Targeting MCT1 Blocks Tumor Angiogenesis Figure 6. Targeting MCT1 inhibits lactate-induced endothelial cell migration and vascular sprouting. (A) Confluent BAECs on coverslips were placed into cell-free dishes in the presence of 10 mM lactate or not. Pictures show cell migration over time. Bar = 50 mm. (B) Quantification of EC migration was performed in a transwell assay in which calcein-labeled BAECs migrated during 24-h through a Matrigel-coated membrane towards serum-free medium containing 10 ng/ml VEGF, 10 mM lactate, or lactate and 5 mM CHC. Pictures show the membranes where pores occupied by endothelial cells (detected using a caveolin-1 antibody) are labeled with a red bullet. Bar = 50 mm. The graph shows calcein fluorescence in the bottom well. **p,0.01, ***p,0.005 compared to control; ###p,0.005 compared to lactate; n = 3. (C) Consecutive rings of a mouse aorta were cultured during 10 days in collagen gels containing 10 mM lactate and/or 2 mM CHC. Pictures show endothelial sproutings (arrowhead point at typical linear structures) and fibroblast seeding (scattered cells). Bar = 500 mm. The graph shows the number of sprouts per ring. ***p,0.005 compared to control; ###p,0.005 compared to lactate; n = 6–11. (B and C) Error bars reflect mean 6 SEM. doi:10.1371/journal.pone.0033418.g006 as a pro-angiogenic agent. In a second set of experiments, we tested the anti-angiogenic activity of CHC in tumors. Comparison of size-matched mouse Lewis Lung carcinoma (LLc) tumors showed that chronic MCT1 inhibition reduced the endothelial density at vascular hotspots by ,2-fold compared to vehicle treatment (Figure 8B). In this model, CHC may interfere with the metabolic activity of MCT1-expressing tumor cells [26]. To better discriminate between direct and indirect anti-angiogenic effects, we repeated the experiment using MCT1-negative hepatocarci- noma (TLT) cells that were previously shown to be insensitive to the metabolic regulation by CHC [26]. Intravital microscopy revealed that chronic MCT1 inhibition completely prevented tumor angiogenesis in this model (Figure 8C). While new vascular structures continuously developed during the growth of saline- treated tumors, the angiogenic phenotype was lost in response to CHC administration (see pictures at day +12). Hemorrhagic foci were already observed after 8-days of treatment. Discussion The two main findings of our study are (i) that lactate stimulates HIF-1 activity directly in normoxic ECs resulting in the increased expression of relevant pro-angiogenic targets including the prototypical VEGF receptor VEGFR2, and (ii) that the anti- angiogenic effects of MCT1 inhibition include inhibition of HIF-1 activation in ECs. This activity was initially identified in vitro using two different EC lines in which MCT1 was targeted pharmaco- logically or using RNA interference. It was confirmed in vivo by using the experimental MCT1 inhibitor CHC in two different tumor types. Our demonstration is based on the unprecedented identification in ECs of a lactate-signaling pathway leading to HIF-1 activation under normoxia. This pathway involves lactate uptake, the conversion of lactate to pyruvate by LDH-B, a competition between pyruvate and 2-oxoglutarate supporting a pyruvate- mediated inhibition of PHD2, and, consequently, HIF-1 activation independently of hypoxia (Figure 9). Lactate did not increase the oxygen consumption rate of ECs (see Figure S1B), ruling out a contribution of metabolic hypoxia to HIF-1 activation. A similar effect of monocarboxylates including pyruvate and lactate has been previously described in malignant cells by Lu et al. [21,22]. Final effectors are however different. While lactate was shown to promote VEGF production by tumor cells [22], we show here that in ECs, it upregulates VEGFR2 expression without modulating PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33418