Angiogenesis and the tumour microenvironment.
It is now well accepted that tumours are highly complex tissues in which cancer cells have conscripted and subverted normal cell types to serve as active collaborators in their neoplastic process creating an aberrant organ made of specific cells (cancer cells), not all genetically identical, and supporting or stromal cells . To complete the organ-like structure, there are soluble factors: cytokines, chemokines and blood vessels. Solid tumours require new blood vessel growth (neovascularisation) if they are to grow beyond 2-3 mm in diameter . These new vessels not only provide the oxygen and the nutrients/metabolic demands of the tumour, but also provide potential routes for tumour dissemination and metastasis.
What we call the tumour microenvironment comprises both a cellular component and a soluble component. The cellular component is made up of stromal cells (fibroblasts and macrophages), inflammatory cells [macrophages, lymphocytes, natural killer (NK) cells, dendritic cells (DCs)] and endothelial cells, whereas the soluble component contains cytokines, chemokines and stromal, inflammatory and angiogenic factors (both pro- and anti-angiogenic).
Cancer cells produce and induce multiple regulators of angiogenesis from the surrounding stromal cells. As cancer is an inflammatory process, many factors are released in the cross-talk between cancer cells, their microenvironment and the host response/immune cells (see Panel 1). Some of these factors are proangiogenic whereas others are anti-angiogenic. When the 'angiogenic switch' is triggered, the pro-angiogenic factors outnumber the endogenous anti-angiogenic factors and thus tumoral angiogenesis occurs (Figure 1). The vascular endothelial growth factor (VEGF) pathway is well established as one of the key angiogenesis regulators . Activation of the VEGF receptor (VEGFR) triggers a network of signalling processes that promote endothelial cell growth, migration and survival.
The VEGF family includes VEGF-A, VEGF-B, VEGF-C and VEGF-D and placenta growth factor (PlGF). It is now clear that angiogenesis is a very complex process and that other factors [including angiopoietins and platelet-derived growth factor (PDGF)] participate in the assembly and maturation of the vessel wall .
[FIGURE 1 OMITTED]
One of the targeted approaches most widely studied for the treatment of colorectal cancer has been the inhibition of angiogenesis. As for all tissues, tumour cells require oxygen and nutrients (i.e. a blood supply) to survive and grow. The observation that tumour growth can be accompanied by increased vascularity led to the suggestion in the 1990s that anti-angiogenesis might be an effective anticancer strategy . It is known that tumour growth is dependent on angiogenesis. Not only does neovascularisation permit further growth of the primary tumour, but it also provides a pathway for migrating tumour cells to establish distant metastases.
For the organism, cancer constitutes an inflammatory process, where host cells surround and infiltrate the tumour. This low-intensity but continuous inflammation has important effects on the development and future growth of tumours . Experimental data have now demonstrated a role in promoting tumour growth and progression for the individual host-cell components, including endothelial cells [5-7], macrophages [8-10] and cancer-associated fibroblasts . It is interesting that most cells of the immune system are endowed with potential dual functions; while able to reject tumours on the one hand, they are also recruited and become 'mercenary cells' for the same cancer they should destroy. Immune cells within a tumour can be considered as 'double players'; under specific stimulation they can produce anti-tumour cytokines, directly to destroy cancer cells and indirectly to target the supporting host cells.
Primary tumours contain fibroblasts/myofibroblasts (carcinoma-associated fibroblasts; CAFs) which are routinely identified by markers such as alpha-smooth muscle actin, vimentin, S100A4 protein/fibroblast specific protein-1 and type I collagen. These cells have been well studied, particularly in invasive breast carcinomas, and found to promote the growth of mammary carcinoma cells and to enhance tumour angiogenesis more effectively than comparable cells derived from tissue adjacent to these tumours. CAFs are capable of secreting elevated levels of stromal cell-derived factor 1 (SDF-1), also called CXCL12, which plays a central role in the promotion of tumour growth and angiogenesis; CAF-derived SDF-1 not only stimulates carcinoma growth directly through the CXCR4 receptor expressed on tumour cells, but also serves to recruit endothelial progenitor cells into tumours [11,12]. Fibroblasts, which are components of the tissue stroma, are an example of how cancer cells subvert normal cell types to serve as active collaborators for neoplastic growth and progression.
