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Tumor-associated macrophages (TAMs) as new target in anticancer therapy
Drug Discovery Today: Therapeutic Strategies
Vol. 3, No. 3 2006
Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Inflammation
Tumor-associated macrophages (TAMs) as new target in anticancer therapy Alberto Mantovani1,2, Chiara Porta1, Luca Rubino1, Paola Allavena1, Antonio Sica1,* 1 2
Fondazione Humanitas per la Ricerca, Istituto Clinico Humanitas, 20089 Milan, Italy Institute of Pathology, State University of Milan, 20133 Milan, Italy
The proposed connection between inflammation and cancer has recently found new molecular confirmations suggesting that the inflammatory circuits
Section Editor: Martin Braddock – AstraZeneca R&D Charnwood, Loughborough, UK
expressed at the tumor site are potential targets of antitumor therapy. Tumor-associated macrophages (TAMs) represent the major component of the inflammatory infiltrate of tumors and several lines of evidence suggest that these cells express protumoral functions amenable of therapeutic intervention. These include TAM activation, recruitment, angiogenesis and survival. Introduction
TAMs are active players in the process of tumour progression and invasion. In several experimental tumour models, the activation of an inflammatory response (most frequently mediated by macrophages) is essential for full neoplastic transformation and progression [4]. Accordingly, in many but not all human tumors, a high frequency of infiltrating TAMs is associated to poor prognosis and genes associated to leukocyte or macrophage infiltration (e.g. CD68) are part of molecular signatures which herald poor prognosis in lymphomas and breast carcinoma [5]. The strategic location of TAMs also suggests that these cells are important regulators of anti-tumour immunity [6,7]. All together, these results support the idea that TAMs represent a therapeutic target in neoplastic diseases and characterization of the phenotype of TAMs appears to be essential to the understanding of tumour-derived signals guiding activation and polarisation of innate and adaptive immunity. Here, we discuss evidence supporting the view that TAM functions, including activation, recruitment, angiogenesis and survival, represent promising targets in anticancer therapy.
Experimental and clinical studies have revealed that chronic inflammation predisposes to different forms of cancer, including colon, prostate and liver cancer, and that usage of non-steroidal anti-inflammatory agents can protect against the emergence of various tumors. This evidence strongly supports the idea that cancers originate at sites of chronic inflammation and several efforts have been made to identify cellular and molecular links of such association. In the late 1970s, it was found that a major leukocyte population present in tumors, the so called tumor-associated macrophages (TAMs), promote tumor growth (reviewed in [1–4]). Over the years it has become increasingly clear that
Activation programs of TAMs
*Corresponding author: A. Sica ([email protected])
The capability to express distinct functional programs in response to different microenvironmental signals is a biological
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DOI: 10.1016/j.ddstr.2006.07.001
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feature of macrophages, which is typically manifested in pathological conditions such as infections and cancer [2,8– 10]. Chronic infections can tightly regulate the immune responses, being able to trigger highly polarized type I or type II inflammation and immunity. Classical or M1 macrophage activation in response to microbial products or Interferon-g are characterized by: high capacity to present antigen; high interleukin-12 (IL-12) and IL-23 production and consequent activation of a polarized type I response; and high production of toxic intermediates [nitric oxide (NO), reactive oxygen intermediates (ROI)]. Thus, M1 macrophages are generally considered potent effector cells that kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. By contrast, various signals (e.g. IL-4, IL-13, glucorticoids, IL-10, immunoglobulin complexes/ TLR ligands) elicit different M2 forms, able to tune inflammatory responses and adaptive Th2 immunity, scavenge debris, promote angiogenesis, tissue remodelling and repair [8]. Microenvironmental signals expressed at the tumor
Box 1. Role of NF-kB and HIF-1 in TAM biology NF-kB The nuclear factor-kB (NF-kB) family is a key player in controlling both innate and adaptive immunity [10]. NF-kB proteins are present in the cytoplasm in association with inhibitory proteins that are known as inhibitors of NF-kB (IkBs). After activation by different stimuli, the IkB proteins become phosphorylated, ubiquitylated and, subsequently, degraded by the proteasome. The degradation of IkB allows NF-kB proteins to translocate to the nucleus and bind their cognate DNA binding sites to regulate the transcription of a large number of genes, including antimicrobial peptides, cytokines, chemokines, stress-response proteins and anti-apoptotic proteins. Clinical evidence has long suggested that cancers arise at sites of chronic inflammation and this hypothesis has recently received molecular confirmations in inflammation-associated cancer models [11,12], which provide in vivo evidence supporting a causal relationship between NF-kB-mediated inflammation and tumorigenesis. NF-kB induces several cellular alterations associated with tumorigenesis and more aggressive phenotypes, including the following: self-sufficiency in growth signals; insensitivity to growth inhibition; resistance to apoptotic signals; immortalization; angiogenesis; tissue invasion and metastasis [13]. Constitutive NF-kB activation often observed in cancer cells may be promoted by either microenvironmental signals, including cytokines, hypoxia and ROI, or by genetic alterations [13]. In particular, proinflammatory cytokines (e.g. IL-1 and TNF), expressed by infiltrating leukocytes, can activate NF-kB in cancer cells and contribute to their proliferation and survival. These findings propose NF-kB as a possible target for development of anti-cancer treatments and clinical trials with drugs that block NF-kB are currently in progress with promising results [13]. To the extent, they have been investigated TAMs display defective NF-kB activation in response to different pro-inflammatory signals [14]. Detective NF-kB activation in TAMs correlates with impaired expression of NF-kBdependent inflammatory functions (e.g. expression of cytotoxic mediators, such as NO and cytokines, TNFa, IL-1 and IL-12) observed in these cells [2,14,15]. These observations were obtained in TAMs isolated from tumors characterized by advanced stages [14] and are in apparent contrast with a protumor function of inflammatory reactions and TAMs in particular [11,12]. This discrepancy may reflect a dynamic change of the tumor microenvironment during the transition from early neoplastic events toward advanced tumor stages, which would result in progressive modulation of NF-kB
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microenvironment have the capacity to pilot recruitment, maturation and differentiation of infiltrating leukocytes and play a central role in the activation of specific transcriptional programmes expressed by tumor-associated macrophages [8]. To the extent that they have been investigated, differentiated mature TAMs have phenotype and functions similar to type II or M2 macrophages. Analyses of the molecular basis of the TAM phenotype have identified the transcriptional factors NF-kB and HIF-1 as master regulators of their transcriptional programmes and indicate these factors as central regulators of tumor progression and metastasis. Box 1 reports main observations of NF-kB and HIF-1 as pivotal transcription factors driving the functional cross-talk between TAMs and cancer cells. Table 1 lists classes of NF-kB and HIF-1 inhibitors.
