Tumor microenvironment: a main actor in the metastasis process
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- Spano, D. & Zollo, M. Clin Exp Metastasis (2012) 29: 381. doi:10.1007/s10585-012-9457-5
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Over recent decades, various studies have argued that the metastatic tissue microenvironment is fully controlled by the intrinsic properties of the cancer cells (growth, motility and invasion, angiogenesis, extracellular matrix remodeling, immune escape) and additional cells types. Overall, the extrinsic factors and determinants mediate the contribution of the host microenvironment to metastasis formation. The tumor microenvironment carries out these functions by secretion of molecules that can influence and modulate its phenotype, making these complex interactions the basis for support for the progression of a cancer. Here, we undertake a summary of the “state of the art” of the functions and actions of these cells, as the main actors in the promotion of the formation of the microenvironment of the metastatic niche, and the associated network of interactions. The unraveling of the relationships between tumorigenic cells and their microenvironment represents an important issue for the development of new therapeutic agents that can fight both initiation and recurrence of cancer.
KeywordsTumor microenvironmentImmune inflammatory cellsCancer-associated fibroblastsHierarchic network of communication
Major histocompatibility complex
Myeloid-derived suppressor cells
Monocyte chemotactic protein 1
Vascular endothelial growth factor
Transforming growth factor-β
Hepatocyte growth factor
Stromal-cell-derived factor 1
Tissue inhibitors of MMP
Polyoma middle T
Epidermal growth factor
Epidermal growth factor receptor
Urokinase plasminogen activator
Colony stimulating factor 1
Lewis lung carcinoma
Regulatory T cells
Stem cell factor
Nitric oxide synthase
Reactive oxygen species
Serum amyloid A3
Fibroblast growth factor-2
Granulocyte colony stimulating factor
Macrophage colony stimulating factor
Tumor metastasis is responsible for approximately 90% of all cancer-related deaths. It is well established that to form a metastasis from a primary tumor, the cancer cells need to acquire additional properties that enable invasion of the extracellular matrix (ECM), intravasation, travel via the blood vessels, migration to and invasion into a secondary site, and finally the formation of metastatic nodules .
Over the past decade, solid tumors have increasingly been recognized as ‘organs’ that show a complexity that approaches, and may even exceed, that of normal healthy tissue . Solid tumors comprise not only the malignant cells, but also several other non-malignant cell types, which together constitute the stromal tissue. The latter is the supportive and connective tissue of the host. It can be composed of many different types of cells, including fibroblasts, resident epithelial cells, pericytes, myofibroblasts, vascular and lymphovascular endothelial cells, and infiltrating cells of the immune system.
Recent evidence has shown that stromal tissue is much more than a passive bystander in the development and progression of cancers. Instead, there is a bidirectional, dynamic and intricate complex of interactions between the cells of the stromal tissue and the cancer cells. Altogether, the non-malignant cells of stromal tissue produce a unique microenvironment that can modify the neoplastic properties of the tumor cells. In turn, during the course of multistep tumorigenesis, the tumor cells contribute to the generation and modification of the tumor microenvironment. Therefore, the tumor microenvironment is a dynamic network that includes the cancer cells and the stromal tissue, as well as the all-surrounding ECM. Many aspects of these dynamic interactions have been defined through numerous studies, but a full understanding of the underlying molecular mechanisms and the hierarchical network of connections awaits further studies in the near future.
The first malignant cells that are generated within a tissue will represent the first actors within this hierarchical network. Furthermore, it is reasonable that during early tumorigenesis, the first cells that are recruited by an incipient neoplasia are fibroblasts, as these represent the main constituent of stromal tissue. Such cancer-associated fibroblasts (CAFs) have a fundamental role at this time, for the secretion of factors that act on the tumor cells in both paracrine and autocrine fashions, which can result in a more aggressive cancer phenotype. Then the CAFs and cancer cells secrete factors that contribute to the recruitment of immunosuppressive cells.
It is still not known which immune cell type is the earliest to be recruited, or which immune cell type acts first within the interplay with the other cells of the tumor microenvironment. If this issue can be dissected out this would shed light on which cells are acting first, thus defining the precise timing of their actions within the network of communication between immune inflammatory cells and within the tumor microenvironment. This knowledge, in turn, can help us to better define the targets for clinical intervention.
To help in the deciphering of these multistep processes, a useful future source of studies will be those performed on animal models that can be depleted of each specific immune cell population. Once recruited into the tumor microenvironment, the immune inflammatory cells can contribute to the malignant progression of the cancer-cell phenotype. This thus leads them to establish a complex network of interplay that contributes to the promotion and maintenance of an immunosuppressive microenvironment, which itself promotes immune escape, and as a consequence, enhances tumor progression.
At the present, we can categorize the tumor immune escape mechanisms into two major types. One category of escape mechanism is active at the level of the tumor, and can be ascribed to defects in the expression of major histocompatibility complex (MHC) class I, in the processing of MHC class I restricted antigens, or in the presentation machinery of the antigens. These abnormalities can be the result of activation of oncogenes within tumor cells, with one example seen in Her2/neu, which enables tumors to take on a ‘stealth’ phenotype and thus to hide from detection by cytotoxic T lymphocytes . The second category of immune escape mechanism arises from the ability of the progressing tumor to interfere with the host immune system. To this end, as mentioned above, the tumor induces and/or recruits immunosuppressive cells, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), which normally serve as safeguards against overwhelming inflammation or autoimmunity. By turning the host immune system against itself, tumors can gain an impressive arsenal of weapons to hamper the induction and progression of anti-tumor immune activity.
