Cell culture and model system

Under experimental conditions, cells were grown in RPMI-1640 (Sigma-Aldrich). For control conditions RPMI-1640 was supplemented with 5% FBS, 10 mM glucose, 5 mM NaCl, 1% penicillin/streptomycin and 24 mM HCO₃ ⁻ to reach a pH of 7.4 when equilibrating with 5% CO₂ (control (ctrl) medium). To mimic tumor microenvironment (TME) conditions RPMI-1640 was supplemented with 1% FBS, 2.5 mM glucose, 10 mM NaCl, 7.5 mM Sodium Lactate (NaL), 1% penicillin/streptomycin and 3 mM HCO₃ ⁻ to equilibrate to a pH of 6.5 when incubated with 5% CO₂ (TME medium). For experiments, cells were grown in 1% gelatine-coated dishes in regular growth medium, washed with PBS and incubated with either control or TME medium for 24 or 48 h. Control cells were kept at 37 °C with 5% CO₂ and 95% humidity. Cells in TME medium were incubated in a computer-controlled workspace/incubator system (Xvivo G300C, Biospherix) at 37 °C with 5% CO₂ and 94% humidified Nitrogen (N₂) and 1% O₂, essentially as previously described [37]. For hypoxia alone, cells in control medium were exposed to 5% CO₂ and 94% N₂ and 1% O₂ as described for the TME cells. For functional inhibition of NHE1, cells were treated with 10 μM cariporide (a kind gift from Sanofi Aventis). Cariporide was dissolved at 10 mM in ddH₂O. MCF-7 human breast cancer cells (a kind gift from Dr. Lone Ronnov-Jessen, University of Copenhagen) were grown in standard low glucose (5.5 mM) DMEM 1885 (SSC, University of Copenhagen, Cat. 22–2-24, #015) supplemented with 6% FBS (Gibco), 1% Pen/Strep (Invitrogen), and 1% MEM Non-Essential Amino Acids 100X (Gibco/Invitrogen), at 37 °C, 95% humidity, 5% CO₂. For TME experiments, cells were seeded in culture dishes and exposed to TME medium as above for 24 or 48 h.