Metabolomic and Stable Isotope Analysis

Cells were treated 24 hours with everolimus then incubated 2 hours with 4 mM glutamine (13C5, 15N2, 99% purity) label from Cambridge Isotope (# CNLM-1275-H-0.5). Samples were then washed with phosphate buffered saline and metabolites extracted with pre-chilled 80% high performance liquid chromatography (HPLC) grade methanol (MeOH).

Tumors were treated with 5 mg/kg everolimus or vehicle 6 hours before being sacrificed. Tumors were flash frozen in liquid nitrogen and ground with mortar and pestle. Fifty to 100 milligrams of tumor powder was added to 5 bed volumes of pre-chilled 80% HPLC grade MeOH.

Samples were centrifuged at 14000 × rpm for 10 minutes at 4°C, and the supernatants were transferred to glass insert liquid chromatography vials. Analyses occurred on an Agilent 1290 liquid chromatography system coupled to an Agilent 6520 quadrupole time of flight mass spectrometer. Samples (5 µL) were injected and separated on a Waters Acquity UPLC BEH (bridged ethyl hybrid) Amide 1.7 µm 2.1 × 100 mm HILIC (hydrophilic interaction liquid chromatography) column with a flow rate of 0.3 mL/minute. Mobile phases consisted of A (water +0.1% formic acid) and B (acetonitrile+0.1% formic acid). The column was equilibrated at 2.5/97.5 (A/B) and maintained for 1 minute post injection. Mobile-phase A increased in a linear gradient from 2.5% to 65% from 1 to 9 minutes post injection then stepped to 97.5% A from 9 to 11 minutes to wash the column. Column was equilibrated in starting condition for 3 minutes before the next injection. The mass spectrometer, equipped with a dual electrospray ionization source, was run in negative ion and then positive ion mode. The scan range was 50–1600 m/z. The source settings consisted of: drying gas flow rate: 11 L/min; nebulizer: 40 pounds per square inch gauge; gas temp: 350ºC; capillary voltage: 3000 V (neg), 2500 V (pos).

Liquid chromatography–mass spectrometry data were analyzed using Agilent Qualitative Analysis B.07.00 and Metabolomic Analysis and Visualization ENgine (MAVEN).⁴³ Metabolite identification was determined using standards and fragmentation. Cell based experiments were normalized based on total ion chromatogram, and in vivo based experiments were normalized based on weight of tumor sample extracted.