The role of macrophages in tumour-promoting growth is well established [9,10,13]. Under certain conditions they can adopt different phenotypes: the M1 phenotype associated with tissue microbial killing, and the M2 phenotype associated with tissue remodelling and angiogenesis. The latter is the predominant phenotype in tumour tissues and they have been named tumour-associated macrophages (TAMs). It is now clear that macrophages can produce a series of angiogenic factors, including VEGF. In hypoxic conditions, TAMs promote upregulation of molecules related to angiogenesis . These include VEGF, fibroblast growth factor (FGF)2, PDGF, hepotocyte growth factor (HGF), PIGF, cyclooxygenase-2 and angiopoietin 1. Macrophages (M2/TAM) also produce matrix metalloproteinase (MMP)9 and MMP12  and the invasive signature of hypoxic macrophages is characterised by upregulation of MMP1 and MMP7. Other macrophage-related cytokines induced under hypoxic conditions are tumour necrosis factor (TNF)[alpha], interferon (IFN)[gamma], interleukin (IL)-10 and arginase [16,17] and the chemokines CXCL1, CCL3, CXCL8 (IL-8), and CXCL12 with its receptor CXCR4. Furthermore, TAMs express VEGFR1 (flt1)  and respond to its activation by migrating and modulating their biological activity in response to VEGFR1 ligands  such as VEGF produced by tumour cells. This fact may explain their switching to a pro-tumour phenotype.
These immune cells, which act as co-factors in microbial killing, have recently been shown to play an important role in angiogenesis in both physiological and pathological conditions. In many in vitro as well as in vivo models, neutrophils are primary regulators of angiogenesis [20-24]. Neutrophil-dependent angiogenesis in vivo has been shown to require VEGF-A released by neutrophils during recruitment and stimulation by CXCL1  or granulocyte colonystimulating factor . Neutrophils have also been found to have a role in angiogenesis mediated by IL-8, which is important in ras oncogene-driven tumour progression [27,28]. In addition, neutrophils are capable of producing multiple angiogenic factors [21,26,29] and an equally wide range of anti-tumour and anti-angiogenic factors [29-31].
Recruitment of leukocytes and NK cells inside the tumour mass may be explained by the high levels of VEGF inside the tumour itself. It is known that endothelial cells exposed to VEGF upregulate the expression of the intercellular adhesion molecule ICAM-1, which plays a key role in CD18-mediated neutrophil adhesion and recruitment [21,32]. In general, NK cells are considered to be potent negative regulators of angiogenesis, in particular in response to IL-12. However, here again we find a dual effect of these cells. The so-called dNK cells, a subset of NK cells, appear to promote angiogenesis. These cells, rather than being cytotoxic, secrete VEGF, PIGF and IL-8 . Transforming growth factor (TGF)-[beta], which is a cytokine frequently found in the tumour microenvironment, is able to switch peripheral NK cells to a secretory dNK phenotype . As in the case of TAMs, neutrophils also form part of the tumour microenvironment (dNK) and they may contribute to angiogenesis.
Although DCs are the prototype of antigen-presenting cells (APCs), they have a role in regulating angiogenesis as they are able to produce TNF[alpha], IFN[gamma], type I IFN, IL-12 and TGF-[beta]. However, there are data to support a strong inhibitory effect of VEGF family members on DC maturation . VEGFR1, which is mainly expressed on immature DCs, is the key receptor for this VEGF inhibitory effect. It has been shown in vitro that angiostatic factors, such as thrombospondin-1 and the chemokines CXCL4 and CXCL14, appear to enhance DC maturation and APC function. On the other hand, osteopontin, a cytokine that favours angiogenesis, enhances DC function at many stages. DCs may contribute to angiogenesis either by releasing pro-angiogenic factors or by transdifferentiation into endothelial-like cells. A direct contribution to the expanding vasculature can be postulated, where a population of CD11c+ myeloid cells exhibits co-expression of endothelial cells and DCs markers.
