Abstract
Breast cancer is the most common cancer type for women in the western world. Despite decades of research, the molecular processes associated with breast cancer progression are still inadequately defined. Here, we focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithelial cells to an isogenic series of mammary tumor cell lines with increasing metastatic potentials. Our results suggest a two-step metabolic progression hypothesis during the acquisition of tumorigenic and metastatic abilities. Metabolite changes accompanying tumor progression are identified in the intracellular and secreted forms in several pathways, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, fatty acid and nucleotide biosynthesis, and the GSH-dependent antioxidative pathway. These results suggest possible biomarkers of breast cancer progression as well as opportunities of interrupting tumor progression through the targeting of metabolic pathways.
Highlights
Breast cancer is the most common cancer type for women in the western world
We focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithelial cells to an isogenic series of mammary tumor cell lines with increasing metastatic potentials
Intracellular Metabolite Changes Clustering Cell Lines—To identify intracellular metabolic changes during tumorigenesis and metastasis, we used liquid chromatography (LC)-MS/MS to profile the levels of 157 different small molecule metabolites [13] in the well characterized 4T1 series of cell lines of a murine mammary cancer model [14]
Summary
Cell Culture—All cell lines used were cultured in Dulbecco’s modified Eagle’s medium supplemented with 7.5% dialyzed fetal bovine serum (Thermo Fisher Scientific HyClone). Metabolism was quenched, and metabolites were extracted by aspiration of media and immediate addition of 4 ml of 80:20 methanol:water at Ϫ80 °C to simultaneously lyse cells and quench metabolism. After 15 min of incubation at Ϫ80 °C, cell remnants were scraped from the tissue culture dish and transferred, along with the methanol:water, into a 15-ml conical centrifuge tube. Ten microliters of the extract were injected into each of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) separations. Extraction of metabolites in conditioned media was performed by combining 1 volume of 24-h conditioned media with 4 volumes of cold methanol, spinning down, and taking supernatant for analysis. Metabolite signals of conditioned media were normalized to the geometric mean for the day the samples were run and log2-transformed
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