TAMs as a therapeutic target Activation Defective NF-kB activation in TAMs correlates with impaired expression of NF-kB-dependent inflammatory functions activity expressed by infiltrating inflammatory cells. While full activation of NF-kB in inflammatory leukocytes resident in preneoplastic sites may exacerbate local inflammation, thus favoring tumorigenesis, tumor growth may results in the progressive inhibition of NF-kB in infiltrating leukocytes, as observed in both myeloid [14,15] and lymphoid [16] cells associated with solid tumors. Additional studies are needed to clarify this part and to understand whether NF-kB activity may play different roles during different stages of tumor progression. HIF-1 Hypoxia is a common feature of solid tumors that has been associated with decreased therapeutic response, malignant progression, local invasion and distant metastasis [17]. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a major regulator of cell adaptation to hypoxic stress and therefore a potential target for anticancer therapies [17]. HIF-1 mediates switch from aerobic to anaerobic metabolism thus conferring a glycolitic phenotype to cancer cells and ensuring their energy requirements, thereby allowing their survival in a hostile environment. TAMs accumulate preferentially in the poorly vascularized region of tumors which are characterized by low oxygen tension [18]. Such environment promotes TAM adaptation to hypoxia, which is achieved by the increased expression of hypoxia inducible and pro-angiogenic genes, such as VEGF, bFGF and CXCL8, as well as glycolitic enzymes, whose transcription is controlled by the transcription factors HIF-1 and HIF-2 [18,19]. The in vivo relevance of this metabolic adaptation to hypoxia by macrophages was recently demonstrated by Cramer et al. [20]. Ablation of the hypoxia responsive transcription factor HIF-1a resulted in impaired macrophage motility and citotoxicity, in low oxygen conditions. This evidence highlights the relevance that the hypoxia-HIF-1 pathway may play in the recruitment and activation of TAMs into solid tumors and may be instrumental for TAMmediated angiogenesis and tumor metastasis. In support of this, we have recently described that hypoxia can influence the positioning and function of cancer and stromal cells, including TAMs, by selectively upregulating expression of the chemokine receptor CXCR4 [21]. Moreover, a recent work has shown that HIF-1 activation may play a role in the induction of the CXCR4 ligand, CXCL12 [22], a chemokine involved in cancer metastasis [23]. Similarly to NF-kB, inhibition of HIF-1a is considered a promising therapeutic approach against cancer and in fact some of its inhibitors (e.g. farnesyl transferase inhibitors, PI3K inhibitors) are now in clinical trials as antitumor drugs [17].
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Table 1. Agent(s)
Molecular target(s)
Classes of NF-kB inhibitors [13] -Anti-oxidants (e.g. N-Acetyl-L-cysteine; Vitamin C) -Proteasome inhibitors (e.g. bortezomib) -Non-steroidal anti-inflammatory drugs (NSAIDs; e.g. aspirin, salicylates) -IKK inhibitors (e.g. Indolecarboxamide derivatives; quinazoline analogs) -Miscellaneous inhibitors of NF-kB (e.g. Thalidomide, prostaglandin metabolites)
IkBa phosphorylation IkB degradation Non-specific IKK inhibitors IKK activity IkBa degradation
Inhibitors of HIF-1 activity [17] -2ME2 -17-AAG -Camptothecin, Topotecan -Pleurotin, 1-methylpropyl; 2-imidazolyl disulphide -YC-1
Microtubule polymerization HSP90 Topoisomerase I Thioredoxin 1 Non-determined
(e.g. expression of cytotoxic mediators, NO) and cytokines, including Tumor-Necrosis Factor (TNFa), IL-1 and IL-12 [2,14,15]. Restoration of NF-kB activity in TAMs is therefore a potential strategy to restore M1 inflammation and intratumoral cytotoxicity. In agreement, recent evidence indicate that restoration of an M1 phenotype in TAMs may provide therapeutic benefit in tumor bearing mice. In particular, combination of CpG plus an anti-IL-10 receptor antibody switched infiltrating macrophages from M2 to M1 and triggered innate response debulking large tumors within 16 h [24]. It is likely that this treatment may restore NF-kB activation and inflammatory functions by TAMs. Moreover, TAMs from STAT6 / tumor bearing mice display an M1 phenotype, with low level of arginase and high level of NO. As a result, these mice immunologically rejected spontaneous mammary carcinoma [25]. These data suggest that switching the TAM phenotype from M2 to M1 during tumor progression may promote antitumor activities. In this regard, the SHIP1 phosphatase was shown to play a critical role in programming macrophage M1 versus M2 functions. Mice deficient for SHIP1 display a skewed development away from M1 macrophages (which have high inducible nitric oxide synthase levels and produce NO), toward M2 macrophages (which have high arginase levels and produce ornithine) [26]. Finally, recent reports have identified a myeloid M2-biased cell population in lymphoid organs and peripheral tissues of tumor-bearing hosts, referred to as the myeloid suppressor cells (MSC), which are suggested to contribute to the immunosuppressive phenotype [27]. These cells are phenotipically distinct from TAMs and are characterized by the expression of the Gr-1 and CD11b markers. MSC use two enzymes involved in the arginine metabolism to control T cell response: inducile nitric oxide synthase and arginase 1, which deplete the milieau of arginine, causing peroxinitrite generation, as well as lack of CD3z chain expression and T cell apoptosis. In prostate cancer, selective antagonists of these two enzymes were proved beneficial in restoring T cell-mediated cytotoxicity [27].