Myeloid-derived suppressor cells
It is well known that bone marrow-derived stem cells are highly plastic and that they can colonise many organs following tissue injury or inflammation . However, there are other tumour-associated cells of myeloid origin that can be involved in the promotion of tumour angiogenesis such as the myeloid-derived suppressor cells (MDSCs). These cells are composed of immature macrophages, granulocytes and progenitors of DCs. They inhibit tumour-specific T-lymphocytes through different complex molecular mechanisms [37,38] and also promote angiogenesis . In mice, this subpopulation of myeloid cells can be identified by simultaneous expression of CD11b and Gr-1 surface markers and their levels are significantly increased in the spleen and bone marrow of cancer patients. These cells are able to produce and release angiogenic factors such as MMP9 and VEGF, and act directly by differentiating into endothelial cells themselves. In two animal models, the MC26 colorectal carcinoma and the 3LL Lewis lung carcinoma, MDSCs have been shown to participate in accelerating tumour growth by promoting tumour angiogenesis . This process is mediated by high MMP9 expression. It is interesting that MMP9 deletion in these cells completely abolished their tumour-promoting ability. In addition to their pro-angiogenic activity, these cells may play a role in maintaining T-cell tolerance in the tumourbearing host. Recruitment and expansion of MDSCs can occur as a result of the high levels of granulocyte/macrophage colony-stimulating factor and/or IL-1 found in chronic inflammatory processes. In addition, MDSCs produce the cytokines IL-10 and TGF-[beta], both of which are involved in the generation of activated DCs and in induction of T-regulatory cells.
Thus MDSCs and, in particular, immature macrophages and DCs may participate in the inhibition of CD4+ and CD8+ T cells and the inhibition of their potential anti-angiogenic effect mediated by the type 1 cytokines IFN[gamma], CXCL10 and CXCL9.
Tumours as aberrant organs are composed of heterogeneous cell populations which include tumour cells and the tumour microenvironment (supportive/stromal cells, host immune/inflammatory cells and soluble factors). There is a low level of inflammation between cancer cells and the surrounding host tissues with a continuous cross-talk among different cell populations that creates a milieu of many soluble factors, some pro- and some anti-angiogenic. A better understanding of the multiple players within the tumour microenvironment, cellular and humoural, will help to determine the relative contribution of each of these factors to tumour formation and to better design the appropriate means to control tumour-induced angiogenesis.
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Correspondence to: Pere Gascon (email: email@example.com)
Vanessa Almendro and Pere Gascon
Division of Medical Oncology, Department of Hematology-Oncology, Hospital Clinic, Barcelona University, Spain
PANEL 1: Factors involved in angiogenesis Tumour cells express: * ELR+ CXC chemokines (*): stimulate angiogenesis * IL-8 * GRO[alpha] * GRO[beta] * GRO[gamma] * ENA-78 stimulate tumour cell growth * GCP-2 * PBP-2 * CCR2 ligands: recruitment of monocycles and macrophages * MCP-1 * MCP-3 * MCP-4 * ELR- CXC chemokines: recruitment of T and NK cells which can destroy vasculature - they block angiogenesis * PF-4 * IP-10 * MIG * I-TAC * 6ckine Monocytes/macrophages express: * CCR2 ligands: positive feedback loop for recruitment of monocytes and macrophages * Angiogenic factors * stimulate angiogenesis bFGF VEGF ECM modulators Proteases * Enhance the production of IL-8, bFGF and BEGF, which promote angiogenesis and tumour growth TNF[alpha] IL-[alpha] Hypoxic tissues express: * IL-8 * VEGF stimulate angiogenesis by upregulating MMP-2 and MMP-9 which results in destruction of extracellular matrix so that blood vessels can infiltrate these areas and grow * ELR+ CXC chemokines are chemoquines bearing the glutamic acid-leucine-arginine (ELR) motif. PBP-2, penicilin-binding protein 2; MCP, monocyte chemotactic protein; IL-8, interleukin-8.
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|Title Annotation:||Feature Article|
|Author:||Almendro, Vanessa; Gascon, Pere|
|Publication:||Advances in Gastrointestinal Cancer|
|Date:||Jun 1, 2008|
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