Cell types in the microenvironment of primary tumor
Fibroblasts are found in various proportions across the spectrum of carcinomas, and in many cases they constitute the main cell population of the tumor stroma. CAFs have important roles not just in the initiation of tumorigenesis and in malignant progression, but also in the facilitation of proliferation, invasion and motility of malignant cells . These cells have a high proliferation rate, and through the secretion of growth factors and chemokines they communicate with cancer cells, resident epithelial cells, endothelial cells, pericytes and inflammatory cells. Fibroblasts deposit high amounts of ECM components, such as collagen types I and III and tenascin C (TNC). This induces an altered ECM microenvironment, which potentially provides additional oncogenic signals that can lead to an acceleration of cancer progression . Fibroblasts mediate inflammatory responses by secreting chemokines, such as monocyte chemotactic protein 1 (MCP1/CCL2) and the interleukins (ILs), such as IL-1. In particular, CCL2 has been shown to be overexpressed in a wide variety of cancer types, it is a potent chemoattractant for monocytes, memory T lymphocytes, and natural killer cells, and it stimulates cell proliferation, migration and invasion . In addition, CAFs constitute a source of matrix metalloproteinases (MMPs), which promote tumor invasion through degradation of the ECM, and vascular endothelial growth factor (VEGF), which promotes angiogenesis. Fibroblasts also provide potent oncogenic signals to resident epithelia, such as through transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF). They can also stimulate cancer-cell proliferation and invasion directly by secreting growth factors, which include the above-mentioned TGF-β and stromal-cell-derived factor 1 (SDF1) .
In the tumor stroma, together with the other stroma cells, the fibroblasts are a source of S100A4, a calcium-binding protein of the S100 protein family, the members of which have important roles in metastasis . High levels of S100A4 expression correlate with negative prognosis in several types of cancer . This protein acts as an angiogenic factor, and it stimulates cell survival and tumor-cell migration, up-regulates MMPs, and down-regulates tissue inhibitors of MMPs (TIMPs). Moreover, S100A4 activates the epidermal growth factor receptor (EGFR), the transcription factor NF-κB, and the mitogen-activated protein (MAP) kinase pathway, which leads to the activation of downstream genes [7, 8]. Forst and collaborators  generated S100A4−/− MMTV-polyoma middle T (PyMT) mice as a mammary tumor model  to analyze the role of S100A4 in premalignant tumor development. They showed that fibroblast secretion of S100A4 into the tumor milieu is stimulated by the tumor-cell-derived cytokine RANTES and occurs via microparticle shedding from the plasma membrane of stroma cells. S100A4, in turn, induces up-regulation of the cytokines (including RANTES), production of fibronectin, and stimulation of cell motility and tumor metastases in vivo . Moreover in the same animal model, S100A4 induces a massive infiltration of T cells , which can then produce cytokines and growth factors. The main T-cell-produced cytokines are eotaxin-2 (also known as CCL24) and granulocyte colony stimulating factor (G-CSF). Eotaxin-2 is a potent chemoattractant for eosinophils, which have been shown to suppress the immune response and promote proliferation in squamous cells and colorectal carcinomas [12, 13]. G-CSF is another proinflammatory cytokine that promotes tumor progression and metastasis . Thus G-CSF mediates mobilization of CD11b+ Gr1+ myeloid cells , which can then stimulate the metastatic spread of tumorigenic cells.
Immune inflammatory cells
Infiltrating immune cells are normal constituents of tumors. They operate in conflicting ways, by either antagonizing or promoting tumors. Indeed, it has been hypothesized that the balance between the tumor antagonizing inflammatory cells and the tumor promoting inflammatory cells modulates tumor progression. The tumor-promoting inflammatory cells include macrophage subtypes, mast cells and neutrophils, as well as T and B lymphocytes [2, 16–18]. These cells can secrete several signaling molecules that serve as effectors of their tumor-promoting actions. These include epidermal growth factor (EGF), the angiogenic growth factor VEGF, other proangiogenic factors, such as fibroblast growth factor-2 (FGF2), and several chemokines and cytokines that amplify the inflammatory state. In addition, these infiltrating immune cells can produce proangiogenic and/or proinvasive matrix-degrading enzymes, including MMP9 and other MMPs, cysteine cathepsin proteases, and heparanase [18, 19]. As a consequence of the expression of those effectors, these cells induce and support tumor angiogenesis, which stimulates cancer-cell proliferation, facilitates tissue invasion, and sustains metastatic dissemination [16, 18–22].
Within the tumor mass, in addition to fully differentiated immune cells, a variety of partially differentiated myeloid progenitors have been identified . These cells are intermediates between the circulating cells of bone marrow origin and the fully differentiated immune cells in normal and inflamed tissues, and they show tumor-promoting activities. Of particular interest for their multiple functions, the MDSCs are characterized by the expression of the macrophage marker CD11b and the neutrophil marker Gr1, and they show significant immunosuppressive activity [19, 23]. These attributes raise the possibility that the recruitment of certain myeloid cells can be doubly beneficial for a developing tumor: by directly promoting angiogenesis and tumor progression, while at the same time providing a means to evade immune destruction.
