The Inflammatory Milieu of Tumors: Cytokines and Chemokines that Affect Tumor Growth and Metastasis

Tumor cells making up the primary tumor and those in metastatic lesions are genetically and phenotypically different. It is safe to assume that the phenotype and functions of non-tumor cells such as endothelial cells, fibroblasts or macrophages residing in the microenvironment of primary tumors differ from those of similar non-tumor cells residing in metastatic microenvironments. It is therefore to be expected that the patho-biologic consequences of interactions between tumor cells and the microenvironments of the primary tumor or of the metastatic lesions will also be different.

Tumor-microenvironment interactions should, thus, be studied in the context of the site. This short review summarizes state of the art with respect to interactions between the brain microenvironment with brain-metastasizing tumor cells.

The capability of the immune system to generate a rapid and effective immune response aimed to eliminate aberrant cells is the key to protect the organism by the development of tumours.

However, tumours are able to evade the mechanisms of immune surveillance. Several lines of experimental and epidemiological evidence indicate that a “smouldering” inflammation is associated with most of, if not all, tumours and supports their progression.

Indeed, tumours promote a constant influx of myelomonocytic cells that play as key orchestrators of cancer-related inflammation, supporting proliferation and survival of malignant cells, subversion of adaptive immune response, angiogenesis, stroma remodeling and metastasis formation.

The connection between inflammation and cancer is unequivocal, but some of the mechanisms at the basis of such association are still unknown. Recent advances in the understanding of the cellular and molecular circuits of cancer-related inflammation as well as their potential relevance as diagnostic, prognostic and therapeutic targets are herein discussed.

Tumor Necrosis Factor (TNF) is a pleiotropic cytokine with a central role in immune homeostasis, inflammation and host defence. Depending on the cellular context, TNF can induce diverse effects such as necrosis, apoptosis, differentiation, angiogenesis, cell migration and immune cell activation. Accordingly, TNF is believed to play a significant role in tumor development and progression; on the other side, this cytokine has potent antineoplastic activity both in experimental and clinical models. In the present chapter we provide a particular insight in the biology of TNF, emphasizing its tumor-related features, and discuss the experimental and clinical evidence available in the current literature supporting anticancer as well as cancer-promoting effects. Recent findings that may provide new alternatives to further exploit the anticancer power of TNF are also discussed.

Interleukin-1 (IL-1) is a major “alarm” upstream pro-inflammatory cytokine that also affects immunity and hemopoiesis, by inducing cytokine cascades. In the tumor arena, IL-1 is produced by malignant or microenvironmental cells. As a pleiotropic cytokine, IL-1 is involved in tumorigenesis and tumor invasiveness, but also in the activation of anti-tumor immunity. In the tumor milieu, IL-1 represents a feasible candidate cytokine for modulation, in order to tilt the balance between inflammation and immunity towards favorable induction of anti-tumor responses. IL-1α and IL-1β are the major agonists of IL-1, while IL-1Ra is a physiological inhibitor of pre-formed IL-1. In their secreted form, IL-1α and IL-1β bind to the same receptors and induce the same biological functions. However, IL-1α and IL-1β differ in their compartmentalization within the producing cell or the microenvironment. IL-1β is only active in its secreted form and mediates inflammation, which promotes carcinogenesis, tumor invasiveness and immunosuppression. On the other hand, IL-1α is mainly cell-associated; in the context of tumors, host- and tumor cell-derived IL-1α stimulates antitumor immunity, rather than inflammation. Recent breakthroughs in inflammasome biology and IL-1β processing/secretion, have spurred the development of novel anti-IL-1 agents which are being used in clinical trials in patients with diverse diseases with inflammatory manifestations. IL-1Ra is already FDA-approved and has been shown to be safe and efficient in alleviating symptoms of rheumatoid arthritis. In experimental cancer, IL-1Ra attenuates tumor-mediated inflammation and invasiveness. Better understanding of the integrative role of IL-1α and IL-1β in the malignant process will enable the application of novel IL-1 modulation approaches at the bedside, in cancer patients with minimal residual disease (MRD), as an adjunct to conventional approaches to reduce the tumor burden.

