Matrigel

The cells were dissociated using 0.05% trypsin-EDTA and pelleted by centrifugation. For Matrigel culture, the cells were washed with PBS and then resuspended in Matrigel (Corning, 356231) (1000 cells per 1 μl of Matrigel), 20 μl of the cell suspension was plated per single well of 8-well ibidi μ-plate (80826-90, Ibidi). The plate was placed in a cell culture incubator at 37 °C, 5% CO2 atmosphere in air for 10 min for Matrigel to solidify. After that, each well was filled with 300 μl of TSC medium and cultured at 37 °C, 5% CO2 atmosphere in air. For suspension culture, the cells were cultured on a 96-well low attachment plate (CLS3474, Corning), 2 × 103 cells per 200 μl of TSC medium in each well and kept at 37 °C, 5% CO2 atmosphere in air. After 24 h of suspension culture, the TSC clumps were collected, washed with PBS and then embedded in 20 μl of Matrigel or 20 μl agarose (11408.01, SERVA) and plated in 8-well ibidi μ-plates. After the gels solidified, each the wells were filled with TSC medium (control) or TSC medium supplemented with 10 μM of cilengitide (V2806, InvivoChem) or 0.5 μg / ml of Ha2/5 blocking antibody (561796, BD Sciences) or a combination of cilengitide and Ha2/5 antibody.