Encyclopedia of Cancer. Springer (2015)
Tumor-associated macrophages (TAMs) define a subset of myeloid cells that highly infiltrate solid tumors. Accumulating evidence clearly demonstrates, in various mouse and human malignancies, including colon, breast, lung, and prostate cancer, a strict correlation between increased numbers and/or density of TAM and poor prognosis. Based on this, recruitment and activation of TAM are regarded as pivotal steps of tumor progression, and TAM are putative targets for therapeutic intervention.
Experimental and clinical studies have revealed that chronic inflammation predisposes to different forms of cancer, including colon cancer, prostate cancer, and liver cancer, and that usage of nonsteroidal anti-inflammatory drugs can protect against the emergence of various tumors.
In the late 1970s, it was found that a major leukocyte population present in tumors, the so-called TAM, promotes tumor growth. Over the years it has become increasingly clear that TAMs are active players in the process of tumor progression and invasion. In several experimental tumor models, the activation of an inflammatory response (most frequently mediated by macrophages) is essential for full neoplastic transformation and progression. This evidence strongly supports the idea that cancers originate at sites of chronic inflammation and suggests that the inflammatory circuits activated at the tumor microenvironment may represent suitable targets of novel anticancer therapies.
Macrophages (Mϕ) play an indispensable role in the immune system with decisive functions in both innate immunity and acquired immunity. In innate immunity, resident Mϕ provide immediate defense against foreign pathogens and coordinate leukocyte infiltration. Mϕ contribute to the balance between antigen availability and clearance through phagocytosis and subsequent degradation of senescent or apoptotic cells, microbes, and possibly neoplastic cells. Their role is essential for triggering, instructing, and terminating the adaptive immune response. Mϕ collaborate with T and B cells, through both cell–cell interactions and fluid phase-mediated mechanisms, based on the release of cytokines, chemokines, enzymes, arachidonic acid metabolites, and reactive radicals. Mϕ activation can be either proinflammatory or anti-inflammatory, thus contributing to tissue–cell destruction or to tissue regeneration and wound healing. These polar phenotypes are not expressed simultaneously but regulated in such a manner that Mϕ display a balanced, harmonious pattern of functions.
Mϕ are critical effector cells in the acute innate response, for delayed-type hypersensitivity reactions and T cell-mediated immunity. In 1986 Mosmann et al. described two polarized sets of mouse T helper (Th) cells – Th1 and Th2 – with distinct cytokine secretion patterns. Th1 cells secreted interleukin 2 (IL-2), interferon- g (IFN-g), and lymphotoxin (LT, TNF-b). Th2 cells secreted IL-4, IL-5, and IL-6 and promoted B-cell proliferation and antibody secretion. Moreover, additional studies clarified that Th1 and Th2 cells may play opposite roles in pathological conditions, including infections and cancers.
Although excluded from the original “type I–type II” paradigm, Mϕ’s role in the balance of polarized immune responses is being increasingly appreciated. Mϕ are able to secrete either IL-12 or IL-10, cross-regulatory cytokines crucial for the elicitation of IFN-g production and development of Th1 cells and IL-4/IL-13 secretion and Th2 cells proliferation correspondingly. The preferential production of IL-12 and IL-10 sets the basis for the M1/M2 Mϕ polarization paradigm, elsewhere defined as the elicitation of functionally distinctMϕ populations, in response to the factors that dominate the inflammatory scene. In analogy with the Th1 and Th2 dichotomy, macrophages can be phenotypically polarized by the microenvironment to mount specific M1 or M2 functional programs. 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 IFN-g is characterized by high capacity to present antigen; high expression of proinflammatory cytokines, such as interleukin 12 (IL-12) and tumor necrosis factor-a (TNF-a) and consequent activation of a polarized type I response; and high production of toxic intermediates (nitric oxide (NO), reactive oxygen species). Thus, M1 macrophages are generally considered potent effector cells that kill microorganisms and tumor cells and produce copious amounts of proinflammatory cytokines. In contrast, various signals (e.g., IL-4, IL-13, glucocorticoids, IL-10, immunoglobulin complexes/TLR ligands) elicit different M2 forms, able to tune inflammatory responses and adaptive Th2 immunity, scavenge debris, and promote angiogenesis, tissue remodeling, and repair. Figure 1 shows selected functions of M1 and M2 polarized macrophages. Microenvironmental signals expressed at the tumor microenvironment have the capacity to pilot recruitment, maturation, and differentiation of infiltrating leukocytes and play a central role in the activation of specific transcriptional programs expressed by TAM.
