t with CHC significantly decreased tumor growth similar 21278739 to siMCT1 22564524 in xenograft models. These results warrant further investigation of MCT1 inhibition as an anti-tumor treatment option. It has already been reported that MCT1 inhibition can lead to cancer cell death via a lethal decline in pHi with blockade of endogenous lactic acid exportation. We hypothesized that pharmacological inhibition of exogenous purchase BIRB796 lactate metabolism with CHC could 
elicit cell death by preventing exogenous lactate entry and utilization in glucose-deprived conditions. Our cell-based studies focused on four cell lines: Human mammary epithelial cells, MCF7, MDA-MB-231, and R3230Ac cells. HMEC cells were included to compare a normal cell response to exogenous lactate with cancer cell responses to lactate. Catabolism studies were also conducted on HUVEC cells as a second normal tissue line. The R3230Ac rat mammary carcinoma was used as our in vivo model, because we have studied glucose uptake and its conversion to lactate previously in this model previously. Both MCF7 and MDA-MB-231 cells were included as breast cancer models for two primary reasons. First, we wanted to represent a luminal and a basal-like breast cancer subtype, as these subtypes are known to be considerably different clinically and pathologically. Second, it has previously been reported that MCT1 is silenced in MDA-MB-231 cells. By including both MDA-MB-231 and MCF7 cells in our experiments, we could compare lactate uptake and metabolism in breast cancer cells lacking and expressing MCT1. Though our studies did not focus on p53 signaling and lactate metabolism, it is important to mention that the p53 status in each of these cell lines differ considerably: R3230Ac and MCF7 cells are p53 WT while MDA-MB231 cells are p53 null. This may be an important avenue for future investigation because p53 influences many metabolic pathways including glycolysis, oxidative phosphorylation and mTOR signaling. In this study, we expanded on our prior work on lactate metabolism, focusing on breast cancer. The main goals were: 1) to establish the tolerance of breast cancer cells to a range of lactate concentrations typical of that seen in human breast cancer, 2) to investigate lactate catabolism in vitro and in vivo and 3) to examine whether treatment with CHC elicits cell death in a lactatedependent manner. We used two doses of CHC sufficient to inhibit MCT1; one concentration chosen was based on previous studies, and the other concentration chosen was based on reported Ki values for the compound. To investigate lactate metabolism in vivo, we employed the R3230Ac tumor model, which has been shown to exhibit regions of high lactate in the absence of measureable glucose. Results Lactate accumulation occurs in locally advanced breast cancer with a median concentration range of 0.6 8.0 mmol/g, and lactate accumulation shows high intratumoral variation Lactate concentrations have been measured in human head and neck, cervical and colorectal cancers by bioluminescent technology. Lactate levels in breast cancer have not previously been measured. We sought to define the range of lactate concentrations found in LABC, to guide our cell-based assays. Twenty-three frozen breast-core biopsies from 21 patients with locally advanced breast cancer were made available to us from an Institutional Review Board -approved phase I/II clinical trial conducted at Duke University. Lactate concentrations were measured with bioluminescence imaging t with CHC significantly decreased tumor growth similar to siMCT1 in xenograft models. These results warrant further investigation of MCT1 inhibition as an anti-tumor treatment option. It has already been reported that MCT1 inhibition can lead to cancer cell death via a lethal decline in pHi with blockade of endogenous lactic acid exportation. We hypothesized that pharmacological inhibition of exogenous lactate metabolism with CHC could elicit cell death 19232718 by preventing exogenous lactate entry and utilization in glucose-deprived conditions. Our cell-based studies focused on four cell lines: Human mammary epithelial cells, MCF7, MDA-MB-231, and R3230Ac cells. HMEC cells were included to compare a normal cell response to exogenous lactate with cancer cell responses to lactate. Catabolism studies were also conducted on HUVEC cells as a second normal tissue line. The R3230Ac rat mammary carcinoma was used as our in vivo model, because we have studied glucose uptake and its conversion to lactate previously in this model previously. Both MCF7 and MDA-MB-231 cells were included as breast cancer models for two primary reasons. First, we wanted to represent a luminal and a basal-like breast cancer subtype, as these subtypes are known to be considerably different clinically and pathologically. Second, it has previously been reported that MCT1 is silenced in MDA-MB-231 cells. By including both MDA-MB-231 and MCF7 cells in our experiments, we could compare lactate uptake and metabolism in breast cancer cells lacking and expressing MCT1. Though our studies did not focus on p53 signaling and lactate metabolism, it is important to mention that the p53 status in each of these cell lines differ considerably: R3230Ac and MCF7 cells are p53 WT while MDA-MB231 cells are p53 null. This may be an important avenue for future investigation because p53 influences many metabolic pathways including glycolysis, oxidative phosphorylation and mTOR signaling. In this study, we expanded on our prior work on lactate metabolism, focusing on breast cancer. The main goals were: 1) to establish the tolerance of breast cancer cells to a range of lactate concentrations typical of that seen in human breast cancer, 2) to investigate lactate catabolism in vitro and in vivo and 3) to examine whether treatment with CHC elicits cell death in a lactatedependent manner. We used two doses of CHC sufficient to inhibit MCT1; one concentration chosen was based on previous studies, and the other concentration chosen was based on reported Ki values for the compound. To investigate lactate metabolism in vivo, we employed the R3230Ac tumor model, which has been shown to exhibit regions of high lactate in the absence of measureable glucose. Results Lactate accumulation occurs in locally advanced breast cancer with a median concentration range of 0.6 8.0 mmol/g, and lactate accumulation shows high intratumoral variation Lactate concentrations have been measured in human head and neck, cervical and colorectal cancers by bioluminescent technology. Lactate levels in breast cancer have not previously been measured. We sought to define the range of lactate concentrations found in LABC, to guide our 18753409 cell-based assays. Twenty-three frozen breast-core biopsies from 21 patients with locally advanced breast cancer were made available to us from an Institutional Review Board -approved phase I/II clinical trial conducted at Duke University. Lactate concentrations were measured with bioluminescence imaging