For colony formation assays, all six-well culture plates containing the bottom and soft layers were used. The cells were plated in soft agarose as follows: cells were harvested from monolayer culture, washed and resuspended at 4×104 cells/ml in fully supplemented RPMI 1640 culture medium and molten 1.5% agarose (to a final concentration of 0.3%) on Day 0, then 0.5 ml of the cellular suspension was applied to the base layer (1×104 cells/well) and allowed to set at 4°C for 6 min. Duplicate soft agarose cultures were established to assess colony formation. Cultures were placed in an incubator at 37°C, 5% CO2 and 100% relative humidity for 12 days. The number of colonies containing ≥50 cells was counted using a light microscope.