To the extent that they have been investigated, differentiated mature TAM has a phenotype and function similar to type II or M2 macrophages. TAM has been shown to exert a negative effect on antitumor immune responses.
TAMs are protumoral cells that are derived from circulating monocytes (Fig. 2) and are recruited to the tumor by a tumor-derived chemotactic factors, originally identified as CC-chemokine ligand 2 (CCL2; also known as MCP-1). Following this observation, other chemokines active on TAM were detected in neoplastic tissues as products of either tumor cells or stromal elements. These molecules have an important role in tumor progression by directly stimulating neoplastic growth, promoting inflammation, and inducing angiogenesis. Evidence supporting a pivotal role for chemokines, in addition to CCL2, in the recruitment of monocytes to neoplastic tissues includes a direct correlation between chemokine production and monocyte infiltration in mouse and human tumors. Molecules other than chemokines can also promote TAM recruitment. In particular, tumor-derived cytokines such as vascular endothelial growth factor (VEGF) and macrophage colony-stimulating factor (M-CSF) promote macrophage recruitment, as well as macrophage survival and proliferation, and their expression correlates with tumor growth.
TAM express selected M2 protumoral functions
The cytokine network expressed at the tumor site plays a central role in the orientation and differentiation of recruited mononuclear phagocytes, thus contributing to direct the local immune system away from antitumor functions. Immunosuppressive cytokines IL-10 and tumor growth factor-b (TGF-β) are produced by both cancer cells (ovary) and TAM. IL-10 promotes the differentiation of monocytes to mature macrophages and blocks their differentiation to dendritic cells (DC). Thus, a gradient of tumor-derived IL-10 may account for differentiation along the DC versus the macrophage pathway in tumors, resulting in tumor promotion. IL-10 promotes the M2c alternative pathway of macrophage activation and induces TAM to express M2-related functions. Under many aspects, TAM summarizes a number of functions expressed byM2 macrophages involved in tuning inflammatory responses and adaptive immunity, scavenges debris, and promotes angiogenesis, tissue remodeling, and repair. The production of IL-10, TGF-β, and PGE2 by cancer cells and TAM contributes to a general suppression of antitumor activities.
TAMs are poor producers of nitric oxide (NO), and, in situ in ovarian cancer, only a minority of tumors and, in these, a minority of macrophages localized at the periphery scored positive for iNOS. Moreover, in contrast to M1-polarized macrophages, TAM has been shown to be poor producers of ROI, consistent with the hypothesis that these cells represent a skewed M2 population. Moreover, TAM was reported to express low levels of inflammatory cytokines (e.g., IL-12, IL-1b, TNF-a, IL-6). Activation of the transcriptional factor NF-kB is a necessary event promoting transcription of several proinflammatory genes. TAM displays defective NF-kB activation in response to M1-polarizing signals lipopolysaccharide (LPS) and TNF-a. Thus, in terms of cytotoxicity and expression of inflammatory cytokines, TAM resembles the M2 macrophages.
Angiogenesis is an M2-associated function that represents a key event in tumor growth and progression. In several studies in human cancer, TAM accumulation has been associated with angiogenesis and with the production of angiogenic factors such as VEGF and platelet-derived endothelial cell growth factor. Additionally, TAM participates to the proangiogenic process by producing the angiogenic factor thymidine phosphorylase (TP), which promotes endothelial cell migration in vitro and whose levels of expression are associated with tumor neovascularization. Moreover, TAM accumulates in the hypoxic regions of tumors, and hypoxia triggers a proangiogenic program in these cells. Therefore, macrophages recruited in situ represent an indirect pathway of amplification of angiogenesis, in concert with angiogenic molecules directly produced by tumor cells. On the antiangiogenic side, in a murine model, GM-CSF released from a primary tumor upregulated TAM-derived metalloelastase and angiostatin production, thus suppressing tumor growth of metastases. Finally, TAM expresses molecules that affect tumor cell proliferation, angiogenesis, and dissolution of connective tissues. These include epidermal growth factor (EGF), members of the FGF family, TGF-β, VEGF, and chemokines. In lung cancer, TAM may favor tumor progression by contributing to stroma formation and angiogenesis through their release of PDGF, in conjunction with TGF-β1 production by cancer cells. Macrophages can produce enzymes and inhibitors that regulate the digestion of the extracellular matrix, such as MMPs, plasmin, urokinase-type plasminogen activator (uPA), and the uPA receptor. Direct evidence has been presented that MMP-9 derived from hematopoietic cells of host origin contributes to skin carcinogenesis. Chemokines have been shown to induce gene expression of various MMPs and, in particular, MMP-9 production, along with the uPA receptor. Evidence suggests that MMP-9 has complex effects beyond matrix degradation including the promotion of the angiogenesis switch and release of growth factors.
