Collagen type 1 gel

Spheroids were generated by seeding LLC cells (1 × 10³) or MDA-MB231 cells (1 × 10³) in each well of a non-adherent, round-bottomed 96 well-plates (Greiner BioOne, Kremsmünster, Autria) in DMEM containing 1% FBS and 20% high-viscosity methyl cellulose (Sigma Aldrich) or 10% FBS lipid depleted and 25% high-viscosity methyl cellulose (MDA-MB231 spheroids). After 24 h (LLC spheroids) and 48 h (MDA-MB231 spheroids) of culture, spheroids were collected, embedded in type I collagen gels at 2 mg/ml (BD Biosciences, San Jose, CA, USA) in 12 well-plates, and maintained in DMEM 1% FBS or DMEM 1% FBS lipid depleted (for MDA-MB231 spheroids) at 37 °C for 24 h. FABP4 and SCD1 inhibitors were diluted into 500 μl of DMEM and added over solid collagen containing spheroids. Cells were examined under a Zeiss Axiovert 25 microscope equipped with an Axiocam Zeiss camera and KS 400 Kontron image-analysis software (Carl Zeiss Microscopy, Zaventem, Belgium).

For 3D cell migration assays, 10⁵ cells were embedded in 20 μL of type I collagen gel (2.0 mg/mL) extracted from rat tail (BD Biosciences). After gelling, the plug was embedded in a cell-free collagen gel (2.0 mg/mL) within a 24-well plate. After allowing the surrounding collagen matrix to gel (1 h at 37 °C), 0.5 mL of culture medium was added on the top of the gel and cultured for another 2 d. Invasion distance from the inner collagen plug into the outer collagen gel was quantified. For invasion assays, Transwell cell invasion assays were performed using either 24-well polycarbonate membrane (Corning) with 8-μm pore size, or 24-well FluoroBlok Transwell insert (BD) with 8-μm pore size. Inserts were prepared by coating the upper surface with 1 mg/mL Matrigel (BD Biosciences) for 4–6 h at 37 °C in a 5% CO₂ incubator. BT549 or 4T1 cells (5 × 10⁴) in DMEM containing 1% FBS were seeded into the upper chamber of the insert. The bottom chamber contained DMEM with 10% FBS. After 24 or 48 h, membranes were processed. Polycarbonate membranes were stained with HEMA3 staining kit (Fisher) and then mounted and enumerated based on number of cells per 20× high power field, five fields per insert. For FluoroBlok transwells, luminescence intensity was measured using a FluoStar Optima microplate reader (BMG Labtech) for 10 individual fields on the bottom of each insert.