KrasG12D/+ co-mutation alters the landscape of lung adenocarcinoma
Description
Tumor pieces from Kras-mutant genetically engineered lung adenocarcinoma mouse models with loss Lkb1 and/or Keap1 were assessed. Steady-state LC-MS analysis (M03818) was performed on tumor pieces; or normal lung pieces. Location is annotated. 13C,15N-Glutamine 15 minute pulse-labeling (M04050) of mice was performed at defined timepoints when tumor burden is known to be significantly progressed. Tumor pieces and plasma were isolated from n=5 mice/genotype. Related to manuscript: Best et al., Glutaminase inhibition impairs CD8 T cell activation in STK11/Lkb1 deficient lung cancer. Cell Metabolism, 2022
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Glutamine Tracing Analysis KL and KKL tumor-bearing mice and uninfected littermates (UN) (n = 5/arm) received intra-peritoneal injection of 14.4 mg U-13C,U-15N-glutamine (Sigma-Aldrich), 15 minutes prior to mandible bleed (into lithium/heparin tube) and sacrifice. Tumor pieces or normal lung tissue (n = 4/mouse) were immediately harvested into Eppendorf tubes and flash-frozen in liquid nitrogen for metabolomics analysis. Tissue pieces were homogenized to powder using Covaris CP02 tissue pulverizer (Covaris, Woburn, MA). Tissue was weighed and extracted using 80/20 methanol/water at -20 °C at a ratio of 40 μL/mg tissue. Samples were vortexed, centrifuged and supernatant evaporated using a Genevac (SP Scientific, Warminster, PA). Plasma samples were thawed and 10 μL were aliquoted and extracted with 90 μL of 80/20 methanol/water at -20 °C. Samples were vortexed, centrifuged and supernatant evaporated using a Genevac. Samples were reconstituted in water for metabolite analysis. Positive and negative mode metabolite analyses were performed with reversed-phase ion-pairing liquid chromatography mass spectrometry (LC-MS) on a Thermo Vanquish Flex pump coupled to a QExactive orbitrap mass spectrometer using electrospray ionzation (Thermo Fisher Scientific, San Jose, CA). Chromatography for negative mode ionization, the stationary phase was an ACQUITY UPLC HSS T3 (1.8 μm 2.1x150 mm) column. LC separation was achieved with a gradient elution of solvent A (97/3 H2O/methanol with 10 mM tributylamine, 15 mM acetic acid at a pH of 4.9), and solvent B (methanol). The gradient was 0 min, 0 % B; 3 min, 20 % B; 5.5 min, 20 % B; 11 min, 55 % B; 13.5 min, 95 % B; 16.5min, 95 % B; 17 min, 0 % B. The flow rate was 200 µL/min. Positive mode LC separation was achieved with a gradient of solvent A (0.025 % heptafluorobutyric acid, 0.1 % formic acid in H2O) and solvent B (acetonitrile) at 400 μl/min. The stationary phase was an Waters Atlantis T3, 3 μm, 2.1 mm × 150 mm column. The gradient was 0 min, 0 % B; 4 min, 30 % B; 6 min, 35 % B; 6.1 min, 100 % B; 7 min, 100 % B; 7.1 min, 0 % B. For both ionization modes, the injection volume was 10 µL and the QExactive Mass Spectrometer scanned in negative mode from m/z 70-1,000 at a resolving power of 70,000.