22 g needle

Next, 5 × 10⁴ cells/well OSCC cells were seeded in 6-well nonadherent plates (Corning Inc., Corning, NY, USA) in serum-free DMEM/F12 medium (11330057; Thermo Fisher Scientific, Bartlesville, OK, USA) supplemented with basic fibroblast growth factor (bFGF) (20 ng/mL, Invitrogen Life Technologies, Carlsbad, CA, USA), B27 supplement (Invitrogen Life Technologies), 5 μg/mL insulin (91077C; Sigma Aldrich, St. Louis, MO, USA), and epidermal growth factor (EGF) (20 ng/mL, Millipore, Bedford, MA, USA). After 12–15 days, orospheres were counted using an inverted phase-contrast microscope. Next, primary orospheres consisting of ≥50 μm spheres were counted, and images were taken with the microscope. For secondary orosphere generation, primary orospheres were dissociated through trypsinization and then pipetted to obtain a single-cell suspension using a 22-G needle (Thermo Fisher Scientific, Bartlesville, OK, USA). After 12–15 days of culture, secondary orospheres consisting of ≥50 µm were counted, and images were taken under the microscope.

HCC tumorspheres were generated from scrambled shRNA or shMALAT1 SK-Hep1 and HepG2 single-cell suspension. SK-Hep1 or HepG2 cells were seeded at a density of 5 × 10⁴ per well into Corning^® Costar^® ultra-low attachment 6-well plates (Corning, NY, USA) containing growth factors for CSCs enrichment stem cell medium (Nutristem-XF, Biological Industries, Israel) and incubated at 37 °C in humidified 5% CO₂ incubator for 5–7 days, followed by visualization of the primary tumorspheres (diameter ≥100 μm) under an inverted microscope. Secondary tumorspheres were generated by dissociating the primary tumorspheres by trypsinization, pipetting dissociated primary tumorspheres through a 22G needle (Thermo Fisher Scientific Inc., Bartlesville, OK, USA) to obtain single-cell suspension. After dissociation of the primary tumorspheres, cells were seeded as earlier described for primary tumorspheres. After 5–7 days of culture, secondary orospheres consisting of ≥100 µm were counted, and images taken under microscope. Tumorsphere size, quantity and formation efficiency were evaluated. Tumorsphere formation efficiency (TFE) was evaluated using the formula: TFE = (number of sphere formed/number of single cells plated) × 100.