Modulation of adaptive immunity by TAM
It has long been known that TAM have poor antigen-presenting capacity and can actually suppress T cell activation and proliferation. The suppressive mediators produced by TAM include prostaglandins, IL-10, TGF-β, and indoleamine dioxygenase (IDO) metabolites. Moreover, TAM is unable to produce IL-12, even upon stimulation by IFN-g and LPS. With this cytokine profile, which is characteristic of M2 macrophages, TAM is unable to trigger Th1-polarized immune responses but rather induce T regulatory cells (Treg). Treg cells possess a characteristic anergic phenotype and strongly suppress the activity of effector T cells and other inflammatory cells, such as monocytes. Suppression of T cell-mediated antitumor activity by Treg cells is associated with increased tumor growth and hence, decreased survival. For instance, in patients with advanced ovarian cancer, an increase in the number of functionally active Treg cells present in the ascites was predictive of reduced survival.
The complex network of chemokines present at the tumor site can play a role also in the induction of the adaptive immunity. Chemokines also regulate the amplification of polarized T cell responses. Some chemokines may enhance specific host immunity against tumors, but on the other hand, other chemokines may contribute to escape from the immune system, by recruiting Th2 effectors and Treg cells. Figure 2 summarizes symbiotic relationship between TAM and cancer cells.
Though the presence of TAM has been long considered as evidence for a host response against the growing tumor, it has become increasingly clear that TAMs are active players in the process of tumor progression and invasion. Molecular and biological studies have been supported by a large number of clinical studies that found a significant correlation between the high macrophage content of tumors and poor patient prognosis. TAM shares many similarities with prototypic polarized M2 mononuclear phagocyte population, in terms of gene expression and functions. In line with the known properties of M2 macrophage populations, several lines of evidence suggest that TAM promotes tumor progression and metastasis by activating circuits that regulate tumor growth, adaptive immunity, stroma formation, and angiogenesis. This hypothesis is now receiving new supporting evidence indicating that in vivo functional switching of infiltrating M2 macrophages toward an M1 phenotype provides therapeutic benefit in mice-bearing tumor xenograft. Identification of mechanisms promoting functional diversion of macrophages toward an M2 direction may disclose new valuable therapeutic targets against tumors.
Fig. 1. Monocytes differentiate into polarized macrophage subsets when exposed to different cytokine milieu. In the presence of GM-CSF, IFN-g, LPS, and other microbial products, monocytes differentiate into M1 macrophages. In the presence of M-CSF, IL-4, IL-13, IL-10, and immunosuppressive agents (corticosteroids, vitamin D3, prostaglandins, and immunocomplexes (IG) in combination with IL-1 or TLR ligands), monocytes differentiate into M2 macrophages. M1 and M2 subsets differ in terms of phenotype and functions. M1 cells have high microbicidal activity, immunostimulatory functions, and tumor cytotoxicity. M2 cells have high scavenging ability, promote tissue repair and angiogenesis and immunosuppression, and favor tumor progression
Fig. 2. Tumor-derived chemotactic factors (CC-chemokines, e.g., CCL2, macrophage colony-stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF)) actively recruit circulating blood monocytes at the tumor site. In the tumor microenvironment, monocytes differentiate into tumor-associated macrophages (TAMs) that establish a symbiotic relationship with tumor cells. The above tumor-derived factors positively modulate TAM survival. From their own, TAM secretes growth factors that promote tumor cell proliferation and survival; regulates matrix deposition and remodeling, thus favoring metastasis formation; activates neoangiogenesis; and promotes immunosuppression
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