3D culture and cell viability assay

3D cultures were performed growing cells on an Algimatrix™ (ThermoFisher Scientific, Waltham, MA, USA) scaffold or in growth factor-reduced Matrigel® (BD Biosciences, Bedford, MA), essentially as described [25]. E-cadherin-silenced or control-transfected cells (1 × 10³) were suspended in Matrigel® and then seeded in 48-well culture plates. Plates were first incubated for 30 min at 37 °C and then complete medium was added. To evaluate cell viability of MCAs grown in Algimatrix™ (ThermoFisher Scientific) scaffolds, mitochondrial activity was measured with the CellTiter-Glo® Luminescent Cell Viability Assay performed according to the manufacturer’s instructions (Promega, Madison, WI). Cell viability of MCAs grown in Algimatrix™ was also evaluated with LIVE/DEAD™ Viability/Cytotoxicity Kit for mammalian cells performed according to the manufacturer’s instructions (ThermoFisher Scientific).