Tumor-associated macrophages (IL-10high IL-12low IL-1rahigh and IL-1 decoyRhigh)
There is a growing body of evidence that indicates that macrophages in a primary tumor promote tumor progression to a metastatic phenotype . This evidence comes from two sources: (1) clinical correlative data that show that in over 80% of cases, strong macrophage infiltration is correlated with poor prognosis, which includes increased lymph-node involvement and decreased patient survival ; and (2) mouse models where genetic ablation of macrophages results in inhibition of tumor progression and a reduced rate of metastasis formation . Lin et al.  demonstrated that when MMTV-PyMT mice are crossed with mice lacking monocytes/macrophages through genetic depletion of the key macrophage growth factor CSF-1 (mice homozygous for the Csf1op null allele), the density of TAMs is significantly reduced, with a significant delay in tumor progression to metastasis. These data indicate that TAMs are required for efficient tumor metastasis formation.
Macrophages constitute an extremely heterogeneous population. They originate from blood monocytes that are not fully differentiated cells, and they are profoundly susceptible to several environmental stimuli. When recruited into peripheral tissues from the circulation, monocytes can differentiate rapidly into distinct, mature macrophages, which have specific immunological functions. TAMs originate from blood monocytes that are recruited at a tumor site by molecules that are produced by neoplastic and stromal cells. In the tumor milieu, TAMs carry on their pro-neoplastic role by influencing fundamental aspects of tumor biology: they can produce molecules that affect neoplastic cell growth directly (e.g., EGF), enhance neoangiogenesis, tune inflammatory responses and adaptive immunity, and catalyze structural and substantial changes in the ECM compartment [22, 27, 28]. Several chemokines are players in this process, including CCL2, CCL5, CCL7, CXCL8 and CXCL12, as well as cytokines such as VEGF, PDGF and the growth factor macrophage colony stimulating factor (M-CSF) [29, 30]. In particular, CCL2 is the main molecule that is involved in monocyte recruitment. Its expression correlates with TAM abundance in many human tumors, including those of ovarian, breast and pancreatic cancer . Furthermore, TAMs can themselves produce CCL2, which suggests that they can act as an amplification loop. Within the tumor mass, the monocytes are surrounded by several signals that can induce their differentiation towards mature M2-polarized macrophages. These macrophages express IL-10high, IL-12low, IL-1rahigh and IL-1 decoyRhigh, and they have a pivotal role in tumor growth and progression. The M2 polarization factors are IL-4, IL-6, IL-10 and IL-13, M-CSF, glucocorticoids, TGF-β and prostaglandin E2 (PGE2), and these can be produced within the tumor mass by neoplastic cells and fibroblasts (e.g. IL-10, TGF-β), and by Th2 lymphocytes (e.g. IL-4, IL-13) .
The role of TAMs in angiogenesis is emphasized by the correlation between their high numbers and the high vascular grades in many tumors, including gliomas, squamous cell carcinomas of the esophagus, and breast, bladder and prostate carcinomas . They release the growth factors VEGF, PDGF, TGF-β, and members of the FGF family . TAMs secrete the angiogenic factor thymidine phosphorylase, which promotes endothelial cell migration in vitro , and they also produce several angiogenesis-modulating enzymes, such as MMP2, MMP7, MMP9, MMP12 and cyclooxygenase-2 (COX-2) [22, 28, 34]. In particular, the preferential accumulation of TAMs in tumor hypoxic regions promotes activation of a hypoxia-induced specific proangiogenic program that leads to the overexpression of proangiogenic molecules like VEGF  and CXCL12, a potent chemoattractant for endothelial cells, and its receptor CXCR4 . In addition to CXCL12, TAMs release several chemokines that are involved in angiogenic processes, including CCL2, CXCL8, CXCL1, CXCL13 and CCL5 . Moreover, TAMs secrete lymphatic endothelial growth factors that strongly promote peritumoral lymphangiogenesis .
Within the primary tumor microenvironment, at least two mechanisms have been proposed by which TAMs facilitate tumor metastasis. The first relates to the secretion of proteases within the tumor microenvironment, such as urokinase plasminogen activator (uPA), cathepsins B and D , MMP2 and MMP9 , which can digest the tumor basement membrane, thus facilitating tumor-cell escape. A second mechanism is through direct enhancement of an early stage of the metastatic cascade . Macrophage-produced EGF increases the motility and invasiveness of malignant cells. Indeed, in a genetic model of a breast cancer in monocyte-deficient mice, the tumors developed normally, although in this absence of macrophage-produced EGF, the tumors did not form pulmonary metastases . An in vivo invasion assay showed that TAMs promote carcinoma-cell motility and invasion through a paracrine signaling loop between the tumor cells and the TAMs. Within this loop, the macrophages express EGF, which promotes formation of elongated protrusions and cell invasion by the carcinoma cells. In addition, EGF promotes the expression of CSF-1 by the carcinoma cells. This CSF-1 promotes the expression of EGF by macrophages, thereby generating a positive-feedback loop. Disruption of this loop by blockade of the signaling of either the EGFR or the CSF-1 receptor is sufficient to inhibit both macrophage and tumor-cell migration and invasion . Furthermore, in vitro evidence has demonstrated that the production and secretion of the Wnt-ligand Wnt5a by macrophages can stimulate the planar-cell-polarity noncanonical Wnt signaling pathway in carcinoma cells, with the consequent promotion of cell invasion . Indeed, the gene expression profile of invasion-promoting TAMs isolated from MMTV-PyMT mice, as compared to that of control isolated TAMs, showed that these macrophages are specifically enriched in molecules involved in Wnt signaling. There is thus extensive evidence that the Wnt signaling pathway is involved in this TAM-mediated tumor-cell motility .
