distinct role of macrophages in different tumor microenvironments

Distinct Role of Macrophages in Different Tumor Microenvironments

Claire E. Lewis1and Jeffrey W. Pollard

2

1Academic Unit of Pathology, Division of Genomic Medicine, University of Sheffield Medical School, Sheffield, United Kingdom and 2Centerfor the Study of Reproductive Biology and Womens Health, Departments of Developmental and Molecular Biology and Obstetrics &Gynecology and Womens Health, Albert Einstein College of Medicine, Bronx, New York

Abstract

Macrophages are prominent in the stromal compartment ofvirtually all types of malignancy. These highly versatile cellsrespond to the presence of stimuli in different parts of tumorswith the release of a distinct repertoire of growth factors,cytokines, chemokines, and enzymes that regulate tumorgrowth, angiogenesis, invasion, and/or metastasis. The dis-tinct microenvironments where tumor-associated macro-phages (TAM) act include areas of invasion where TAMspromote cancer cell motility, stromal and perivascular areaswhere TAMs promote metastasis, and avascular and perine-crotic areas where hypoxic TAMs stimulate angiogenesis. Thisreview will discuss the evidence for differential regulation ofTAMs in these microenvironments and provide an overview ofcurrent attempts to target or use TAMs for therapeuticpurposes. (Cancer Res 2006; 66(2): 605-12)

Introduction

Compelling evidence has emerged in recent years for macro-phages playing an important role in tumor cell invasion intosurrounding normal tissues, proliferation and survival, andmetastasis to local and distant sites. The onset and maintenanceof tumor angiogenesis also seems to be driven, in part, by thesecells. Macrophages are derived from CD34+ bone marrowprogenitors that continually proliferate and shed their progenyinto the bloodstream as promonocytes. They then develop intomonocytes and extravasate into tissues where they differentiateinto a specific type of resident tissue macrophage (1). Thephenotype of these fully differentiated, resident macrophages canvary markedly within tissues, from that of microglial cells in thebrain, Kupffer cells in the liver, and Langerhans cells in the skin.Despite these regional differences, resident macrophages share aset of common functions, including their ability to guard againstmicrobial infections, to regulate normal cell turnover and tissueremodeling, and to help repair sites of injury (1).

Macrophages also form a major component of the inflammatoryinfiltrate seen in both primary and secondary tumors (2), where, aswill be seen below, they also exhibit a distinct phenotype and aretermed tumor-associated macrophages (TAM). Monocytes entertumors through blood vessels throughout the life span of tumors,from early-stage tumor nodules that are beginning to vascularize tolate-stage tumors that are invasive and metastatic. A number of

tumor-derived chemoattractants are thought to ensure this ongoingrecruitment, including colony-stimulating factor-1 (CSF-1 alsoknown as M-CSF), the CC chemokines, CCL2, CCL3, CCL4, CCL5,and CCL8, and vascular endothelial growth factor (VEGF). Thelevels of many of these proteins in human tumors correlatepositively with the numbers of TAMs present in those tumors (3).

That these cells might help to drive tumor progression has beeninferred over a number of years from studies looking at the linkbetween TAM levels and prognosis. Although a few have correlatedhigh TAM numbers with good prognosis, the majority have linkedsuch numbers to reduced patient survival (Table 1). Indeed, highTAM numbers have been shown to be an independent prognosticfactor in many forms of cancer (2). These observations accord wellwith the results of animal studies using macrophage-depletedmice to investigate the role of macrophages in tumor progressionin vivo . For example, we used mice homozygous for a nullmutation in the CSF-1 gene to deplete macrophages in a mousemodel for breast cancer. In this model, mammary tumors areinitiated by the mammary epithelial restricted expression of thepolyoma virus middle T oncoprotein (PyMT). Macrophagedepletion resulted in slower progression of preinvasive lesions tomalignant lesions and reduced formation of lung metastases (4).Similarly, a recent study using small interfering RNA to inhibitCSF-1 expression by MCF-7 xenografts confirmed these findings,showing that lower numbers of TAMs are accompanied by amarked reduction in tumor growth and angiogenesis (5).