The tumor microenvironment is extremely complex and depends on tumor cell interaction with the responding host cells. Angiogenesis, or new blood vessel growth from pre-existing vasculature, is a preeminent feature of successful tumor growth of all solid tumors. While a number of factors produced by both the tumor cells and host responding cells have been discovered that promote tumorassociated angiogenesis, increasing evidence is growing to support the important role of CXC chemokines in this process. As a family of cytokines, the CXC chemokines are pleiotropic in their ability to regulate tumor-associated angiogenesis. In this chapter we will focus on the ELR+ CXC chemokines and their contribution to tumor-associated angiogenesis.

Many inflammatory mediators including chemokines, cytokines, growth factors and eicosanoids are expressed in the tumor milieu and contribute to both tumor growth and tumor control. This chapter will summarize our current understanding of one chemokine/chemokine receptor pair; the G-protein-coupled seven transmembrane chemokine receptor CXCR3 and three ligands, CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC). Endogenous CXCL9-11 can be detected at the mRNA and protein level in many tumors. In some malignancies, the presence of intratumor ligand is associated with the presence of infiltrating CXCR3+ T lymphocytes and/or NK cells and correlates with a better prognosis. Antiangiogenic properties of CXCL9-11 have also been demonstrated and likely contribute to the better prognosis associated with ligand detection. Endogenous expression of CXCR3 ligands is not always beneficial to the host. Direct stimulation of tumor cell proliferation has been demonstrated in some tumor models. Forced overexpression or intratumor injection of each ligand also has demonstrable antitumor activity in many cancer models. These therapeutic effects are often dependent on actions of CXCR3+ immune effector cells or CXCR3+ vascular endothelial cells. The CXCR3 receptor is expressed by many malignant cells and, in some cases, is shown to promote invasion and metastasis to distant sites where ligand is expressed. Multiple variants of CXCR3 exist; CXCR3-A appears to be more important in promoting metastasis whereas CXCR3-B may inhibit cell proliferation. Pharmacologic antagonism or gene-silencing of CXCR3 inhibits metastasis in preclinical models of malignancies of the colon, breast, and in osteosarcoma, and malignant melanoma, but the contribution of individual CXCR3 variants to this mechanism remains to be elucidated.

Cancer diseases are driven by intrinsic properties of cells that have undergone malignant transformation, aided by a supporting microenvironment. Often, the tumor milieu contains inflammatory cells and soluble inflammatory factors whose activities facilitate the growth of the tumor at the primary site, and also metastasis formation. This chapter will describe the roles of inflammatory members of the CC family of chemokines in cancer, focusing on CCL2 (MCP-1) and CCL5 (RANTES). In line with their activities in the immune setting, there are cases in which the two chemokines induce the recruitment of leukocytes with anti-tumor activities to tumors. However, this potential activity of CCL2 and CCL5 is overcome in the majority of malignant diseases by their very potent tumor-promoting functions. By using breast cancer as the test case, this chapter will describe the large variety of manners and mechanisms by which CCL2 and CCL5 promote malignancy, including: (1) The ability of both chemokines to change the type of leukocyte infiltrates in tumors so that the equilibrium is shifted towards the presence of cells that promote malignancy, such as tumor-associated macrophages (TAM) and Th17 cells. These functions of CCL2 and CCL5 take place in parallel to reduced anti-tumor T cell activities. (2) The angiogenic activities of CCL2 and its ability to “condition” the bone microenvironment towards “seeding” by the tumor cells. (3) The roles of CCL2 in promoting the presence of mesenchymal stem cells (MSC) in tumors, and the ability of CCL5 to mediate the promalignancy activities of these cells. (4) The direct activity of CCL5 (and to some extent also of CCL2) on the tumor cells, leading to their increased motility and invasion, and the stimulation of matrix metalloproteinase (MMP) production by the chemokine. These activities of the chemokines can together enhance metastatic dissemination. Therefore, the currently available information suggests that CCL2 and CCL5 should be considered as potential therapeutic targets and as diagnostic/prognostic tools in breast cancer and possibly in other malignant diseases.