Recruitment Chemokines and chemokine receptors are a prime target for the develoment of innovative therapeutic strategies in the control of inflammatory disorders. Recent results suggest that chemokine inhibitors could affect tumour growth by reducing macrophage infiltration. Preliminary results in MCP-1/ CCL2 gene targeted mice suggest that this chemokine can indeed promote progression in a Her2/neu-driven spontaneous mammary carcinoma model [28]. Thus, available information suggests that chemokines represent a valuable therapeutic target in neoplasia. CSF-1 was identified as an important regulator of mammary tumor progression to metastasis, by regulating infiltration and function of TAMs. Transgenic expression of CSF-1 in mammary epithelium led to the acceleration of the late stages of carcinoma and increased lung metastasis, suggesting that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects [6,29]. Recent results have shed new light on the links between certain TAM chemokines and genetic events that cause cancer. The CXCR4 receptor lies downstream of the vonHippel/Lindau/hypoxiainducible factor (HIF) axis. Transfer of activated ras into a cervical carcinoma line, HeLa, induces IL-8/CXCL8 production that is sufficient to promote angiogenesis and progression. Moreover, a frequent early and sufficient gene rearrangement that causes papillary thyroid carcinoma (Ret-PTC) activates an inflammatory genetic program that includes CXCR4 and inflammatory chemokines in primary human thyrocytes [30]. The emerging direct connections between oncogenes, inflammatory mediators and the chemokine system provide a strong impetus for exploration of the anticancer potential of anti-inflammatory strategies. It was further demonstrated in non-small cell lung cancer (NSCLC) that mutation of the tumor suppressor gene PTEN results in upregulation of HIF-1 activity and ultimately in HIF-1-dependent transcription of the CXCR4 gene, which provides a mechanistic basis for the upregulation of CXCR4 expression and promotion of metastasis formation [31]. It appears therefore that targeting HIF-1 activity may disrupt www.drugdiscoverytoday.com
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the HIF-1/CXCR4 pathway and affect TAM accumulation, as well as cancer cell spreading and survival. The HIF-1-inducible vascular endothelial growth factor (VEGF) is commonly produced by tumors and elicits monocyte migration. There is evidence that VEGF can significantly contribute to macrophage recruitment in tumors. Along with CSF-1, this molecule also promotes macrophage survival and proliferation. Because of the localization of TAMs into the hypoxic regions of tumours, viral vectors were used to transduce macrophages with therapeutic genes, such as IFNg, that were activated only in low oxygen conditions [32]. These works present promising approaches which use macrophages as vehicles to deliver gene therapy in regions of tumor hypoxia.
Angiogenesis VEGF is a potent angiogenic factor as well as a monocyte attractant that contributes to TAM recruitment. TAMs promote angiogenesis and there is evidence that inhibition of TAM recruitment plays an important role in anti-angiogenic strategies. We found that in addition to VEGF, the angiogenic program established by hypoxia may rely also on the increased expression of CXCR4 by TAMs and endothelial cells [21]. Intratumoral injection of CXCR4 antagonists, such as the the bicyclam AMD3100, may work as in vivo inhibitors of tumor angiogenesis. Box 2 lists new CXCR4 antagonists [33]. Linomide, an anti-angiogenic agent, caused significant reduction of the tumour volume, in a murine prostate cancer model, by inhibiting both recruitment and the stimulatory effects of TAMs on tumour angiogenesis [34]. On the basis of this, the effects of Linomide, or other anti-angiogenic drugs, on the expression of pro- and anti-angiogenic molecules by TAMs may be considered valuable targets for anticancer therapy.
Survival Anti-tumor agents with selective cytotoxic activity on monocyte-macrophages would be ideal therapeutic tools for their combined action on tumor cells and TAMs. We recently reported that Yondelis (Trabectedin), a natural product derived from the marine organism Ecteinascidia turbinata, with potent anti-tumor activity [35] is specifically cytotoxic to macrophages and TAMs, while sparing the lymphocyte sub-set. This compound inhibits NF-Y, a transcription factor of major importance for mononuclear phagocyte differentia-
Box 2. CXCR4 antagonists [33] -
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Small-molecule bicyclams (e.g. AMD3100, AMD1498) ALX40-4C (9 D-Amino acid polycationic peptide) T22 (18 Amino acid peptide; polyphemusin) T140 (14 Amino acid peptide) CGP64222 (9 Amino acid basic peptide) KRH-1636 (Low molecular weight molecule)
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tion. In addition, Yondelis inhibits the production of CCL2 and IL-6 both by TAM and tumor cells [36]. These antiinflammatory properties of Yondelis may be an extended mechanism of its anti-tumor activity. Finally, proinflammatory cytokines (e.g. IL-1 and TNF), expressed by infiltrating leukocytes, can activate NF-kB in cancer cells and contribute to their proliferation, survival and metastasis (1,4), thus representing potential anticancer targets.