In this review, we will review how the general phenotype ofmacrophages in tumors differs from that seen in nonmalignanttissues, and then how their migration into distinct tumor sitesexposes them to different microenvironmental signals thateducate them to perform functions that are required by tumorcells in those areas. We will then discuss the possible therapeuticimplications of these and the ways in which TAMs could betargeted in new anti-inflammatory therapies.

TAM Phenotypes

TAMs have been studied in some detail with some studiesdescribing their phenotype as relatively immature, characterizedby low expression of the differentiation-associated macrophageantigens, carboxypeptidase M and CD51, high constitutive expres-sion of interleukin (IL)-1 and IL-6, and low expression of tumornecrosis factor-a (TNF-a; refs. 6, 7). However, it is not known yetwhether the expression of these markers by TAMs varies betweendifferent tumors and within specific areas of tumors.

Macrophages derived from healthy or inflamed tissues arecapable of lysing tumor cells, presenting tumor-associatedantigens to T cells, and expressing immunostimulatory cytokinesto stimulate the proliferation and antitumor functions of T cellsand natural killer (NK) cells in vitro . In stark contrast, macro-phages from experimental or human tumors show greatly reducedlevels of these activities, possibly due in part to their exposure to

Note: J.W. Pollard is the Sheldon and Betty E. Feinberg Senior Faculty Scholar inCancer Research.

Requests for reprints: Claire E. Lewis, Academic Unit of Pathology, Division ofGenomic Medicine, Floor E, The Henry Wellcome Laboratories for Medical Research,University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UnitedKingdom. Phone: 44-114-271-2903; Fax: 44-114-226-1464; E-mail: [email protected] American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-4005

www.aacrjournals.org 605 Cancer Res 2006; 66: (2). January 15, 2006

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such tumor-derived molecules as IL-4, IL-10, transforming growthfactor-h1 (TGF-h1), and prostaglandin E2 (PGE2; ref. 8). Moreover,Mantovani et al. (9) have proposed that exposure to IL-4 and IL-10in tumors may actually induce TAMs to develop into polarizedtype II (alternatively activated) or M2 macrophages. These cellshave poor antigen-presenting capability, produce factors thatsuppress T-cell proliferation and activity, and are generally betteradapted to scavenging debris, promoting angiogenesis, andrepairing and remodeling wounded/damaged tissues. This pheno-type contrasts markedly with the phenotype of classically activatedtype I or M1 macrophages that are efficient immune effector cellsable to kill microorganisms and tumor cells, present antigen, andproduce high levels of T-cell stimulatory cytokines (9).

Roles of TAM in Tumor Progression

Tumor invasion. In PyMT-induced mammary tumors, macro-phages are present in areas of basement membrane breakdownand invasion during the development of early-stage lesions(Fig. 1A ; ref. 4). This finding, together with other results showingthe up-regulation of proteolytic enzymes like cathepsin B inmacrophages present at the same locations (10), indicates thatTAMs could be involved in the invasion of tumor cells intosurrounding normal tissue. Indeed, Hagemann et al. (11) havereported that coculturing macrophages with tumor cells enhancestheir invasive properties in a manner dependent on TNF-a andmatrix metalloproteinases (MMP). Subsequently, it was shown inMatrigel invasion assays that TNF-a acted by stimulation of thec-Jun-NH2-kinase and nuclear factor-nB signaling pathways (12).Downstream targets included such proinvasive target genes asMIF and extracellular MMP inducer (EMMPRIN) in tumor cells(12). These proteins then act in turn to enhance the local releaseof MMPs by macrophages and possibly other stromal cells suchas fibroblasts (12, 13). In other coculture experiments, tumor cellinvasion into a collagen matrix was increased by macrophagesthat synthesized epidermal growth factor (EGF) in response totumor-derived CSF-1, leading to induction of several genes thatincreased directional movement and invasion of the tumor cells