The chemokine superfamily and its 48 ligands have been divided into two groups: inflammatory and homeostatic [1]. The inflammatory chemokines were initially defined as those whose production was strongly induced in various leukocytes or lymphocyte populations upon activation. This definition seemed accurate because many of the inflammatory chemokines were known to participate in the development of inflammatory responses. Some inflammatory chemokines represent some of the highest expressed proteins produced by activated leukocytes or lymphocytes (for example, CCL3 and CCL4, previously known as MIP1α and MIP1β). We now know that these ligands play very important roles in the control of inflammatory responses.

In contrast, homeostatic chemokines were defined as those that are constitutively expressed in certain tissues or by certain cells, in the apparent absence of a triggering stimulus. This definition (and subsequent division of the chemokines) became generally accepted after the biology of a few key homeostatic ligands became understood. Probably the best ‘prototype’ of an early homeostatic chemokine is CCL21. This chemokine is unique in other ways; it exhibits 6 cysteines instead of the classic 4 characteristic of most chemokines (in fact, because of this feature we called it 6Ckine when we first described it [2]). It was initially difficult to find a tissue with a strong expression of CCL21. This was because it is almost exclusively expressed by the high endothelial venules of the lymph nodes (in both the mouse and the human). This expression pattern represented an important clue to its function.

TGF-βs are pleiotropic regulatory proteins that are often highly overexpressed in the microenvironment of advanced tumors. Since nearly all cell types respond to TGF-β, TGF-βs are wellpositioned to integrate cellular responses in the parenchymal and stromal compartments of normal tissues and of tumors, and to mediate cross-talk between the two. Pre-clinical studies have uncovered complex roles for TGF-βs in tumorigenesis, with tumor suppressive and tumor promoting effects on both compartments. In this review, we discuss the biological activities of TGF-β that underlie these effects and address some of the mechanisms that determine which type of effect dominates at different stages of the tumorigenic process. Finally, we consider the prospects for therapeutic targeting of TGF-β in cancer, as a way to simultaneously normalize both the microenvironmental and parenchymal compartments of the tumor, and thus prevent or reverse tumor progression.

Clinical and epidemiologic studies have suggested an association between chronic inflammation and cancer development and have estimated that chronic inflammation accounts for 25% of malignancies worldwide [1-6]. However, the molecular and cellular mechanisms linking chronic inflammation to tumorigenesis remain largely unresolved. Tumor outgrowth and progression are multifactorial and complex processes which are dependent on the ability of the transformed tumor cells to organize their microenvironment. Similarly to every normal tissue, the tumor tissue is an organized, albeit abnormal structure that will support the growth of “functional cells,” i.e., tumor cells. Unlike normal tissue, the tumor cells and tissues are not functional and do not contribute to the well-being of the organism. In fact, the novel tumor tissue will damage other tissues and will spread to various functional microenvironments eventually leading to the collapse of the entire organism. Tumor cells are powerful tissue organizers capable of controlling and organizing multiple cell types, such as fibroblasts, vasculature cells (endothelial cells, pericytes and smooth muscle cells), and immune cells (lymphocytes, macrophages, dendritic cells, mast cells and neutrophils), to support their survival, outgrowth, and metastasis. The interactions of cancer cells with components of their tumor microenvironment are bidirectional and are crucial for cancer progression [7-9]. Metastasis of tumor cells is the major source of morbidity and mortality associated with cancer in humans. Metastasis is a complex process that requires the detachment of tumor cells from the primary lesion, invasion into vascular or lymphatic vessels or bloodstream, trafficking and homing of tumor cells to destination organs and finally survival and outgrowth of metastasized cells in their new microenvironment [10, 11]. Due to its multifactorial nature, metastasis formation is a rare event critically dependent on the interaction between a specific target organ and the arriving tumor cells [12]. In order to organize their microenvironment, tumor cells communicate with their surrounding microenvironment via a network of secreted growth factors, cytokines, and chemokines [13, 14]. These players are critical for the development, outgrowth, and progression of tumor cells. Therefore, gaining an understanding and control on their production, communication network and function keeps great promise for the development of novel and specific preventive and therapeutic treatments for cancer. In this review, we will summarize current knowledge, therapeutic approach and drugs in development.