Lipid Profiling of Tumor Tissues

Lipid standards were purchased from Avanti Polar Lipids (Avanti Polar Lipids Inc., AL, USA). Solvents for MS analysis and for tissue extraction were LC-MS grade and obtained from Sigma-Aldrich Co. 20 mg of tumor and non-neoplastic stroma tissues were homogenized in water using a bullet blender homogenizer (Bullet Blender Gold, Next Advance, Inc., Averill Park, NY, USA) in the presence of zirconium oxide beads (0.5 mm) at speed 8 for 5 min at 4° C. A portion of the homogenate (corresponding to 2 mg wet weight) was immediately subjected to a one-phase methanolic lipid extraction [40 (link)]. The homogenate was sonicated in 1 ml MeOH containing 0.001% butylated hydroxytoluene (as antioxidant) in a bath sonicator for 5 min, then shaken for 5 min and centrifuged at 10,000 g for 5 min. The supernatant was transferred into a new Eppendorf tube. The extracts were stored at –20° C. MS analysis was performed by an LTQ-Orbitrap Elite instrument (Thermo Fisher Scientific, Bremen, Germany) equipped with a robotic nano ion source TriVersa NanoMate (Advion BioSciences, Ithaca, NY, USA). Lipid classes and species were annotated using the lipid classification system [41 (link)] and individual lipids were identified by LipidXplorer software [42 (link)].