Targeting MCT1 Blocks Tumor Angiogenesis Figure 8. MCT1 inhibition blocks tumor angiogenesis. (A) Two Matrigel plugs were implanted subcutaneously in the same mouse, one containing 10 mM lactate (left flank) and the other one an equal volume of saline (right flank). Plugs were microdissected 7 days after implantation. Pictures show CD31 immunostainings (bar = 100 mm; inset: a vascular structure carrying red blood cells), which were quantified in the graph. ***p = 0.0007; n = 4. Error bars reflect mean 6 SEM. (B and C) On Day 0, mice were randomly assigned to treatment groups consisting of daily i.p. injections of CHC (1 mmol/Kg) or vehicle. (B) Mice carrying LLc tumors (461 mm) were treated. Pictures show CD31 immunostainings of size- matched tumors (bar = 20 mm), which were quantified in the graph. **p = 0.0051; n = 4. Error bars reflect mean 6 SEM. (C) Intravital microscopy pictures were captured over time in treated mice carrying tumors generated with MCT1-negative TLT tumor cells. Bar = 1 mm. doi:10.1371/journal.pone.0033418.g008 proliferation [42]. Second, as documented in a previous study, MCT1 inhibition can block lactate-induced NF-kB activation in ECs, with a subsequent downregulation of autocrine IL-8 signaling and IL-8-mediated angiogenesis [23]. Interestingly, this latter effect was also in part dependent on a direct competition between 2-oxoglutarate and lactate for PHD2. Given this mode of action, other targets may be anticipated. For example, 2-oxoglutarate is a necessary cofactor for factor inhibiting HIF-1 (FIH-1), a 2- oxoglutarate-dependent dioxygenase that inactivates HIF-1 through HIF-1a asparagine hydroxylation [47]. Because PHDs and FIH-1 are simultaneously inhibited by compounds such as dimethyloxalylglycine that displace 2-oxoglutarate from the active enzyme sites [48], our data suggest that lactate-dependent dioxygenase inhibition may extend to FIH-1. Both PHD2 and FIH-1 also control HIF-2 activity through regulating HIF-2a hydroxylations [49], suggesting that the activity of lactate could further include HIF-2 activation in ECs. HIF-1 and HIF-2 control both different and overlapping sets of genes modulating angio- genesis and vascular stability and maturation, respectively [50– 53]. Whether the detection of mature blood vessels in lactate- PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33418