Matrix remodelling TAMs produce several matrix-metalloproteases (e.g. MMP2, MMP9) which degrade proteins of the extra-cellular matrix and also produce activators of MMPs, such as chemokines [7]. Inhibition of this molecular pattern may prevent degradation of extracellular matrix, as well as tumor cell invasion and migration. The biphosphonate zoledronic acid is a prototipical MMP inhibitor. In cervical cancer, this compound suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing angiogenesis and cervical carcinogenesis [37]. The halogenated bisphosphonate derivative chlodronate is a macrophage toxin which depletes selected macrophage populations. Given the current clinical usage of this and similar agents it will be important to assess whether they have potential as TAM toxins. The secreted protein acidic and rich in cysteine (SPARC) has gained much interest in cancer, being either up-regulated or down-regulated in progressing tumors. SPARC produced by macrophages present in tumor stroma can modulate collagen density, leukocyte and blood vessel infiltration [38].
Effector molecules Cyclooxygenase (COX) is a key enzyme in the prostanoid biosynthetic pathway. COX-2 not only is up regulated by activated oncogenes (i.e., ß-catenin, MET) but is also produced by TAMs in response to tumor-derived factors like mucin in the case of colon cancer. The usage of COX-2 inhibitors in the form of nonsteroidal antiinflammatory drugs is associated with reduced risk of diverse tumors (colorectal, oesophagus, lung, stomach and ovary). Selective COX2 inhibitors are now thought as a part of combination therapy [39]. The IFN-g-inducible enzyme indoleamine 2,3-dioxygenase is a well-known suppressor of T cell activation. It catalyzes the initial rate-limiting step in tryptophan catabolism, which leads to the biosynthesis of nicotinamide adenine dinucleotide. By depleting tryptophan from local microenvironment, indoleamine 2,3-dioxygenase (IDO) blocks activation of T lymphocytes. Recently, it was shown that inhibition of IDO may cooperate with cytotoxic agents to elicit regression of established tumors and may increase the efficacy of cancer immunotherapy [40]. Fig.1 summarizes therapeutic approaches to prevent TAM protumoral functions.
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Figure 1. Multifaceted therapeutic approaches to prevent TAM protumoral functions. TAMs promote tumor progression by favoring angiogenesis, suppression of adaptive immunity, matrix remodeling, tumor progression and metastasis. The figure summarizes strategies impairing selective TAM protumoral functions ( ) or restoring antitumor activities (+). Cytotoxic drugs (e.g. Yondelis) may decrease TAM number and prevent protumoral functions. A similar result may be obtained by limiting TAM recruitment (Linomide). Restoration of M1 immunity (STAT-3 and -6 inhibitors; anti-IL-10 plus CpG; IDO inhibitors) would provide cytotoxic activity and re-activation of Th1 specific antitumor immunity. Inhibition of both pro-inflammatory cytokines and growth factors expression (NF-kB inhibitors) may disrupt inflammatory circuits supporting tumor growth and progression. MMP inhibitors would prevent cancer cell spread and metastasis. Finally, inhibitors of TAM-mediated angiogenesis (HIF-1 inhibitors) would restrain blood supply and inhibit tumor growth. M-CSF (Macrophage-Colony Stimulating Factor); VEGF (Vascular Endothelial Growth Factor); CSFs (Colony-Stimulating Factors); IL- (Interleukin); TGF-b (Transforming Growth Factor-b); IDO (indoleamine 2,3-dioxygenase); MMP inhibitors (Matrix metalloproteinase inhibitors); TLR agonists (TollLike Receptor agonists); STAT (signal transducer and activator of transcription); NF-kB (nuclear factor-kB).
Concluding remarks Macrophages are key cell components of the inflammatory reactions expressed at the tumor site. Epidemiological and experimental evidence show that TAM express pro-tumoral functions, suggesting these cells as valuable target for novel anticancer strategies. Indeed, increased number and density of TAMs correlate with poor prognosis in different human cancers, while experimental evidence, ranging from adoptive transfer of cells to genetic manipulations, suggests that myelomonocytic cells can promote tumor invasion and metastasis. Box 3 indicates experimental approaches used to inhibit TAM functions. Box 4 summarizes the general status of knowledge on TAM biology and points out additional aspects to be addressed. Several studies have displayed key molecules and pathways driving recruitment and activation of TAMs and, more recently, the TAM transcriptome was provided in a murine fibrosarcoma [10]. Thus, a global effort is undergoing to fully characterize the molecular mechanisms supporting
TAM-mediated tumor progression. Despite these efforts, TAM functions have been significantly characterized only in animal models, and up today their phenotypic characterization remains only partial in human cancers. Moreover, it is still unknown whether different tumor microenvironments, likely established by different tumor types, may drive different functional phenotypes of TAM and contribute to specific pro- or anti-tumor activities. Along with TAM recruitment, activation Box 3. Anticancer strategies targeting TAM functions - Macrophage transduction with therapeutic genes (e.g. IFN-g) - Antisense oligonucleotides against CSF-1 (prevent recruitment and survival of TAMs) - Yondelis-Trabectedin (Cytotoxic drugs to myelomonocytic cells) - Linomide (prevents recruitment and angiogenic activity by TAMs) - Biphosphonate zoledronic acid (MMP inhibitor) - Anti-IL-10 plus CpG (restoration of M1 innate immunity) - Methyl-thiohydantoin-tryptophan (prevents the immunosuppressive activity of IDO)
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Box 4. Established and required knowledge on TAM biology Established Epidemiological evidence: correlation between TAM number and vessel density and prognosis. Pro-tumoral functions expressed by TAMs: angiogenesis, production of growth factors for tumor cells and blood vessels, matrix remodelling, immune suppression. To be provided Definitive in vivo demonstration of TAM protumoral functions in human tumors. Characterization of possible heterogeneity of TAM phenotype in different tumors. Definition of therapeutic strategies targeting TAMs in human neoplasia.
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and polarization mechanisms, the functional hetereogenicity of TAM should be viewed as an additional level of investigation to develop innovative anticancer strategies.
Acknowledgements
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This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC), Italy; by European Community (DC-Thera integrated project, number: LSHB-CT-2004-512074) and by Ministero Istruzione Universita` Ricerca (MIUR), Italy; Istituto Superiore Sanita’ (ISS).
