The anchorage-independent growth ability of the cells was evaluated in soft agar, as described [30]. Briefly, 3 mL of 0.5% agar (214010; Becton Dickinson) in basal modified Eagle’s medium supplemented with 10% FBS was layered onto each well of 6-well tissue culture plates. Cells (3 × 104) suspended in 1 mL normal medium (B9638; Sigma-Aldrich) were mixed with 2 mL of 0.5% agar in basal modified Eagle’s medium supplemented with 10% FBS, and 1 mL of the mixture was added into each well on top of the 0.5% agar layer. Plates were incubated at 37 °C in 5% CO2 for 3 weeks, and the colonies were counted. The data is presented as the number of colonies/104 cells [30].

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