Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA213

Poster Communications

Acidic extracellular pH increases cholesterol ester, sphingomyelin and hexosylceramide levels in metastatic breast cancer cells

E. Pedraz-Cuesta1, L. K. Holland2, K. Maeda2, S. F. Pedersen1

1. Section of Cell and Developmental Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark. 2. Membrane Integrity Group, Unit for Cell Death and Metabolism, Danish Cancer Society Research Center, Copenhagen, Denmark.

Cancer cells in solid tumors exhibit profound changes in lipid metabolism and dysregulation of pH homeostasis, including a very acidic extracellular pH (pHe) (1, 2). Both changes play a causal role in cancer development, however, the possible role of the altered pHe in controlling lipid metabolic pathways is poorly understood. The aim of this study was to determine whether changes in pHe impact overall lipid metabolism, and to investigate how this in turn may impact the phenotype of metastatic breast cancer cells. Metastatic breast cancer cells (triple negative, MDA-MB-231) were cultured under acidic (pH=6.5) and alkaline (pH=7.6) pHe for 24 or 72 h. Medium pH was adjusted by changing HCO3- at 5% CO2. Under these conditions, intracellular pH (pHi) was 6.6 ± 0.1 and 6.7 ± 0.1 (pHe 6.5) compared to 7.2 ± 0.1 and 7.3 ± 0.1 (pHe 7.6) after 24 and 72 h, respectively, measured by live imaging of BCECF fluorescence (n=3). Quantitative shotgun lipidomics were employed to analyze the cellular lipid profile. Notably, the cholesterol ester content was 140 ± 11 % higher at pH 6.5 than at 7.6 after 24 h, while the total amount of cholesterol did not change (n=3). The content of sphingomyelins and hexosylceramides was also elevated at acid pH, whereas the total content of ceramides did not change. Changes were qualitatively similar after 72 h. To determine whether lipid location was affected under acidic pHe, lipid droplets, cholesterol, and sphingomyelin, using Oil Red O (lipid droplets), fluorescent reporters of outer and inner leaflet cholesterol, and lysenin were used to assess the specific location of these lipid classes. To determine the possible impact of lipidomic changes, cellular cholesterol, cholesterol ester, and sphingomyelin content were manipulated and cell proliferation, migration and invasion quantified. Total cholesterol was decreased 35 ± 15 % and increased 235 ± 50 % after 16 h incubation with methyl β cyclodextrin (MβCD) or MβCD saturated with cholesterol, respectively (n=3). This was associated with corresponding changes in lipid droplet content and plasma membrane cholesterol level. Proliferation (BrdU assay) was reduced by 62 ± 24% under cholesterol depletion, and recovered upon cholesterol enrichment (24 h, n=4). Invasion (Boyden chamber assay) was decreased by 40 ± 6 % under cholesterol depletion and increased to 127 ± 25 % after cholesterol enrichment (24 h, n=4). Depletion of sphingomyelin (myriosin or neutral sphingomyelinase) had similar effect showing a decrease of 57 ± 8%. Ongoing studies address the exact metabolic pathways affected. We conclude that the acidic tumor microenvironment may contribute to the metastatic breast cancer phenotype by impacting lipid metabolism.

Where applicable, experiments conform with Society ethical requirements