(14). Further experiments are required to identify the relationship,if any, between EGF and TNF-a signaling in cross-talk betweenTAMs and tumor cells and to show whether TAMs modulatetumor cell invasion in the same manner in vivo .Tumor growth. TAM infiltration positively correlates with

tumor cell proliferation as measured by MIB-1 levels in breastcarcinomas (15), Ki67 levels in endometrial carcinomas (16), ormitotic index in renal cell carcinoma (17). Indeed, various studieshave shown that TAMs express a number of factors that stimulatetumor cell proliferation and survival, including EGF (14, 18),platelet-derived growth factor, TGF-h1, hepatocyte growth factor,and basic fibroblast growth factor (bFGF; Fig. 2; ref. 19).Accordingly, when cocultured with tumor cells, macrophagessecrete substances that stimulate tumor cell proliferation (20, 21).Moreover, macrophage depletion studies indicate that the presenceof TAMs is essential for the growth of various types of experimentaltumors in vivo (22). The production of such factors by macro-phages in wounds may also help to explain why some tumors(e.g., tumors induced by injections of the Rous sarcoma virus) formmore efficiently at sites of wounding or tissue injury. Thisphenomenon is known to involve factors like TGF-h1 beingexpressed by inflammatory cells in wounded tissues (23). However,it remains to be seen whether TGF-h1 and/or other moleculesspecifically expressed by macrophages in these sites are centrallyinvolved in the development of wound-induced tumors.

Other proteins secreted by TAMs have also been implicated intumor growth, such as MMP-9, which is essential for the growth ofhuman ovarian tumors in nudemice (24). However, these effectsmaybe indirect rather than direct via stimulation of tumor angiogenesis(see below). Also, the role of TAMs in tumor growth may varyby tumor type, because in CSF-1 knockout mice neither theincidence nor the early growth of PyMT-induced benign mammarytumors is adversely affected by macrophage depletion, althoughgrowth of more advanced carcinoma stages is impeded (4).3

Tumor angiogenesis. It is now widely accepted that the growthand spread of malignant tumors requires angiogenesis, the processby which new blood vessels sprout from the existing vasculature.Considerable evidence indicates that TAMs play an important part inregulating angiogenesis. The possibility that macrophages might becapable of modulating angiogenesis was first proposed by Sunder-kotter et al. (25) in 1991. We and others have since elaborated on thisto provide a detailed picture of the possible mechanisms used bymacrophages to regulate angiogenesis in tumors (Fig. 2; ref. 26).

TAMs release a number of potent proangiogenic cytokines andgrowth factors, such as VEGF, TNF-a, IL-8, and bFGF. Addition-ally, they express a broad array of angiogenesis-modulatingenzymes, including MMP-2, MMP-7, MMP-9, MMP-12, and cyclo-oxygenase-2 (COX-2; refs. 2527). TAM production of MMP-9 hasbeen shown to be crucial for angiogenesis in a mouse model ofhuman cervical carcinogenesis (estrogen-treated K14-HPV16female transgenic mice; ref. 28). Interestingly, as an importantsource of MMP-2 and MMP-9 in tumors, TAM may also contributeto the vascular normalization that occurs in tumors shortly aftertheir treatment with inhibitors of VEGF signaling (29). The manyproangiogenic functions of TAM may help explain reportedcorrelations between increased TAM numbers and high vasculargrades of many tumor types, including in breast carcinoma(22, 30), malignant uveal melanoma (31), glioma (32), squamous

3 E.Y. Lin and J.W. Pollard, unpublished results.

Table 1. High numbers of TAMs correlate with survival indifferent forms of cancer

Favorableprognosis

Reference Poorprognosis

Reference

Stomach (59) Breast*,c (15, 37)Colorectal (72) Prostate* (35)

Melanoma (73) Endometrial* (36)

Bladder*,c (34)Kidney* (17)

Esophageal

Superficialc (50)

Squamous cell carcinoma* (33)Malignant uveal melanoma* (31)

Follicular lymphoma (75)

*Correlation with increased tumor angiogenesis.

cCorrelation with increased involvement of local lymph nodes. Nocorrelation with survival was found in colon carcinoma (70), high-grade

astrocytomas (71), lung carcinoma (74), or cervical carcinoma (76).

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Cancer Res 2006; 66: (2). January 15, 2006 606 www.aacrjournals.org


cell carcinoma of the esophagus (33), bladder carcinoma (34), andprostate carcinoma (35).