Finally, within the tumor microenvironment, TAMs have strong immunosuppressive activity, not only through their production of IL-10, but also by their secretion of chemokines (e.g., CCL17 and CCL22), which preferentially attract T-cell subsets that are devoid of cytotoxic functions, such as regulatory T (Treg) cells and Th2 lymphocytes . In addition, TAMs secrete CCL18, which recruits naïve T cells, whereby their recruitment into this microenvironment characterized by M2 cells and immature dendritic cells is likely to induce T cell anergy. This is the mechanism that is responsible for the inability of an immune T cell to mount a complete response against its target .
Myeloid-derived suppressor cells (CD11b+ Gr1+)
MDSCs comprise a phenotypically heterogeneous population of immature myeloid cells at different stages of differentiation. They derive from bone-marrow progenitors that have not completed their maturation into granulocytes, monocytes or dendritic cells . Normally, MDSCs are in the bone marrow of healthy individuals, although they preferentially accumulate in the spleen and blood of tumor bearers . In mice, these cells express the membrane antigens Gr1 and CD11b, and based on the expression of different epitopes of Gr1, they can be further subdivided into the granulocytic, as CD11b+ Gr1+ (Ly6G+), and monocytic, as CD11b+ Gr1+ (Ly6C+ Ly6G−) subclasses . In human, MDSCs appear to be more like a ‘family of MDSCs’, which includes different cell populations with varying phenotypes and biological diversities. The role of these cells in human cancer progression is underlined by the correlation between the expansion of the MDSC pool, the tumor stage, and the evidence of metastasis formation .
The major function of MDSCs is to orchestrate other cells of the immune response, to promote an immunosuppressive and anti-inflammatory phenotype, which results in tumor immune escape . For this function in tumor-bearing hosts, MDSCs need to expand in the lymphoid organs and to subsequently be recruited to the primary tumor site [23, 45], processes being directed by tumor-associated inflammation, and by angiogenic and chemoattractant factors.
Proinflammatory cytokines (e.g. IL-1β, IL-6) and bioactive lipids (e.g. PGE2, COX-2) are the major contributors to the inflammatory milieu of a tumor. These molecules can induce MDSCs in tumor-bearing hosts. Indeed, IL-6 can partially restore MDSC induction in IL-1β-deficient mice, thus facilitating tumor progression . COX-2 inhibition blocks MDSC expression of arginase 1 (ARG1) and induces an anti-tumor immune response . Similarly, Sinha et al.  reported that tumor-derived PGE2 and/or COX-2 significantly induce MDSCs from bone-marrow precursor cells through the EP2/4 receptor. They also showed that treatment of tumor-bearing mice with COX-2 inhibitors reduces the frequency of MDSCs in the tumors and blood, and delays tumor growth . Recently, it has been demonstrated that S100A8 and S100A9, pro-inflammatory proteins released by neutrophils, induce MDSCs. In particular, S100A9 blocks differentiation of myeloid precursors into functional dendritic cells or macrophages, through a STAT3-dependent pathway. As a consequence, S100A9-deficient mice have reduced MDSC numbers and spontaneously reject transplanted lymphomas . S100A8 and S100A9 are also highly expressed at tumor sites, thus leading to the strong recruitment of MDSCs . Furthermore, MDSCs produce and secrete these proteins, which can lead to an autocrine loop of engagement.
VEGF released by tumors is one of the main factors responsible for expansion of MDSCs . This effect is partly due to MMP9, whereby its remodeling of the ECM promotes the growth of new blood vessels, thus stimulating VEGF production. Furthermore, Yang and collaborators  showed that MMP9 induces the expansion and maintenance of MDSCs. An important factor within the ECM is tumor-derived stem-cell factor (SCF), which leads to myelopoiesis and the expansion of MDSCs through the inhibition of differentiation of myeloid precursors to functional dendritic cells . The recruitment of MDSCs to tumor sites is also driven by chemoattractant molecules, such as CCL2/CCR2 , SDF1/CXCR4, CXCL5/CXCR2 , and uPA .
The suppressive function of MDSCs is turned on by several factors, including IL-4, IL-13, interferon (IFN)-γ, IL-1β and TGF-β. Activated MDSCs suppress the anti-tumor immune response in the tumor microenvironment directly through the expression of inducible nitric oxide synthase (iNOS) and ARG1. MDSCs that express ARG1 deplete l-arginine from the microenvironment, and thus limit its availability to T cells. Consequently T cells are deficient in the CD3ζ chain of the T-cell receptor (TCR), and they are arrested in the G0–G1 phase of the cell cycle. This results in inhibition of both their function and their proliferation . The high expression of iNOS in MDSCs increases the production of NO and reactive oxygen species (ROS). ROS inhibit MDSC maturation , induce DNA damage in immune cells in the tumor microenvironment, inhibit the differentiation of MDSCs into functional dendridic cells, and recruit MDSCs to tumor sites . Moreover, extracellular ROS catalyze the nitration of the TCR, which consequently inhibits the T cell-peptide-MHC interaction, which results in T-cell suppression . MDSCs also impair T-cell activation by abrogation of the expression of l-selectin on both CD4+ and CD8+ T cells, which thus suppresses the homing of these cells to tumor sites, where they would then be activated . Another mechanism by which MDSCs interfere with T-cell activation is their ability to expand the Treg cells in the tumor microenvironment [61, 64]. Treg cells inhibit T-effector cell proliferation, differentiation and activation, which will subsequently blunt the anti-tumor immune response . Furthermore, MDSCs inhibit natural killer cells and B cells , and induce M2 polarization of TAMs through secretion of high levels of IL-10 . As a consequence, the balance between immunosuppressive cells and anti-tumor immune cells is further tilted towards a dominant immunosuppressive microenvironment. Finally, similar to TAMs, MDSCs promote angiogenesis , thus facilitating metastatic growth through improved delivery of nutrients and oxygen within the tumor microenviroment.