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Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Inflammation
Tumor-associated macrophages (TAMs) as new target in anticancer therapy Alberto Mantovani1,2, Chiara Porta1, Luca Rubino1, Paola Allavena1, Antonio Sica1,* 1 2
Fondazione Humanitas per la Ricerca, Istituto Clinico Humanitas, 20089 Milan, Italy Institute of Pathology, State University of Milan, 20133 Milan, Italy
The proposed connection between inflammation and cancer has recently found new molecular confirmations suggesting that the inflammatory circuits
Section Editor: Martin Braddock – AstraZeneca R&D Charnwood, Loughborough, UK
expressed at the tumor site are potential targets of antitumor therapy. Tumor-associated macrophages (TAMs) represent the major component of the inflammatory infiltrate of tumors and several lines of evidence suggest that these cells express protumoral functions amenable of therapeutic intervention. These include TAM activation, recruitment, angiogenesis and survival. Introduction
TAMs are active players in the process of tumour progression and invasion. In several experimental tumour models, the activation of an inflammatory response (most frequently mediated by macrophages) is essential for full neoplastic transformation and progression [4]. Accordingly, in many but not all human tumors, a high frequency of infiltrating TAMs is associated to poor prognosis and genes associated to leukocyte or macrophage infiltration (e.g. CD68) are part of molecular signatures which herald poor prognosis in lymphomas and breast carcinoma [5]. The strategic location of TAMs also suggests that these cells are important regulators of anti-tumour immunity [6,7]. All together, these results support the idea that TAMs represent a therapeutic target in neoplastic diseases and characterization of the phenotype of TAMs appears to be essential to the understanding of tumour-derived signals guiding activation and polarisation of innate and adaptive immunity. Here, we discuss evidence supporting the view that TAM functions, including activation, recruitment, angiogenesis and survival, represent promising targets in anticancer therapy.
Experimental and clinical studies have revealed that chronic inflammation predisposes to different forms of cancer, including colon, prostate and liver cancer, and that usage of non-steroidal anti-inflammatory agents can protect against the emergence of various tumors. This evidence strongly supports the idea that cancers originate at sites of chronic inflammation and several efforts have been made to identify cellular and molecular links of such association. In the late 1970s, it was found that a major leukocyte population present in tumors, the so called tumor-associated macrophages (TAMs), promote tumor growth (reviewed in [1–4]). Over the years it has become increasingly clear that
Activation programs of TAMs
*Corresponding author: A. Sica ([email protected])
The capability to express distinct functional programs in response to different microenvironmental signals is a biological
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DOI: 10.1016/j.ddstr.2006.07.001
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feature of macrophages, which is typically manifested in pathological conditions such as infections and cancer [2,8– 10]. Chronic infections can tightly regulate the immune responses, being able to trigger highly polarized type I or type II inflammation and immunity. Classical or M1 macrophage activation in response to microbial products or Interferon-g are characterized by: high capacity to present antigen; high interleukin-12 (IL-12) and IL-23 production and consequent activation of a polarized type I response; and high production of toxic intermediates [nitric oxide (NO), reactive oxygen intermediates (ROI)]. Thus, M1 macrophages are generally considered potent effector cells that kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. By contrast, various signals (e.g. IL-4, IL-13, glucorticoids, IL-10, immunoglobulin complexes/ TLR ligands) elicit different M2 forms, able to tune inflammatory responses and adaptive Th2 immunity, scavenge debris, promote angiogenesis, tissue remodelling and repair [8]. Microenvironmental signals expressed at the tumor
Box 1. Role of NF-kB and HIF-1 in TAM biology NF-kB The nuclear factor-kB (NF-kB) family is a key player in controlling both innate and adaptive immunity [10]. NF-kB proteins are present in the cytoplasm in association with inhibitory proteins that are known as inhibitors of NF-kB (IkBs). After activation by different stimuli, the IkB proteins become phosphorylated, ubiquitylated and, subsequently, degraded by the proteasome. The degradation of IkB allows NF-kB proteins to translocate to the nucleus and bind their cognate DNA binding sites to regulate the transcription of a large number of genes, including antimicrobial peptides, cytokines, chemokines, stress-response proteins and anti-apoptotic proteins. Clinical evidence has long suggested that cancers arise at sites of chronic inflammation and this hypothesis has recently received molecular confirmations in inflammation-associated cancer models [11,12], which provide in vivo evidence supporting a causal relationship between NF-kB-mediated inflammation and tumorigenesis. NF-kB induces several cellular alterations associated with tumorigenesis and more aggressive phenotypes, including the following: self-sufficiency in growth signals; insensitivity to growth inhibition; resistance to apoptotic signals; immortalization; angiogenesis; tissue invasion and metastasis [13]. Constitutive NF-kB activation often observed in cancer cells may be promoted by either microenvironmental signals, including cytokines, hypoxia and ROI, or by genetic alterations [13]. In particular, proinflammatory cytokines (e.g. IL-1 and TNF), expressed by infiltrating leukocytes, can activate NF-kB in cancer cells and contribute to their proliferation and survival. These findings propose NF-kB as a possible target for development of anti-cancer treatments and clinical trials with drugs that block NF-kB are currently in progress with promising results [13]. To the extent, they have been investigated TAMs display defective NF-kB activation in response to different pro-inflammatory signals [14]. Detective NF-kB activation in TAMs correlates with impaired expression of NF-kBdependent inflammatory functions (e.g. expression of cytotoxic mediators, such as NO and cytokines, TNFa, IL-1 and IL-12) observed in these cells [2,14,15]. These observations were obtained in TAMs isolated from tumors characterized by advanced stages [14] and are in apparent contrast with a protumor function of inflammatory reactions and TAMs in particular [11,12]. This discrepancy may reflect a dynamic change of the tumor microenvironment during the transition from early neoplastic events toward advanced tumor stages, which would result in progressive modulation of NF-kB
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microenvironment have the capacity to pilot recruitment, maturation and differentiation of infiltrating leukocytes and play a central role in the activation of specific transcriptional programmes expressed by tumor-associated macrophages [8]. To the extent that they have been investigated, differentiated mature TAMs have phenotype and functions similar to type II or M2 macrophages. Analyses of the molecular basis of the TAM phenotype have identified the transcriptional factors NF-kB and HIF-1 as master regulators of their transcriptional programmes and indicate these factors as central regulators of tumor progression and metastasis. Box 1 reports main observations of NF-kB and HIF-1 as pivotal transcription factors driving the functional cross-talk between TAMs and cancer cells. Table 1 lists classes of NF-kB and HIF-1 inhibitors.
