Abstract P6-01-01: Metastatic and non-metastatic isogenic breast cancer cells lines show different metabolic signatures in response to intermittent hypoxia and transient glutamine/glucose deprivation
Authors: Simoes RV , Ackerstaff E , Serganova I , Kruchevsky N , Sukenick G , Blasberg R , Koutcher JA 
Published: August 2014

Abstract
INTRODUCTION. Cancer cells quickly adapt to their microenvironment by reprogramming their metabolism. Specific metabolic responses to defined stress conditions, mimicking tumor microenvironmental changes, may therefore unveil signatures of cancer phenotype, not detected in standard tissue culture.
PURPOSE. To identify distinct metabolic signatures associated with metastatic ability, we investigated in the two isogenic breast cancer cell lines, 4T1 (highly metastatic) and 67NR (non-metastatic), how glutamine supply/deprivation affects cellular glucose metabolism, and vice-versa, in aerobic or hypoxic conditions, and the effects on cell growth.
METHODS. 4T1 and 67NR cells were cultured in medium A, i.e. DME containing 1% P/S, 10% FBS, 25 mM glucose (Glc), and 6 mM glutamine (Gln). Cell growth was determined in tissue culture from cells cultured for 48 h in various media A-D: A, all nutrients available; B: A but no Gln; C: A but 2 mM Gln; D: A but no Glc. Cellular mitochondrial function was also assessed in both 4T1 and 67NR cells using an XF96 Analyzer (Sea Horse Bioscience, Billerica, MA) and the specific inhibitors: oligomycin, FCCP, antimycin and rotenone. Metabolism was studied in cells grown on microcarriers in the various media A-D under aerobic (∼21% O2) and hypoxic conditions (∼1% O2), using our magnetic resonance (MR)-compatible cell perfusion system on a 500 MHz spectrometer. For the cell perfusion studies, Glc or Gln were exchanged for 1–13C-Glc or 3–13C-Gln respectively. The metabolic fate of 13C-labeled nutrients was followed under the various environmental conditions by 13C MR spectroscopy (MRS), while energy metabolism was observed by 31P MRS.
RESULTS. Deprivation of either Glc or Gln for 48 h reduced significantly the cell growth of 4T1 and 67NR cells, resulting in a similar growth rate for the nutrient-deprived 4T1 and 67NR cells. As determined from the MR experiments, each cell line adopted different metabolic strategies to cope when exposed to specific metabolite stress conditions, including on the level of oxygen availability. Unlike in 67NR cells, glycolysis is glutamine-dependent in the more aggressive 4T1 cell line (∼2-fold higher glucose-derived lactate synthesis rate was observed in the presence of glutamine), which decreases when oxygen becomes available to fuel TCA cycle activity, as monitored by glucose-derived glutamate synthesis. The studies further indicate an impaired TCA cycle activity in 67NR cells, reflected in a high accumulation rate of glucose- or glutamine-derived succinate (significantly higher compared to 4T1 cells). This is consistent with the mitochondrial functional analysis, showing higher mitochondrial TCA activity in 4T1 cells than in 67NR, most likely due to impairment of succinate dehydrogenase (SDH, respiratory Complex II) in the latter.
CONCLUSIONS. Our results support the association between increased mitochondrial metabolism and metastatic potential, observed recently. We are investigating the SDH status in these cells lines and in additional metastatic and non-metastatic breast cancer cell lines, to establish whether SDH, and glutamine metabolism, could be potential targets for therapy.
Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-01-01.