Spheroid Invasion Assay Protocol

The spheroid drop invasion assay was conducted in accordance with previously published methods [68 (link)]. A total of 1.5 × 10⁵ cells were centrifuged at 1000× g in a 1.5 mL Eppendorf tube. The supernatant was removed, and the cell pellet resuspended in 30 μL of Geltrex (#A1413202, Thermo Fisher Scientific). An amount of 10 μL of the suspended Geltrex cell solution was seeded onto the well of a 48-well plate (#150687, Thermo Fisher Scientific). The well plate was placed upside down into the incubator to incubate for 20 min at 37 °C and 5% CO₂. Once removed from the incubator, 2 mL of DMEM/F12 with 10% FBS medium was added to each well. The medium was changed every 48 h. The spheroids were imaged daily and the area of the Geltrex spheroid and invading cells were measured in ImageJ.