In PyMT-induced mouse mammary tumors, macrophages arerecruited to premalignant lesions immediately before the angio-genic switch that presages the transition to malignancy. Depletionof these macrophages results in a f50% reduction in vasculardensity even in tumors that progress to advanced stages. Thiseffect is associated with increased necrosis as would be expectedby loss of blood supply. TAMs are sufficient to induce angio-genesis in this model because premature recruitment of macro-phages into benign hyperplastic lesions by CSF-1 overexpressionresults in accelerated vascularization of these normally poorlyvascularized lesions along with accelerated progression tomalignancy.4

Newly formed blood vessels in tumors are often disorganizedand prone to collapse, resulting in areas of inadequate perfusionand hypoxia (low oxygen tension). Rapid tumor cell proliferationalso outstrips the rate of new blood vessel growth, contributing tohypoxia in some areas. A number of studies have used hypoxic

cell markers, like pimonidazole, to highlight the presence of bothtransient (avascular and nonnecrotic) and chronic (perinecrotic)areas of hypoxia in human and experimental tumors. TAMsaccumulate in such hypoxic/necrotic areas in human endometrial,breast, prostate, and ovarian carcinomas (30, 3639). We havealso found TAMs to accumulate in such areas in PyMT-inducedmammary tumors (Fig. 1) and human prostate tumor xenografts(3). This pattern of TAM migration is possibly due to a number ofmechanisms, including the hypoxic induction of such macrophagechemoattractants as EMAPII, endothelin 2, and VEGF (as reviewedin ref. 3). Furthermore, as macrophages are phagocytes, they mayalso be attracted into hypoxic, perinecrotic areas along a trail ofnecrotic debris emanating from such areas. Hypoxia seems toinhibit macrophage migration, thereby immobilizing them in suchareas. Increased numbers of TAMs in these sites correlate withincreased lymph node involvement and/or poor prognosis inbreast cancer (37) and endometrial cancer (38). The increasedlevels of TAMs at such sites may promote tumor progression inpart by stimulating levels of angiogenesis, such as appears tooccur in breast carcinoma (37).

TAMs respond to tumor hypoxia by up-regulating the hypoxia-inducible transcription factors HIF-1 and HIF-2. Although both

Figure 1. Location of different TAM subpopulations in tumors. TAMs were visualized by staining fixed sections of spontaneous PyMT-induced murine mammary tumors(A, C, E , and G) or human breast carcinomas (B, D, F , and H ) using antibodies that specifically recognize the pan-macrophage markers F4/80 (murine) or CD86(human). TAMs are seen to gather in areas of tumor cell invasion where they help to degrade the basement membrane and facilitate the migration of tumor cells into thestroma of the surrounding tissue (A and B ), and in perivascular areas where they seem to promote metastasis by expressing factors like EGF (C and D). Othersubpopulations of TAMs are seen in hypoxic, perinecrotic (E and F ), and stromal (G and H) areas of these tumors where they promote angiogenesis and metastasis,respectively. Bar , 50 Am; V , blood vessel; N , necrosis.

4 E.Y. Lin, J. Li, L. Gnatovskiy, et al., unpublished results.

Macrophages in Different Tumor Microenvironments



factors are up-regulated by macrophages exposed to hypoxiain vitro as well as in hypoxic/necrotic areas of human tumors (40),their relative contribution to the regulation of gene expression byTAMs has yet to be fully elucidated. White et al. (41) usedadenoviral infection to overexpress HIF-2 in human macrophagesand found it to be the primary inducer of genes encodingangiogenic proteins in these cells. However, the repertoire of genesactivated by overexpression of each HIF may differ from those up-regulated in natural settings of hypoxia.

Macrophages also up-regulate VEGF and other proangiogenicfactors in response to hypoxia. TAMs express VEGF almostexclusively in avascular and perinecrotic areas of human breastcarcinomas (42). Hypoxic induction of this cytokine is largelydependent on HIF-1, at least in murine peritoneal macrophages(43). Macrophages also synthesize elevated levels of MMP-7 whenexposed to hypoxia in vitro and in avascular areas of humantumors (44). This multifunctional MMP has many substrates in theextracellular matrix and basement membrane, and is known tostimulate endothelial cell proliferation and migration, which cansupport tumor angiogenesis (45). A recent cDNA array study has

identified up-regulation of messages encoding >30 other proangio-genic genes in primary macrophages exposed to hypoxia, includingCXCL8, angiopoietin, COX-2 (the inducible form of nitric oxidesynthase), and other factors (41).

