Spheroid formation was performed under stem cell-selective culture conditions as previously described.49 (link) Cells were detached and resuspended in 0.8% methylcellulose (MC)-based serum-free medium (Stem Cell Technologies, Vancouver, Canada) containing 20 ng/mL epidermal growth factor (EGF; BD Biosciences, San Jose, CA), basic fibroblast growth factor (bFGF), and 4 μg/mL insulin (Sigma). Cells at the density of 1×104 cell/well were plated onto an ultralow attachment 24-well plate with a total volume of 600 μL and cultured for 4 weeks. EGF and bFGF (20 ng/mL) and insulin (4 μg/mL) were added every three days. The culture medium was replaced with fresh 0.8% MC-based serum-free medium supplemented with growth factors as described above once a week. Spheres were harvested, dissociated into single cell suspensions, and cultured under stem cell-selective conditions to form secondary spheroids. Relative spheroid numbers and areas were calculated by dividing the values of particle-exposed cells by passage control cells