TAMs as a therapeutic target Activation Defective NF-kB activation in TAMs correlates with impaired expression of NF-kB-dependent inflammatory functions activity expressed by infiltrating inflammatory cells. While full activation of NF-kB in inflammatory leukocytes resident in preneoplastic sites may exacerbate local inflammation, thus favoring tumorigenesis, tumor growth may results in the progressive inhibition of NF-kB in infiltrating leukocytes, as observed in both myeloid [14,15] and lymphoid [16] cells associated with solid tumors. Additional studies are needed to clarify this part and to understand whether NF-kB activity may play different roles during different stages of tumor progression. HIF-1 Hypoxia is a common feature of solid tumors that has been associated with decreased therapeutic response, malignant progression, local invasion and distant metastasis [17]. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a major regulator of cell adaptation to hypoxic stress and therefore a potential target for anticancer therapies [17]. HIF-1 mediates switch from aerobic to anaerobic metabolism thus conferring a glycolitic phenotype to cancer cells and ensuring their energy requirements, thereby allowing their survival in a hostile environment. TAMs accumulate preferentially in the poorly vascularized region of tumors which are characterized by low oxygen tension [18]. Such environment promotes TAM adaptation to hypoxia, which is achieved by the increased expression of hypoxia inducible and pro-angiogenic genes, such as VEGF, bFGF and CXCL8, as well as glycolitic enzymes, whose transcription is controlled by the transcription factors HIF-1 and HIF-2 [18,19]. The in vivo relevance of this metabolic adaptation to hypoxia by macrophages was recently demonstrated by Cramer et al. [20]. Ablation of the hypoxia responsive transcription factor HIF-1a resulted in impaired macrophage motility and citotoxicity, in low oxygen conditions. This evidence highlights the relevance that the hypoxia-HIF-1 pathway may play in the recruitment and activation of TAMs into solid tumors and may be instrumental for TAMmediated angiogenesis and tumor metastasis. In support of this, we have recently described that hypoxia can influence the positioning and function of cancer and stromal cells, including TAMs, by selectively upregulating expression of the chemokine receptor CXCR4 [21]. Moreover, a recent work has shown that HIF-1 activation may play a role in the induction of the CXCR4 ligand, CXCL12 [22], a chemokine involved in cancer metastasis [23]. Similarly to NF-kB, inhibition of HIF-1a is considered a promising therapeutic approach against cancer and in fact some of its inhibitors (e.g. farnesyl transferase inhibitors, PI3K inhibitors) are now in clinical trials as antitumor drugs [17].
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Table 1. Agent(s)
Molecular target(s)
Classes of NF-kB inhibitors [13] -Anti-oxidants (e.g. N-Acetyl-L-cysteine; Vitamin C) -Proteasome inhibitors (e.g. bortezomib) -Non-steroidal anti-inflammatory drugs (NSAIDs; e.g. aspirin, salicylates) -IKK inhibitors (e.g. Indolecarboxamide derivatives; quinazoline analogs) -Miscellaneous inhibitors of NF-kB (e.g. Thalidomide, prostaglandin metabolites)
IkBa phosphorylation IkB degradation Non-specific IKK inhibitors IKK activity IkBa degradation
Inhibitors of HIF-1 activity [17] -2ME2 -17-AAG -Camptothecin, Topotecan -Pleurotin, 1-methylpropyl; 2-imidazolyl disulphide -YC-1
Microtubule polymerization HSP90 Topoisomerase I Thioredoxin 1 Non-determined
(e.g. expression of cytotoxic mediators, NO) and cytokines, including Tumor-Necrosis Factor (TNFa), IL-1 and IL-12 [2,14,15]. Restoration of NF-kB activity in TAMs is therefore a potential strategy to restore M1 inflammation and intratumoral cytotoxicity. In agreement, recent evidence indicate that restoration of an M1 phenotype in TAMs may provide therapeutic benefit in tumor bearing mice. In particular, combination of CpG plus an anti-IL-10 receptor antibody switched infiltrating macrophages from M2 to M1 and triggered innate response debulking large tumors within 16 h [24]. It is likely that this treatment may restore NF-kB activation and inflammatory functions by TAMs. Moreover, TAMs from STAT6 / tumor bearing mice display an M1 phenotype, with low level of arginase and high level of NO. As a result, these mice immunologically rejected spontaneous mammary carcinoma [25]. These data suggest that switching the TAM phenotype from M2 to M1 during tumor progression may promote antitumor activities. In this regard, the SHIP1 phosphatase was shown to play a critical role in programming macrophage M1 versus M2 functions. Mice deficient for SHIP1 display a skewed development away from M1 macrophages (which have high inducible nitric oxide synthase levels and produce NO), toward M2 macrophages (which have high arginase levels and produce ornithine) [26]. Finally, recent reports have identified a myeloid M2-biased cell population in lymphoid organs and peripheral tissues of tumor-bearing hosts, referred to as the myeloid suppressor cells (MSC), which are suggested to contribute to the immunosuppressive phenotype [27]. These cells are phenotipically distinct from TAMs and are characterized by the expression of the Gr-1 and CD11b markers. MSC use two enzymes involved in the arginine metabolism to control T cell response: inducile nitric oxide synthase and arginase 1, which deplete the milieau of arginine, causing peroxinitrite generation, as well as lack of CD3z chain expression and T cell apoptosis. In prostate cancer, selective antagonists of these two enzymes were proved beneficial in restoring T cell-mediated cytotoxicity [27].
