß undergo embryonic lethality, as they show a severe deficit in pericyte coverage of blood vessels, microvascular leakage, hemorrhage, and edema formation [139 140 141 ]. In addition, transgenic mice expressing PDGF-D in basal epidermal cells show enhanced recruitment of macrophages in the dermis after wounding [142 ]. One could imagine that PDGF-secreting TAMs participate in the stabilization of blood vessels by recruiting mural cells, which are necessary to provide structural support to blood vessels. Even if TAMs produce soluble factors that participate in blood vessel maturation, it is of importance to state that tumor vessels exhibit an anarchic organization compared with normal blood vessels [79 ]. In addition, endothelial cells can have immature junctions and fenestrations, thus exposing tumor cells to the blood circulation [80 ]. Thus, although TAMs secrete some factors that help to stop endothelial growth and "finalize" vessel maturation, the secretion of products by M2 macrophages is more robust and over-runs the maturation signals.

CONCLUDING REMARKS

The most important function of macrophages is the defense of the body against pathogen aggressions. However, when recruited within neoplastic tissues, tumor-associated macrophages polarize differently and do not predominantly exert their immune function but rather favor tumor growth and angiogenesis. It is thus conceivable to target macrophages to limit tumor expansion by using activated or genetically modified macrophages or by using molecules secreted by macrophages. These could be investigated to limit the proangiogenic and protumor functions of macrophages and move the balance toward their immune function.

Received November 13, 2005; revised June 23, 2006; accepted June 26, 2006.

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