We found recently that when macrophages are coculturedin vitro with human tumor spheroids, they infiltrate deep into thecentral, hypoxic areas of these structures. The release of VEGF bymacrophages-infiltrated spheroids was significantly higher thanthat seen for noninfiltrated spheroids. This increase translated intoa significant stimulation of angiogenesis in vivo when spheroidswere implanted into microcirculation window chambers on theflanks of nude mice for 3 days (46).

A recent report has identified a subset of monocytes thatexpress the angiopoietin receptor Tie-2 as important inducers ofangiogenesis in both spontaneous and orthotopic tumor models(47). Knockout of these Tie-2-expressing cells in vivo markedlyreduced angiogenesis in human glioma xenografts and promptedsubstantial tumor regression. The authors of this study proposethat Tie-2-expressing monocytes/macrophages may account formost of the proangiogenic activity of myeloid cells that are

Figure 2. The roles of different subpopulations of TAMs in tumor progression. 1, invasion: TAMs secrete a variety of proteases to breakdown the basement membranearound areas of proliferating tumor cells (e.g., ductal carcinoma in situ in the breast), thereby prompting their escape into the surrounding stroma where theyshow deregulated growth. 2, angiogenesis: In areas of transient (avascular) and chronic (perinecrotic) tumor hypoxia, macrophages cooperate with tumor cells toinduce a vascular supply for the area by up-regulating a number of angiogenic growth factors and enzymes. These diffuse away from the hypoxic area and, together withother proangiogenic stimuli in the tumor microenvironment, stimulate endothelial cells in neighboring, vascularized areas to migrate, proliferate, and differentiateinto new vessels. 3, immunosuppression: Macrophages in hypoxic areas secrete factors that suppress the antitumor functions of immune effectors within the tumor.4, metastasis: A subpopulation of TAMs associated with tumor vessels secretes factors like EGF to guide tumor cells in the stroma toward blood vessels wherethey then escape into the circulation. In the stromal compartment (both the acellular regions and others where they are in close contact with tumor cells), TAMs secretegrowth factors to stimulate tumor cell division and/or undefined factors that promote tumor cell motility.

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present in tumors. In support of this proposal, we have notedthat a subset of CD14+ human peripheral blood monocytes alsoexpresses Tie-2.5

Hypoxic areas of tumors produced by inadequate vascularperfusion are likely to generate additional adverse conditions inthe tumor microenvironment, such as low pH and elevated levelsof lactate, which might stimulate proangiogenic gene expression inTAMs (48, 49). Moreover, as macrophages are likely to experience acombination (if not all) of these cellular stresses simultaneously inischemic areas of tumors, an investigation of their combined effecton macrophage function is clearly warranted.

An interesting recent paper by Ohno et al. (36) confirmed thathigh numbers of TAMs locating to areas of chronic hypoxia(i.e., necrosis) in endometrial carcinomas were associated withreduced relapse-free survival and increased myometrial invasion,as might be expected, but also that the close proximity of TAMsto tumor cell nests in the same tumors correlated with improvedsurvival. Furthermore, TAMs in the stromal compartment ofthese tumors correlated positively with lymph node metastasis.Together, these observations suggest that TAMs respond todifferent signals in different areas of a tumor, promoting tumorprogression in hypoxic/necrotic areas (although angiogenesiswas not examined per se in this study), facilitating metastasis instromal areas, and reducing tumor cell activity in areas whereTAMs make close contact with tumor cells. The identity of thesignals present in these three tumor zones regulating thesedifferent TAM functions have yet to be determined. However, inmodel systems, macrophage responses can be influenced throughcell-to-cell contact by the type of CSF-1 expressed. For example,in xenograft models of malignant glioma, mice died rapidly ifthey were engrafted with cells transfected with the secreted formof CSF-1 but survived if the cells expressed the cell surface-bound form of CSF-1. The survival of the mice was associatedwith macrophage binding to the glioma cells followed bysubsequent killing (50, 51).