Recruitment Chemokines and chemokine receptors are a prime target for the develoment of innovative therapeutic strategies in the control of inflammatory disorders. Recent results suggest that chemokine inhibitors could affect tumour growth by reducing macrophage infiltration. Preliminary results in MCP-1/ CCL2 gene targeted mice suggest that this chemokine can indeed promote progression in a Her2/neu-driven spontaneous mammary carcinoma model [28]. Thus, available information suggests that chemokines represent a valuable therapeutic target in neoplasia. CSF-1 was identified as an important regulator of mammary tumor progression to metastasis, by regulating infiltration and function of TAMs. Transgenic expression of CSF-1 in mammary epithelium led to the acceleration of the late stages of carcinoma and increased lung metastasis, suggesting that agents directed at CSF-1/CSF-1R activity could have important therapeutic effects [6,29]. Recent results have shed new light on the links between certain TAM chemokines and genetic events that cause cancer. The CXCR4 receptor lies downstream of the vonHippel/Lindau/hypoxiainducible factor (HIF) axis. Transfer of activated ras into a cervical carcinoma line, HeLa, induces IL-8/CXCL8 production that is sufficient to promote angiogenesis and progression. Moreover, a frequent early and sufficient gene rearrangement that causes papillary thyroid carcinoma (Ret-PTC) activates an inflammatory genetic program that includes CXCR4 and inflammatory chemokines in primary human thyrocytes [30]. The emerging direct connections between oncogenes, inflammatory mediators and the chemokine system provide a strong impetus for exploration of the anticancer potential of anti-inflammatory strategies. It was further demonstrated in non-small cell lung cancer (NSCLC) that mutation of the tumor suppressor gene PTEN results in upregulation of HIF-1 activity and ultimately in HIF-1-dependent transcription of the CXCR4 gene, which provides a mechanistic basis for the upregulation of CXCR4 expression and promotion of metastasis formation [31]. It appears therefore that targeting HIF-1 activity may disrupt www.drugdiscoverytoday.com
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the HIF-1/CXCR4 pathway and affect TAM accumulation, as well as cancer cell spreading and survival. The HIF-1-inducible vascular endothelial growth factor (VEGF) is commonly produced by tumors and elicits monocyte migration. There is evidence that VEGF can significantly contribute to macrophage recruitment in tumors. Along with CSF-1, this molecule also promotes macrophage survival and proliferation. Because of the localization of TAMs into the hypoxic regions of tumours, viral vectors were used to transduce macrophages with therapeutic genes, such as IFNg, that were activated only in low oxygen conditions [32]. These works present promising approaches which use macrophages as vehicles to deliver gene therapy in regions of tumor hypoxia.
Angiogenesis VEGF is a potent angiogenic factor as well as a monocyte attractant that contributes to TAM recruitment. TAMs promote angiogenesis and there is evidence that inhibition of TAM recruitment plays an important role in anti-angiogenic strategies. We found that in addition to VEGF, the angiogenic program established by hypoxia may rely also on the increased expression of CXCR4 by TAMs and endothelial cells [21]. Intratumoral injection of CXCR4 antagonists, such as the the bicyclam AMD3100, may work as in vivo inhibitors of tumor angiogenesis. Box 2 lists new CXCR4 antagonists [33]. Linomide, an anti-angiogenic agent, caused significant reduction of the tumour volume, in a murine prostate cancer model, by inhibiting both recruitment and the stimulatory effects of TAMs on tumour angiogenesis [34]. On the basis of this, the effects of Linomide, or other anti-angiogenic drugs, on the expression of pro- and anti-angiogenic molecules by TAMs may be considered valuable targets for anticancer therapy.
Survival Anti-tumor agents with selective cytotoxic activity on monocyte-macrophages would be ideal therapeutic tools for their combined action on tumor cells and TAMs. We recently reported that Yondelis (Trabectedin), a natural product derived from the marine organism Ecteinascidia turbinata, with potent anti-tumor activity [35] is specifically cytotoxic to macrophages and TAMs, while sparing the lymphocyte sub-set. This compound inhibits NF-Y, a transcription factor of major importance for mononuclear phagocyte differentia-
Box 2. CXCR4 antagonists [33] -
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Small-molecule bicyclams (e.g. AMD3100, AMD1498) ALX40-4C (9 D-Amino acid polycationic peptide) T22 (18 Amino acid peptide; polyphemusin) T140 (14 Amino acid peptide) CGP64222 (9 Amino acid basic peptide) KRH-1636 (Low molecular weight molecule)
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tion. In addition, Yondelis inhibits the production of CCL2 and IL-6 both by TAM and tumor cells [36]. These antiinflammatory properties of Yondelis may be an extended mechanism of its anti-tumor activity. Finally, proinflammatory cytokines (e.g. IL-1 and TNF), expressed by infiltrating leukocytes, can activate NF-kB in cancer cells and contribute to their proliferation, survival and metastasis (1,4), thus representing potential anticancer targets.
Matrix remodelling TAMs produce several matrix-metalloproteases (e.g. MMP2, MMP9) which degrade proteins of the extra-cellular matrix and also produce activators of MMPs, such as chemokines [7]. Inhibition of this molecular pattern may prevent degradation of extracellular matrix, as well as tumor cell invasion and migration. The biphosphonate zoledronic acid is a prototipical MMP inhibitor. In cervical cancer, this compound suppressed MMP-9 expression by infiltrating macrophages and inhibited metalloprotease activity, reducing angiogenesis and cervical carcinogenesis [37]. The halogenated bisphosphonate derivative chlodronate is a macrophage toxin which depletes selected macrophage populations. Given the current clinical usage of this and similar agents it will be important to assess whether they have potential as TAM toxins. The secreted protein acidic and rich in cysteine (SPARC) has gained much interest in cancer, being either up-regulated or down-regulated in progressing tumors. SPARC produced by macrophages present in tumor stroma can modulate collagen density, leukocyte and blood vessel infiltration [38].