Mast cells are an additional category of cells that colonize the metastatic niche; they are derived from bone-marrow hematopoietic progenitors. While still immature, mast cells migrate from vascular to peripheral tissues, where their maturation depends on the microenvironmental conditions. From a physiological perspective, mast cells participate in tissue remodeling, wound healing, and angiogenesis . These cells have pathological effects in acute disorders, in chronic allergic disorders  and autoimmune diseases .
There is a lot of evidence that implicates mast cells in cancer proliferation and metastasis. In humans, there are increased numbers of mast cells in different types of tumors. Clinical studies have shown that the number of tumor-infiltrating mast cells correlates with increased intratumoral microvessel density, enhanced tumor growth and invasion, and poor clinical outcome. Moreover, in pancreatic cancer, hepatocellular carcinoma and intrahepatic cholangiocarcinoma, mast-cell infiltration increases in comparison with normal tissues and with the corresponding benign diseases [69, 70]. Furthermore, mast cell numbers are higher in specimens from patients with advanced disease and with metastases to the lymph nodes, than in specimens from patients with early stages of disease . In vitro, mast cells increase proliferation, migration and invasion of thyroid cancer , pancreatic ductal adenocarcinoma  and breast-cancer cells . In addition, metastases are reduced in mast-cell-deficient animal models .
Mast-cell infiltration and activation in tumors is mainly mediated by tumor-derived SCF and the receptor c-KIT, which is mainly found on these cells . Once activated, mast cells can release several mediators that are involved in the processes of tumor microenvironment remodeling, thus facilitating tumor metastasis. Indeed, mast cells also produce proteases, such as tryptase, chymases, MMP9 and cathepsin, which together promote inflammation, modulate immune responses by hydrolyzing chemokines and cytokines, and degrade the ECM . Mast-cell-derived leukotrienes promote the recruitment of neutrophils , induce vascular permeability, trigger chemotaxis in various cells, and increase mucus production . Other released mediators include vasoactive factors (e.g. histamine, IL-8, VEGF, prostaglandin D and substance P), which lower endothelial barriers , and proangiogenic factors (e.g. VEGF, PDGF, MMP9 and PGE2), which together induce angiogenesis. In addition, mast cells contribute to the establishment of inflammatory and immunosuppressive conditions in the tumor microenvironment, by secretion of pro-inflammatory and immunosuppressive cytokines, and of chemokines. The pro-inflammatory cytokines (e.g. IL-1β, IL-6, IL-8, and TNF-α) increase the interstitial fluid volume by plasma effusion, and extend the distance that oxygen needs to be delivered to the oxygen-hungry cells, thus leading to hypoxia-induced metastasis . The immunosuppressive cytokines (e.g. TGF-β and IL-10 ) favor immune suppression. The chemokines (e.g. CCL5 and CXCL8 ) act as chemoattractants for additional effector immune cells, thus remodeling the immune and inflammatory microenvironment of the tumor. Finally, mast cells secrete growth factors (e.g. EGF, IGF, NGF and bFGF) that favor tumor-cell survival and proliferation, thus again facilitating metastasis .
There is a complex interplay between mast cells and the other immune cells within the tumor microenvironment. The factors secreted by mast cells lead to CCL2 production and IL-17 up-regulation in MDSCs. CCL2 signaling recruits more MDSCs, leading to more IL-17 production, which further exacerbates the inflammatory tumor microenvironment. IL-17 leads to the up-regulation of IL-9, IL-10, IL-13, CCL17, CCL22, CD39 and CD73. This results in various actions: CCL17 and CCL22, in turn, are chemoattractants that bring more Treg cells to tumor sites; CD39 and CD73 enhance the suppressor function of Treg cells; IL-9 produced by Treg cells helps to maintain the survival of mast cells; and IL-10 and IL-13 induce ARG1 expression by MDSCs. Mast cell modulation of the recruitment and the suppressor function of MDSCs and Treg cells represents another mechanism by which they promote the tumor immune escape.
The pre-metastatic niche: a collaboration between different cell types
During the metastatic process, tumor cells detach from the primary tumor, invade and intravasate the vasculature, and come to rest in local capillaries in secondary organs. Here they need to extravasate, invade, survive and proliferate. The pioneering studies by Paget first described the “seed and soil” hypothesis , which suggested that microenvironment factors and mechanical forces in the circulation are both important determinants of site-specific metastatic spread. Recently, this hypothesis has been revisited, which has lead to a metastatic-niche model. This model suggests that a suitably conducive microenvironment (the pre-metastatic niche) must evolve for tumor cells to engraft (metastatic niche) and proliferate at secondary sites (micrometastatic to macrometastatic transition). Therefore, this model assumes that both the intrinsic properties of the metastatic cells (genetic and epigenetic changes) and the host microenvironment are important determinants in metastasis dissemination . This model also assumes that the tumor cell does not solely dictate its own fate, but that this formation of a hospitable microenvironment is essential—not just permissive—for a disseminating tumor cell to spawn secondary tumor growth.
