Matrigel invasion assay

Invasion assays were performed using a 24-well BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Bedford, MA, USA) in accordance with the manufacturer’s protocol. A total of 2×10⁵ ASPS-KY cells in 0.2% FBS-containing medium were placed in the upper wells of the chamber. The lower chamber contained medium with 10% FBS. The chambers were treated with DMSO as a control or with different doses of TKIs. After 48 h of incubation, non-invading cells were removed from the upper chamber with cotton swabs. The invaded cells were stained and visualized using a Diff-Quick reagent (Sysmex International Reagents, Kobe, Japan). The cells were digitally photographed (×200 magnification) and counted. The invasion index was calculated by comparing the percent invasion of the untreated cells with that of those treated with a TKI.