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 (Mphi) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mphi and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity.


METHODS
Mphi 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). Mphi 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 (TNFalpha) when stimulated by lipopolysaccharide/interferon gamma (LPS/IFNgamma); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, Mphi 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 Mphi 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 Mphi to switch from M1 Mphi 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/IFNgamma stimulation, an increased secretion of IL-10, a decreased secretion of TNFalpha in response to LPS/IFNgamma 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 Mphi and MDA-MB-231 transformed M1 Mphi 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 Mphi phenotype or cytokine secretion profiles.
Joimel
Ulrich
U
Laboratoire M,E,R,C,I - EA 3829, Faculté de Médecine et de Pharmacie, Université de Rouen, 76183 Rouen cedex, France. ulrich.joimel@gmail.com
Gest
Caroline
C
Soria
Jeannette
J
Pritchard
Linda-Louise
LL
Alexandre
Jérôme
J
Laurent
Marc
M
Blot
Emmanuel
E
Cazin
Lionel
L
Vannier
Jean-Pierre
JP
Varin
Rémi
R
Li
Hong
H
Soria
Claudine
C
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000098:blood supply* / Q000188:drug therapy / Q000473:pathology*; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D002642:Chick Embryo; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008982:Molybdenum / Q000494:pharmacology*; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D011994:Recombinant Proteins
D020533:Angiogenesis Inhibitors; D018925:Chemokines; D017077:Culture Media, Conditioned; D016207:Cytokines; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D008982:Molybdenum; D007371:Interferon-gamma; C020809:tetrathiomolybdate