Spheroid Culture Assay for Cancer Cells

Spheroid culture assay was performed as previously described [10 (link)]. Cells were plated at a density of 2500 cells/well in ultra-low attachment six-well culture plates (Cat# 3471, Corning, NY, USA) in DMEM/F12 spheroid media (Cat# 21331020, Gibco, ThermoFisher Scientific, Waltham, MA, USA) and media supplemented with B27 (Cat#17504044, Gibco, ThermoFisher Scientific, Waltham, MA, USA) and N-2 (Cat# 17502001, Gibco, ThermoFisher Scientific, Waltham, MA, USA). Spheres were counted on day 7 and collected on day 10. Images were captured using a PrimoVert Zeiss light microscope (Carl Zeiss Microscopy, White Plains, NY, USA). The experiment was performed in triplicate and the sphere numbers were counted. Images were analyzed to assess the sphere area using Zeiss ZEN Blue lite software (Carl Zeiss Microscopy, White Plains, NY, USA) (10 spheres per group, when possible; if the number was lower, all spheres were measured).