Targeting MCT1 Blocks Tumor Angiogenesis Figure 4. Lactate triggers HIF-1 induction in normoxic endothelial cells. Lactate was used at 10 mM for 24-h. (A) HIF-1a mRNA was detected using qRT-PCR in HUVECs incubated with lactate. ns, non-significant; n = 4. (B and C). HIF-1a protein expression was detected by western blot in BAECs incubated with lactate and/or (B) 5 mg/ml Actinomycin D (**p,0.01, ***p,0.005, ns, non-significant compared to control, n = 3–4); (C) increasing concentrations of 2-oxoglutarate. *p,0.05, **p,0.01; n = 5–6. (D) ODD-driven luciferase activity was assessed in HUVECs incubated with 150 mM CoCl2 or lactate. **p,0.01; n = 5. (E) Intracellular pyruvate was detected using an enzymatic assay in HUVECs incubated with lactate. **p,0.01; n = 4. (F–H) HIF-1a protein expression was detected by western blot. (F) BAECs were incubated with lactate, oxamate (10 mM), and/or acetate (10 mM). **p,0.01, ***p,0.005, ns, non-significant compared to control cells without lactate; n = 4–16. (G) HUVECs transfected with the indicated siRNA were incubated with lactate. *p = 0.0170, **p = 0.0019 compared to control; ##p = 0.0056 compared to lactate alone; n = 3–9. (H) BAECs were incubated with pyruvate (10 mM), lactate, or not. **p,0.01 compared to control; n = 3–19. (A–H) Error bars reflect mean 6 SEM. doi:10.1371/journal.pone.0033418.g004 HIF-1 activators, nitric oxide (NO) and reactive oxygen species (ROS) [30], in our experimental conditions. Lactate-driven HIF- 1a and VEGFR2 protein expressions were indeed resistant to the NO synthase inhibitor Nv-nitro-L-arginine methyl ester (L- NAME) and to the general antioxidant N-acetyl-L-cysteine (NAC) (Figure 5E). Targeting MCT1 inhibits lactate-induced angiogenesis in tumors We next investigated whether lactate-induced HIF-1 activation conferred an angiogenic phenotype to ECs. We first observed that lactate increased EC motility, as illustrated by the increased capacity of confluent ECs to colonize a cell-free area in the presence of lactate compared to vehicle (Figure 6A). Lactate-induced migration was quantified in transwell experiments in which ECs in the upper chamber had to invade a layer of Matrigel matrix to reach the bottom well containing lactate. In this assay, 10 mM lactate promoted EC migration as efficiently as 10 ng/ml VEGF (Figure 6B). EC migration was partly inhibited by CHC, indicating that lactate uptake is as an integral part of the migratory phenotype. The involvement of MCT1 in lactate-induced endothelial invasion was further tested ex vivo. A 3D-culture of aortic rings in collagen gels was used to evaluate the outgrowth of linear endothelial structures from the preexisting vessel [35]. Both basal and lactate-induced endothelial responses were completely blocked by CHC (Figure 6C). The inhibition was sustained but reversible, as sprouting eventually resumed 14 days after the CHC treatment. The angiogenic conversion of ECs was also assayed on Matrigel [36]. In this assay, lactate alone promoted sustained endothelial tube formation (Figure 7A), a response that was completely inhibited using SU5402 (targeting VEGF and bFGF receptors), echinomycin (an inhibitor of the transcriptional activity of HIF-1), PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33418