In Vitro Invasion Assay of Tumor Cells

The in vitro invasion activity of control and MDA-MB-231 ppFurin-expressing cells was performed using 24-well microchemotaxis chambers precoated with 7.5 μg of collagen type IV (Becton Dickinson Labware, Franklin Lake, NJ, USA), as previously described [22 (link),31 (link)]. Tumor cells (1 × 10⁵/^mL) were resuspended in serum-free media and were loaded into the upper chamber of Transwells. Cells were incubated at 37 °C for 24 h, and invasion across collagen layer was initiated by adding 10% FBS (chemoattractant) to the lower chamber. Cells detected in each well were counted and the results were represented as (number of migrated cells/number of total cells) × 100%.