Cell types in the microenvironment of pre-metastatic niche
Myeloid cells (VLA4+ VEGFR1+ c-KIT+ SCA1+ CD11b+)
Evidence has been provided that primary tumors can secrete several factors that mediate the establishment of specific microenvironments in the distant organs that are sites of future metastasis, in terms of the formation of the ‘pre-metastatic niche’ . In response to these soluble factors, tumor-associated cells, such as hematopoietic progenitor cells and macrophages, cluster at these pre-metastatic niches, which then creates an environment that is conducive to tumor-cell adhesion and invasion [85, 86]. Kaplan and collaborators  first showed that a specific population of bone-marrow-derived cells can cluster at pre-metastatic sites. These cells express VLA4 (also known as integrin α4β1) and the hematopoietic progenitor markers VEGFR1, c-KIT, SCA1 and CD11b. The recruitment of these cells to a pre-metastatic niche precedes the arrival of the tumor cells; therefore, these cells initiate and maintain the pre-metastatic niche. In examining the pre-metastatic lung in mice with syngeneic Lewis lung or B16 melanoma tumors implanted intradermally in the flank, Hiratsuka and collaborators  showed that VEGF-A, TNFα and TGF-β secreted by the primary tumors induced the expression of the S100A8 and S100A9 inflammatory proteins within the parenchyma of the lung. These proteins, in turn, induce expression of the chemoattractant serum amyloid A3 (SAA3) protein, which is responsible for the recruitment of tumor cells and CD11b+ myeloid cells to the pre-metastatic lung. The intravenous administration of a neutralizing anti-SAA3 antibody to these tumor-bearing mice was sufficient to inhibit tumor-cell mobilization to the lung and the colonization in the lung parenchyma by bone-marrow-derived cells; this resulted in a remarkable reduction in tumor-cell colonization of the lung. Furthermore, they demonstrated that SAA3 causes its own secretion through a positive-feedback mechanism that is mediated by Toll-like receptor 4 (TLR4). The activation of TLR4 induces NF-κB pathway, which is a hallmark of inflammation . This finding raises the possibility that in the pre-metastatic niche, NF-κB functions in the preparation of the metastatic-like environment for the primary tumor cells. TLR4 has a central role during the inflammatory-like response in the pre-metastatic niche. Indeed, deletion of TLR4 results in a dramatic decrease of the number of metastatic tumor sites and a reduction in the engraftment of CD11b+ myeloid cells at these sites. Additionally, CD11b+ myeloid cell recruitment to future metastatic sites is impaired in TLR4−/− mice, which suggests that TLR signaling has a critical role in the cross-talk between the tumor cells and the bone-marrow-derived cells during pre-metastatic niche formation.
Recently, S100A4 was shown to generate the pre-metastatic niche. Similar to the processes induced at the site of the primary tumor, at the location of secondary tumor development, and in combination with other factors (such as cytokines), the tumor-derived factor RANTES promotes the release of S100A4, which stimulates T-cell attraction and T-cell production of proinflammatory cytokines (e.g. G-CSF); these, in turn, will attract myeloid cells to the pre-metastatic niche [10, 11].
Erler and colleagues  uncovered an important role for lysyl oxidase (LOX) in the formation of the pre-metastatic niche. Once secreted into the circulation from the hypoxic environment of primary breast tumors, LOX can co-localize with fibronectin at sites of future metastasis, where it serves to cross-link collagen IV in the basement membrane of the lung. This increases the adherence of CD11b+ myeloid cells, which produce the MMP2 that cleaves collagen IV and leads to invasion of the lung and further recruitment of bone-marrow-derived cells in a positive feedback loop. This thus culminates in recruitment of the metastasizing tumor cells. Fibronectin deposition also appears to be a critical factor in the regulation of the formation of the pre-metastatic niche, and fibronectin matrices have been shown to provide specific microenvironments for the regulation of LOX catalytic activity . Therefore, it is likely that the initial deposition of fibronectin and the function of LOX during pre-metastatic niche formation give rise to the generation of a suitable form of the ECM to facilitate the recruitment of bone-marrow-derived cells and other mesenchymal cells. The contribution of the ECM to the engagement of tumor cells at the pre-metastatic niche is further underlined by a recent study of Oskarsson and collaborators . They demonstrated that TNC engages the Notch and Wnt pathways, to support the fitness of initiating breast-cancer cells during the establishment of lung metastases. Interference with cancer-cell-derived TNC production results in suppression of the survival and expansion of micrometastatic colonies. They also showed that cancer-cell-derived TNC provides a head-start for the viability of breast cancer cells in the pre-metastatic niche setting, and remains critical until TNC from the infiltrating myofibroblasts or other stromal sources accumulates in the expanding nodules.
Finally, tumor-derived factors, such as VEGF-A, can have angiomodulatory effects that induce changes in the local microvasculature. This can occur before the arrival of the tumor cells at sites of future metastasis, aiding in extravasation and the clustering of tumor-associated myeloid cells, which then activate platelets, finally followed by the seeding of the first tumor cell.
In the pre-metastatic niche, along with the stromal and endothelial cells that reside in the tissue parenchyma, the newly recruited myeloid cells provide a platform of chemokines, growth factors, matrix-degrading enzymes and adhesion molecules, thereby accelerating the assembly of the metastatic lesion . It has been shown that tumor-secreted factors induce myeloid cells to secrete tumor-promoting cytokines such as TNFα. This last cytokine, TNFα, affects several steps in the metastatic process, which include increased tumor-cell proliferation, increased vascular permeability, and recruitment of other host cells . In the pre-metastatic lung, VEGF-A stimulates endothelial cells and CD11b+/VEGFR1+ myeloid cells to express and secrete MMP9 . The consequent MMP9 induction of tissue remodeling can serve both to facilitate tumor-cell invasion and to release growth factors and chemokines, which will include the soluble c-KIT ligand. This will, in turn, further recruit bone-marrow-derived progenitor cells and tumor cells that express the c-KIT receptor . Moreover, the myeloid cells express osteopontin, a protein that has been implicated in tumor-cell adhesion and survival, and in the regulation of MMP activity, which also inhibits the host immune defense [92, 93]. This finding suggests that in the pre-metastatic niche, as in the primary tumor microenvironment, the myeloid cells have an immunoregulatory function, which creates immune sites in which the malignant cells can survive and proliferate without detection.
