Hyperpolarized 13 C MRS detection of reduced pyruvate-lactate conversion following PI 3 K inhibition

Methods The effect of PI3K inhibition was examined in MDA-MB-231 human breast adenocarcinoma and glioblastoma multiforma (GBM) human neurosphere cells following incubation with 50 μM LY294002 for 40 hr. For MRS studies, cells were encapsulated in agarose beads and loaded into a perfusion system, modified from previously described (2). MR studies were performed on a 500 MHz INOVA spectrometer (Varian). P spectra were acquired using a 30° pulse with 1 sec relaxation. C1-pyruvate was hyperpolarized using the Hypersense DNP (Oxford Instruments) polarizer as described previously (3). Sequential injections of 3.0 mL hyperpolarized pyruvate were performed at 2 hr intervals. In experiments with MDA-MB-231 cells, final concentrations of 2 to 10 mM polarized pyruvate were attained in a volume of 8.0 mL. C MR spectra were acquired in 3 sec intervals for 300 secs using a 5° pulse. Experiments with GBM used final concentrations of 1 to 3 mM polarized pyruvate and C spectra acquired using a 13° pulse. The intensities of lactate peaks were quantified by integration and normalized to polarization and cell number determined prior to encapsulation. Maximum lactate values of treated cells were compared to control values of corresponding pyruvate injections. In parallel experiments, the effect of PI3K inhibitor treatment on lactate dehydrogenase (LDH) and HIF-1 levels was determined by standard Western blot techniques. Enzyme activity assays were performed to assess the effect of PI3K inhibition on LDH reaction kinetics.