Effector molecules Cyclooxygenase (COX) is a key enzyme in the prostanoid biosynthetic pathway. COX-2 not only is up regulated by activated oncogenes (i.e., ß-catenin, MET) but is also produced by TAMs in response to tumor-derived factors like mucin in the case of colon cancer. The usage of COX-2 inhibitors in the form of nonsteroidal antiinflammatory drugs is associated with reduced risk of diverse tumors (colorectal, oesophagus, lung, stomach and ovary). Selective COX2 inhibitors are now thought as a part of combination therapy [39]. The IFN-g-inducible enzyme indoleamine 2,3-dioxygenase is a well-known suppressor of T cell activation. It catalyzes the initial rate-limiting step in tryptophan catabolism, which leads to the biosynthesis of nicotinamide adenine dinucleotide. By depleting tryptophan from local microenvironment, indoleamine 2,3-dioxygenase (IDO) blocks activation of T lymphocytes. Recently, it was shown that inhibition of IDO may cooperate with cytotoxic agents to elicit regression of established tumors and may increase the efficacy of cancer immunotherapy [40]. Fig.1 summarizes therapeutic approaches to prevent TAM protumoral functions.
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Figure 1. Multifaceted therapeutic approaches to prevent TAM protumoral functions. TAMs promote tumor progression by favoring angiogenesis, suppression of adaptive immunity, matrix remodeling, tumor progression and metastasis. The figure summarizes strategies impairing selective TAM protumoral functions ( ) or restoring antitumor activities (+). Cytotoxic drugs (e.g. Yondelis) may decrease TAM number and prevent protumoral functions. A similar result may be obtained by limiting TAM recruitment (Linomide). Restoration of M1 immunity (STAT-3 and -6 inhibitors; anti-IL-10 plus CpG; IDO inhibitors) would provide cytotoxic activity and re-activation of Th1 specific antitumor immunity. Inhibition of both pro-inflammatory cytokines and growth factors expression (NF-kB inhibitors) may disrupt inflammatory circuits supporting tumor growth and progression. MMP inhibitors would prevent cancer cell spread and metastasis. Finally, inhibitors of TAM-mediated angiogenesis (HIF-1 inhibitors) would restrain blood supply and inhibit tumor growth. M-CSF (Macrophage-Colony Stimulating Factor); VEGF (Vascular Endothelial Growth Factor); CSFs (Colony-Stimulating Factors); IL- (Interleukin); TGF-b (Transforming Growth Factor-b); IDO (indoleamine 2,3-dioxygenase); MMP inhibitors (Matrix metalloproteinase inhibitors); TLR agonists (TollLike Receptor agonists); STAT (signal transducer and activator of transcription); NF-kB (nuclear factor-kB).
Concluding remarks Macrophages are key cell components of the inflammatory reactions expressed at the tumor site. Epidemiological and experimental evidence show that TAM express pro-tumoral functions, suggesting these cells as valuable target for novel anticancer strategies. Indeed, increased number and density of TAMs correlate with poor prognosis in different human cancers, while experimental evidence, ranging from adoptive transfer of cells to genetic manipulations, suggests that myelomonocytic cells can promote tumor invasion and metastasis. Box 3 indicates experimental approaches used to inhibit TAM functions. Box 4 summarizes the general status of knowledge on TAM biology and points out additional aspects to be addressed. Several studies have displayed key molecules and pathways driving recruitment and activation of TAMs and, more recently, the TAM transcriptome was provided in a murine fibrosarcoma [10]. Thus, a global effort is undergoing to fully characterize the molecular mechanisms supporting
TAM-mediated tumor progression. Despite these efforts, TAM functions have been significantly characterized only in animal models, and up today their phenotypic characterization remains only partial in human cancers. Moreover, it is still unknown whether different tumor microenvironments, likely established by different tumor types, may drive different functional phenotypes of TAM and contribute to specific pro- or anti-tumor activities. Along with TAM recruitment, activation Box 3. Anticancer strategies targeting TAM functions - Macrophage transduction with therapeutic genes (e.g. IFN-g) - Antisense oligonucleotides against CSF-1 (prevent recruitment and survival of TAMs) - Yondelis-Trabectedin (Cytotoxic drugs to myelomonocytic cells) - Linomide (prevents recruitment and angiogenic activity by TAMs) - Biphosphonate zoledronic acid (MMP inhibitor) - Anti-IL-10 plus CpG (restoration of M1 innate immunity) - Methyl-thiohydantoin-tryptophan (prevents the immunosuppressive activity of IDO)
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Box 4. Established and required knowledge on TAM biology Established Epidemiological evidence: correlation between TAM number and vessel density and prognosis. Pro-tumoral functions expressed by TAMs: angiogenesis, production of growth factors for tumor cells and blood vessels, matrix remodelling, immune suppression. To be provided Definitive in vivo demonstration of TAM protumoral functions in human tumors. Characterization of possible heterogeneity of TAM phenotype in different tumors. Definition of therapeutic strategies targeting TAMs in human neoplasia.
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and polarization mechanisms, the functional hetereogenicity of TAM should be viewed as an additional level of investigation to develop innovative anticancer strategies.
Acknowledgements
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This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC), Italy; by European Community (DC-Thera integrated project, number: LSHB-CT-2004-512074) and by Ministero Istruzione Universita` Ricerca (MIUR), Italy; Istituto Superiore Sanita’ (ISS).
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