Tumor Lipid Extraction and Preparation

Tumor samples were extracted by liquid-liquid extraction method, as previously described by [26 (link)]. Briefly, frozen tumors were thawed, weighed (100 mg), and homogenized using the Polytron PT 2100 instrument in pH 7.4 saline (1:4 w/v). Tumor homogenate (100 μL) was then transferred to another tube and the internal standard EHop-0036 (10 μL from 4500 ng/mL stock) was added to the samples followed by vortex (30 s). A hundred microliters (100 μL) of sodium hydroxide 0.5 M were then added to the mixture and samples were mixed by vortex for 5 min. Afterwards, 790 μL of heptane: ethyl acetate mixture (1:1) were added and samples were vortexed again for 10 min. The upper layer was recovered following centrifugation (5 min at 510 × g) and the solvent was evaporated for one hour in a Centrivap console (Labconco, Kansas City, MO, USA) at room temperature. Samples were then reconstituted with 100 μL of methanol, vortexed for ten minutes, and centrifuged at 1000× g for 1 min.