Recently Møller and collaborators  showed that stromal fibroblasts can have important roles in organs where metastases develop, in the support of metastatic organ colonization. In the pre-metastatic niche, the factors produced by disseminated tumor cells, trapped in the peripheral organs, or even released from the primary tumor itself, serve to down-regulate Fibulin-5 (FBLN5), which is mainly present in stromal fibroblasts within the peripheral organs. FBLN5 is a 66 kDa glycoprotein that belongs to the group of modulators of cell–ECM interactions, known as the matricellular proteins, and it is essential for the formation of elastic fibers. Møller and collaborators  demonstrated that FBLN5 expression in fibroblasts suppresses metastasis formation by inhibition of the production of MMP9 and by reduction in the invasive behavior of fibroblasts. Therefore, the down-regulation of FBLN5 in stromal fibroblasts driven by tumor-derived factors results in the up-regulation of MMP9, ECM remodeling, stimulation of angiogenesis, and invasion of fibroblasts. All these events will create a fertile milieu within which tumor cells can develop metastases .
Macrophages (F4/80+ CSF-1R+ CD11b+ Gr1− CX3CR1high CCR2high VEGFR1high)
Recent studies have shown that macrophages are associated with tumors at metastatic sites  and that they have important roles not only in promoting primary tumor progression to the metastatic phenotype, but also in the establishment and growth of metastases. Using three different and independent methods of macrophage depletion, Qian and collaborators  showed that macrophages have a significant role in the extravasation of breast-cancer metastatic cells, as well as in their establishment and growth in the lung. These functions were ascribed to a specific macrophage subpopulation, which is characterized by a cell-surface-marker signature of F4/80+ CSF-1R+ CD11b+ Gr1− CX3CR1high CCR2high and VEGFR1high. The recruitment of these macrophages to pulmonary metastases is independent of the breast-cancer metastatic cell type and the species of origin (mouse or human), and this was also seen in spontaneous metastases derived either from primary autochthonous mammary tumors or from xenotransplants of human breast-cancer cells in immunocompromized mice. The model for macrophage enhancement of metastasis hypothesizes that following arrest of the tumor cells in the capillaries of the metastasis target organ, monocytes are quickly recruited and differentiated in situ into a metastasis-associated macrophage phenotype with a distinctly defined cell-surface-marker signature. This recruitment is at least in part under the influence of locally synthesized CSF-1. In addition these macrophages express receptors for the macrophage chemotactic cytokines CCL2 and VEGF (CCR2 and VEGFR1, respectively); it is likely that such signaling molecules have a role in this recruitment process. These CD11b+ macrophages recognize extravasating tumor cells and interact with them directly, to help them to invade the lung parenchyma, probably through secretion of proteases, and growth, motility and survival factors. In the absence of these newly differentiated macrophages, this process of tumor-cell extravasation is very inefficient and the tumor cells rapidly die by apoptosis; thus, the seeding efficiency is very low. Once extravascular, tumor cells continue to send signals to recruit, and to also possibly influence differentiation of macrophages into trophic types  that further enhance tumor-cell viability and growth. When the tumors attain a certain size, these macrophages provide angiogenic factors, such as VEGF-A and angiopoietins, accelerate recruitment of other inflammatory cells, and secrete proteases. These actions thus further remodel the ECM , and lead to continuous metastasis growth.
Conclusions and future remarks
It is now recognized that the tumor microenvironment undergoes extensive changes during the evolution and progression of cancers, and that this is a major factor in the determination of the survival and growth of disseminated tumor cells at potential metastatic sites. There is an extensive interplay between tumor cells and tumor microenvironment cells, in which the incipient neoplasias recruit and activate stromal cell types that assemble in an initial pre-neoplastic stroma. The latter, in turn, responds by enhancing the neoplastic phenotype of the nearby cancer cells, which again feed signaling back to the stroma, to continue its reprogramming. Therefore, a full understanding of both the tumor biology and the molecular mechanisms underlying this development and malignant progression requires the study of both of these individual specialized cell types within the tumor microenvironment. Several studies have provided insights towards the definition of which molecular components facilitate the communication between these cells and tumor cells. These findings show that it is conceivable that the cells of the tumor microenvironment can serve as novel therapeutic targets in the treatment of cancers.
To this purpose, a few questions need to be addressed. For example, within the tumor microenvironment, which cell type has the major role in the modulation of the functions of other cell types and of cancer cells? Which cell type shows the highest level of cross-talk with both tumor cells and the other cell types? At this time, it will be difficult to answer these questions, unless we produce ‘ad-hoc’ animal models in the near future that can reproduce the system of an interaction network to prompt the formation of the mestatatic niche. We believe that, most probably, fibroblasts are the earliest cells recruited by the incipient neoplasia. CAFs modulate the malignant phenotype of the tumor cells, communicate with the other cell types within the tumor microenvironment, remodel the ECM, contribute to the recruitment of immune cells, such as MDSCs, TAMs and T cells, and concur to the modulation of the immunosuppressive functions of MDSCs.