Together, the data strongly suggest that TAMs are importantmodulators of angiogenesis, being involved both in the formationof new vessels and in their remodeling into a coherent functionalnetwork. They migrate into hypoxic/necrotic areas of the tumorwhere vascularization is urgently needed for tumor cell survival,and are then activated by local signals, like hypoxia, tosynthesize angiogenic regulators. This contributes to theformation of new vessels, facilitating local tumor growth andsurvival (Fig. 2).Metastasis. Mounting lines of evidence also implicate TAMs in

the regulation of metastasis (Fig. 2). High numbers of TAMs inprimary tumors have been correlated with early establishment ofmetastases in a number of tumor types (30, 34). TAMs seem to playroles in both the release of metastatic cells from the primarytumor as well as the establishment of secondary tumors at distantsites.

As early as 1982, Gorelik et al. (52) showed that inoculation ofthioglycollate-elicited peritoneal macrophages reduced the rate oflung clearance of several types of murine tumor cells injected i.v.and increased the formation of metastatic lung nodules by i.v.injected B16 melanoma or Lewis lung carcinoma cells. Further-more, in PyMT-induced murine mammary tumors as well as inxenografts of rat breast cancer cells, intravital imaging has

defined at least two ways that macrophages affect metastasis.First, the motility of tumor cells away from the main body of thetumor almost always occurs in juxtaposition to macrophages(which seem to attract tumor cells). Second, extravasion of tumorcells into blood vessels often occurs at clusters of macrophagesfound attached to the abluminal side of the vessels. Experimentsconducted in vitro and in vivo indicate that each of these celltypes exhibit coordinated movement (14, 53). This movementcritically requires CSF-1 and EGF signaling in the macrophagesand tumor cells respectively, with the ligands being produced bythe reciprocal cell type. Inhibition of either pathway blocks cellmovement of both cell types. Furthermore, the number of tumorcells entering the bloodstream is dramatically reduced withreduction of macrophage number along the vessels or inhibitionof EGF signaling.6 These observations accord with those of Ohnoet al. (36) whose findings show a correspondence between thenumber of macrophages in the tumor stromal compartment andthe metastatic potential of the tumor. The subpopulation of TAMseen in close proximity to some blood vessels in both murinemammary tumors and human breast carcinomas indicate thatthey may play a similar role in these tissues (Fig. 1, C and D).However, although macrophages isolated from human breasttumors express abundant EGF (15), this has yet to be shown inperivascular TAMs. Thus, it remains to be formally establishedwhether this subpopulation of TAMs is essential for metastasis.Interestingly, TAMs have also been shown to express the lym-phatic endothelial growth factor, VEGF-C, suggesting that theymay also promote dissemination of tumor cells by stimulatingthe formation of lymphatic vessels in tumors (54).

In mouse models of PyMT-induced mammary cancer, systemicdepletion of macrophages results in reduced formation of lungmetastases (4), suggesting that systemic macrophages or TAMsare important in the establishment of metastatic tumors. Theability of tumor cells to colonize and grow in the lungs isdependent on VEGF-induced expression of MMP-9 by alveolarmacrophages (and endothelial cells; ref. 55). Also, Oosterling et al.(56) also showed a link between macrophages in metastatic sitesand the growth of metastatic tumors. These investigatorsselectively depleted macrophages in the peritoneal cavity or liverand showed that CC531 colon tumor cells injected into either theperitoneum or liver portal vein formed tumors more slowly thanin control mice. Together, these findings suggest that macro-phages at metastatic sites support tumor growth, and accords wellwith clinical studies showing that increased numbers of macro-phages in regional lymph node metastases correlates with poorpatient survival (57).Immunosuppression. Unlike macrophages from healthy

tissues, which are capable of presenting tumor-associated antigens,lysing tumor cells, and stimulating the antitumor functions ofT cells and NK cells, TAMs in the tumor microenvironment lackthese activities, leaving the host without the ability to mount aneffective antitumor immune response. A number of studies haveshown that tumor-derived molecules, like cytokines, growthfactors, chemotactic molecules, and proteases, influence TAMfunctions (8, 25, 58). For example, tumor cells secrete IL-4, IL-6,IL-10, MDF, TGF-h1, and PGE2, which inhibit the cytotoxic activityof TAMs (8, 58). Moreover, TGF-h1, IL-10, and PGE2 may suppress

