Stimulation of angiogenesis resulting from cooperation between macrophages and MDA-MB-231 breast cancer cells: proposed molecular mechanism and effect of tetrathiomolybdate

Abstract

BackgroundInfiltration by macrophages (Mφ) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mφ and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity.MethodsMφ coincubated with MDA-MB-231 were used as a model to mimic the inflammatory microenvironment. Angiogenesis induced by the culture media was tested in the chick chorioallantoic membrane (CAM). Mφ phenotype was evaluated by 1) expression of the M1 marker CD80, and secretion of interleukin 10 (IL-10), an M2 marker; 2) capacity to secrete Tumour Necrosis Factor α (TNFα) when stimulated by lipopolysaccharide/interferon γ (LPS/IFNγ); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, Mφ and the coculture were characterised by an antibody cytokine array. All experiments were carried out both in presence and in absence of TM.ResultsIncubation of Mφ with MDA-MB-231 induced a pro-angiogenic effect in the CAM. It emerged that the angiogenic activity of the coculture is due to the capacity of Mφ to switch from M1 Mφ towards M2, probably due to an increase in Macrophage Colony Stimulating Factor. This M1-M2 switch was shown by a decreased expression of CD80 upon LPS/IFNγ stimulation, an increased secretion of IL-10, a decreased secretion of TNFα in response to LPS/IFNγ and an inability to potentiate apoptosis. At the molecular level, the angiogenic activity of the coculture medium can be explained by the secretion of CXC chemokines/ELR+ and CC chemokines. Although TM did not modify either the M2 phenotype in the coculture or the profile of the secreted chemokines, it did decrease the angiogenic activity of the coculture medium, suggesting that TM inhibited angiogenic activity by interfering with the endothelial cell signalling induced by these chemokines.ConclusionsCooperation between Mφ and MDA-MB-231 transformed M1 Mφ to an angiogenic, M2 phenotype, attested by secretion of CXC chemokines/ELR+ and CC chemokines. TM inhibited this coculture-induced increase in angiogenic activity, without affecting either Mφ phenotype or cytokine secretion profiles.

Molecular Mechanisms Underlying Lymphovascular Invasion in Invasive Breast Cancer

“…This work provides evidence for a synergistic interaction between macrophages and tumour cells during cell migration in vivo and could explain, at least in part, the increased tendency of basal-like BCs, which overexpress EGF receptor, to be more node negative and show haematogenous rather than lymphatic dissemination to viscera and bone [60,61]. Macrophages are well known to produce interleukin-10 and angiogenic factors [62]. Within the hypoxic environment, growth factors that promote angiogenesis and lymphangiogenesis are up-regulated and secreted by malignant cells [63] and serve as chemo-attractants for macrophages.…”

Behind the Link between Copper and Angiogenesis: Established Mechanisms and an Overview on the Role of Vascular Copper Transport Systems

“…VEGF and angiopoietins) and metallo-proteases [252,253]. It has been shown that TM-induced copper starvation in a co-culture of malignant breast cancer cells and macrophages does not interfere with the local release of angiogenic cytokines, but compromises downstream angiogenic-dependent cell signalling [253]. An in vitro study on RAW264.7 macrophage cell cultures exposed to hypoxia, a condition of a real tumour context, showed relevant changes in the expression and activity of cell copper transport proteins, reflecting a higher delivery of the metal to ceruloplasmin [134], and an obvious elevation of blood-circulating copper in a bioavailable form.…”

“…Copper actively contributes the inflammatory cross-talk between cancer cells and tumour-recruited macrophages which locally stimulate angiogenesis by releasing a wide array of pro-angiogenic factors (e.g. VEGF and angiopoietins) and metallo-proteases [252,253]. It has been shown that TM-induced copper starvation in a co-culture of malignant breast cancer cells and macrophages does not interfere with the local release of angiogenic cytokines, but compromises downstream angiogenic-dependent cell signalling [253].…”

Molecular Pathology of Breast Cancer Metastasis

“…TAMs are well-known to produce Interleukin-10 and angiogenic factors [61], and they aggregate nearby necrotic and hypoxic areas of the tumour microenvironment [62]. Hence, and under this hypoxic environment, growth factors that promote blood and lymph neovascularization are upregulated and secreted by the malignant cells.…”