Lactate adversely affects the in vitro formation of endothelial cell tubular structures through the action of TGF-β1

Abstract

When lactate accumulation in a tumor microenvironment reaches an average concentration of 10–20 mM, it tends to reflect a high degree of malignancy. However, the hypothesis that tumor-derived lactate has a number of partially adverse biological effects on malignant and tumor-associated host cells requires further evidence. The present study attempted to evaluate the impact of lactate on the process of angiogenesis, in particular on the formation of tubular structures. The endothelial cell (EC) network in desmoplastic breast tumors is primarily located in areas of reactive fibroblastic stroma. We employed a fibroblast–endothelial cell co-culture model as in vitro angiogenesis system normally producing florid in vitro tubule formation to analyze this situation. In contrast to previous studies, we found that lactate significantly reduces EC network formation in a dose-dependent manner as quantified by semi-automated morphometric analyses following immunohistochemical staining. The decrease in CD31-positive tubular structures and the number of intersections was independent of VEGF supplementation and became more pronounced in the presence of protons. The number of cells, primarily of the fibroblast population, was reduced but cell loss could not be attributed to a decrease in proliferative activity or pronounced apoptotic cell death. Treatment with 10 mM lactate was accompanied by enhanced mRNA expression and release of TGF-β1, which also shows anti-angiogenic activity in the model. Both TGF-β1 and lactate induced myofibroblastic differentiation adjacent to the EC tubular structures. The lactate response on the EC network was diminished by TGF-β1 neutralization, indicating a causal relationship between lactate and TGF-β1 in the finely tuned processes of vessel formation and maturation which may also occur in vivo within tumor tissue.