5 C. Murdoch and C.E. Lewis, unpublished observations. 6 J. Wyckoff, Y. Wang, E.Y. Lin, et al., unpublished results.




the expression of MHC class II molecules by macrophages in thetumor microenvironment as well as distant sites like the spleenand peritoneum. This effect may limit the ability of TAMs topresent tumor-associated antigens to T cells effectively in theseareas (8). Thus, tumors undergo immunoediting to down-regulatemacrophage functions that are potentially dangerous to the tumoreven in circumstances where recognizable tumor antigens arepresented. It should be noted that the tumoricidal activity of TAMsmay vary markedly in different tumor microenvironments. Forexample, in advanced gastric carcinoma, the number of TAMs inclose proximity to tumor cells in tumor cell nests correlatespositively with tumor cell apoptosis, suggesting that TAMs or TAMeffector cells are actively involved in tumor cell destruction.Notably, tumors that have large numbers of nest TAM areassociated with a significant increase in 10-year disease-freesurvival (59).

Another important aspect of TAM involvement in antitumorimmune mechanisms is the ability of these cells to releaseimmunostimulatory cytokines. For example, macrophage expres-sion of IL-12, a cytokine known to stimulate both the proliferationand cytotoxicity of T cells and NK cells (60), is markedlysuppressed in tumors, possibly by exposure to IL-10, PGE2, andTGF-h1 (8, 61). By coinjecting mice with human prostate tumorcells and macrophages engineered to overexpress IL-12, Satoh et al.(62) showed that it was possible to quickly reestablish enhancedexpression of MHC in TAMs along with increased T-cell infiltrationand an antitumor immune response, resulting in a markedreduction in the growth of both the primary tumor and lungmetastases.

Hypoxia in the tumor microenvironment is likely to contributeto suppressing the antitumor activity of TAMs as it stimulates therelease of the potent immunosuppressive factors PGE2 and IL-10.These factors impair the development of immune cells by actingon the early stages of their development from primitivepluripotent stem cells (i.e., immunopoiesis), inhibiting theantitumor activity of any immune cells that are formed (8, 63).Additionally, they act on TAMs to reduce their cytotoxicity activitytoward tumor cells (8, 63, 64). This may involve reduced oxygenradical formation due to decreased substrate availability.Hypoxia also inhibits the ability of macrophages to phagocytosedead or dying cells and present antigens to T cells. Onemechanism by which this may be achieved is by reduction inthe surface expression of CD80, a costimulatory molecule neededfor the full activation of T-cell responses to antigenic peptides.In contrast, hypoxia can also enhance the direct cytotoxicity ofmacrophages by up-regulating release of TNF-a. As mentionedabove, TAMs also up-regulate the expression of MMP-7 protein inhypoxic areas of tumors (44). MMP-7 is known to cleave the Fasligand from neighboring cells, making tumor cells not only lessresponsive to chemotherapeutic agents, such as doxorubicin (65),but also lysis by NK and T cells (66).

Conclusion

To summarize, analysis of the expression of protumor moleculesby TAMs in human tumors and functional studies in macrophage-depleted murine tumor models have shown TAMs to play animportant part in various key steps in tumor progression (Fig. 2). Itseems that in early preinvasive lesions, tumor cells releasechemokines to attract macrophages (and other inflammatorycells) into stromal areas surrounding the tumor. Macrophages are

also found in dense clusters as part of leukocytic infiltrates at areasof focal breakdown of the basement membrane. Tumor cells arethus able to escape the confines of the basement membranes andmigrate across into the stroma of surrounding healthy tissues,where they, together with macrophages, stimulate angiogenesis.These newly formed vessels sprout into the early tumor, providingnutrients and oxygen for tumor growth and supplying multiple exitroutes for metastasizing cells. The latter may involve a subpop-ulation of TAMs aligned with the outer surface of tumor-associatedblood vessels and cells lying in other regions of the stromalcompartment. These vessel-associated TAMs provide chemotacticsignals for tumor cells that promote migration and intravasationinto the blood and lymphatic vasculature. A separate subpopula-tion of TAMs also gather at sites distant from the vasculature,where the limited diffusion of oxygen (and possibly nutrients likeglucose) activates a quite different TAM phenotype that expressesa broad array of proangiogenic factors. This ensures therevascularization of each hypoxic tumor area and protects tumorcells in these from hypoxia-induced cell death. Growth factorssecreted by TAMs in these (and possibly other) areas of tumorsthen directly promote the proliferation of tumor cells. The conse-quence of these combined TAM-driven processes is progression ofthe tumor toward a more highly angiogenic, malignant phenotype.

