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

Background: Infiltration by macrophages (M phi) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between M phi and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity. Methods: M phi 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). M phi 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 alpha (TNF alpha) when stimulated by lipopolysaccharide/interferon gamma (LPS/IFN gamma); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, M phi and the coculture were characterised by an antibody cytokine array. All experiments were carried out both in presence and in absence of TM. Results: Incubation of M phi 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 M phi to switch from M1 M phi 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/IFN gamma stimulation, an increased secretion of IL-10, a decreased secretion of TNF alpha in response to LPS/IFN gamma 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. Conclusions: Cooperation between M phi and MDA-MB-231 transformed M1 M phi 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 M phi phenotype or cytokine secretion profiles.