These features make the CAFs new potential targets in cancer therapy. Several strategies to inhibit either CAF activation, or CAF-derived factors (e.g. TNC, HGF, uPA, SDF1) have been applied in pre-clinical studies of cancer therapies, and the results have shown efficacy in the inhibition of tumor growth and invasion (for an extensive review, see Gonda et al. ). As summarized in the present review, the extensive interplay between tumor cells and the cells that populate the tumor microenvironment, and their different subcellular types, and the genetic events that modulate the intracellular signaling pathways (mainly the Wnt, NF-κB and Notch signaling pathways) point to the future in the development of tumor microenvironment therapeutics.
Within the tumor microenvironment, the immune inflammatory cells contribute to the generation of an immunosuppressive environment, which thus promotes tumor immune escape. Based on these observations, several immuno-therapeutic approaches have been developed to target the immune cells that infiltrate the tumor. Mast cells have an extensive network of communication, both with tumor cells and with the other immune cells. Through this communication network, mast cells modulate tumor-cell proliferation and invasion, and the development, survival, proliferation, migration, maturation or/and function of the other immune cells. For these reasons, the modulation of mast-cell recruitment, viability, activity, or mediator release patterns at malignant sites might be relevant to control tumor growth and metastasis. The immuno-therapeutic approaches at this time include the use of drugs that inhibit mediator release, mast-cell-derived proteases, and the SCF/c-KIT signaling pathway (for an extensive review, see Groot Kormelink et al. ).
TAMs and MDSCs also have important immunosuppressive functions, and therefore they have been considered as targets for the development of new anti-cancer treatments. Some anti-angiogenic agents (such as thalidomide linomide, pentoxifyline and genistein) can inhibit TAM recruitment and reduce tumor size . Similarly, in a murine breast-cancer model, treatment with Met-CCL5 (a receptor antagonist) led to a decreased number of infiltrating macrophages that was associated with a significantly reduced tumor size . Several immuno-therapeutic strategies that target MDSCs and that can neutralize their immunosuppressive effects have been reported in both animal models and human. These strategies include approaches that are aimed at the induction of differentiation of these immature cells, or of a decrease in their number and tumor infiltration, or at interfering with their immunosuppressive functions. Although these approaches gave encouraging results in vitro and in in vivo preclinical studies, with cultured cells and tumor-bearing animal models, respectively, disappointing or controversial results were then reported in clinical trials (for an extensive review, see Tadmor et al. ). Therefore, although further studies will be needed to determine which cell(s) is/are the best therapeutic target(s) and which drugs are the most efficient and selective, the therapeutic targeting of tumor microenvironment cells in human might represent a valuable strategy to complement conventional anti-cancer strategies.
As described above, the succession of reciprocal cancer-cell to stromal-cell interactions must be repeated in distant tissues as disseminated cancer cells proceed to colonize their new organ sites and to specify the tissue microenvironments that further influence this complex communication effort. These findings support the rationale for targeting the microenvironment of the metastatic lesion in conjunction with targeting the tumor cells directly and the primary tumor microenvironment, to improve the treatment of metastatic disease. Probably the best window of opportunity for the control of metastatic disease will be the time period between metastatic seeding and clinical detection of metastasis. Indeed, at this time, the tumor cells are likely to be vulnerable to therapeutic agents, and patients can be expected to be in their optimal physical condition to endure treatment. The therapeutic strategies can be used to inhibit both the recruitment and function of specific cell populations that contribute to the recruitment, extravasation, establishment and growth of tumor cells, such as myeloid progenitor cells and macrophages, or the molecules involved in these processes. For this purpose, the therapeutic approaches should aim to inhibit the egress of the myeloid progenitors from bone marrow or their setting in the pre-metastatic niche (for example using anti-LOX or anti-fibronectin antibodies). Another approach could be aimed at the neutralizing of these tumor-derived factors that mediate the establishment of the pre-metastatic niche (such as RANTES, VEGF-A, TNFα, TGF-β), or the factors of the host tissue that are involved in the recruitment of myeloid progenitors and tumor cells (such as S100A4, S100A8, S100A9, SAA3), or the signaling pathway(s) involved in the cross-talk between tumor cells and bone-marrow-derived cells during pre-metastatic niche formation (such as TLR signaling). In addition, the therapeutic targeting of TNC might decrease the survival of cancer cells, and thus reduce metastasis formation.
In conclusion, further studies on the mechanisms of the cross-talk between tumor cells and the tumor microenvironment will allow better definition of the nature of these interactions, and will finally unravel the roles of these cells from the tumor microenvironment in cancer progression and metastasis formation. This issue represents the new major challenge in cancer genetics, and it will allow us to harness these relationships for clinical benefit.
We acknowledge European GRANT-FP7-Tumic HEALTH-F2-2008-201662 (MZ). Associazione Italiana per la Ricerca sul Cancro, AIRC (MZ) and Associazione Italiana per la Lotta al Neuroblastoma 2008–2010 (MZ), PRIN 2008 E5AZ5F (MZ). DS is supported by the Department of Biochemistry and Biotechnological Medicine, Università Federico II, Naples, Italy, and CEINGE, Centro di Ingegneria Genetica e Biotecnologie Avanzate, Naples, Italy.
Conflict of interest
The authors declare that they have no competing interests as defined by Clinical & Experimental Metastasis, or other interests that might be perceived as influencing the results and discussion reported in this manuscript.