Thus, it seems that at least four different microenvironmentssites of initial tumor cell invasion, perivascular sites, stromalregions, and hypoxic/necrotic areasmay educate macrophagesto carry out specific functions in support of tumor cell require-ments and activities in those different areas (67). These studiesreflect the pitfalls associated with the interpretation of early TAMstudies in which these cells were isolated from whole animal orhuman tumors and characterized as one phenotype in functionalin vitro assays.

However, apart from hypoxia, little is known of the micro-environmental signals that regulate the activities of subpopula-tions of TAMs in these different tumor sites. In vitro studies haveshown that CSF-1 synthesized locally by tumors stimulates theexpression of EGF by macrophages, causing a two-cell paracrineloop that promotes tumor cell motility (53). It is hoped that recentadvances in the use of laser microdissection to capture cells fromspecific areas of tissues sections (68) will now allow the isolation ofTAMs from such areas (and more specifically, the RNA or proteinsexpressed by these cells), so that their phenotype can be more fullycharacterized using DNA and/or protein microarrays. It is also notknown whether different TAM functions in such tumor sites reflectdifferent final differentiation states of these cells, or whether theyare entirely plastic and/or reversible, so that TAMs can express oneor more phenotypes in a temporal sequence, dependent only onthe presence or absence of local signals.

If such studies generate information on macrophage-specificsurface receptors uniquely expressed by TAMs in the mostaggressive areas of tumors (i.e., the hypoxic, stromal and/orperivascular areas, where TAMs drive angiogenesis and metastasis,respectively), then it might prove possible to target cytotoxictherapies to TAMs in these specific areas, leaving other antitumorsubpopulations of TAMs and macrophages in other tissuesrelatively unaffected. In addition, TAMs, through their phagocyticcapacity, might engulf liposomes carrying cytotoxic drugs andcarry them into the tumor, where, upon their death, these drugswould be released.

Because TAMs migrate into and accumulate in hypoxic areas, ithas been postulated that these cells might be used as vectors to

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carry genes or drugs activated by hypoxia into these sites. Thedevelopment of such a delivery system would be useful becausemost drugs or gene therapy vectors applied systemically fail topenetrate more than a few hundred micrometers from their pointsof entry via blood vessels, such that tumor cells in avascular tumorareas go largely untreated in most forms of therapy. Some limiteddata has been provided recently to suggest that such amacrophage-based system might work. Small breast tumorspheroids have been infiltrated with macrophages infected withadenovirus containing a hypoxia-driven prodrug-activating enzyme(cytochrome P450). When infiltrated, spheroids were then exposedto the corresponding prodrug, cyclophosphamide, the P450enzyme expressed by hypoxic macrophages within spheroidsconverted the prodrug into its active, cytotoxic metabolite(4-hydroxy-cycloposphamide). This cell membranepermeablecytotoxin then diffused from infected macrophages, intercalatedinto the DNA of surrounding tumor cells, causing cell death during

their subsequent mitosis. Because macrophages are terminallydifferentiated and thus nondividing cells, they are refractory to thecytotoxic effects of the active metabolite. As a result, spheroidsinfiltrated with P450-engineered macrophages displayed a signif-icant reduction in volume and gross morphologic damagecompared with mock-engineered controls (69). These results areencouraging but in vivo studies to test the efficacy of this newapproach for targeted gene delivery are now required.

Acknowledgments

Received 11/7/2005; accepted 11/9/2005.Grant support: Biotechnology and Biological Sciences Research Council, Medical

Research Council, UK, and the Yorkshire Cancer Research, UK (C.E. Lewis); andNational Cancer Institute grants CA 94173, CA 100324, and the Cancer Center Coregrant P30 CA 13330 (J.W. Pollard).

We thank Dr. Elaine Lin (Albert Einstein College of Medicine, Bronx, NY) forproviding tissues from the different stages of PyMT tumor progression seen in Fig. 1and Tatyana Harris for her help drawing Fig. 2.



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2006;66:605-612. Cancer Res

Claire E. Lewis and Jeffrey W. Pollard

MicroenvironmentsDistinct Role of Macrophages in Different Tumor

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