Expression data for 3D versus 2D cell culture

Ferroptosis is a regulated non-apoptotic cell death process characterized by iron-dependent lipid peroxidation. This process has recently emerged as a promising approach for cancer therapy. Some evidence indicates that GPX4 may be a useful target for drug development, yet factors that govern GPX4 inhibitor sensitivity in vivo are poorly understood. We find that pharmacological and genetic loss of GPX4 function was sufficient to induce ferroptosis in multiple adherent (“2D”) cancer cell cultures. However, reducing GPX4 protein levels did not affect tumor xenograft growth when these cells were implanted in mice. Furthermore, sensitivity to GPX4 inhibition was markedly reduced when cells were cultured as spheroids (“3D”). We utilized microarray to identify genome-wide expression changes in 3D versus 2D cell culture conditions. 3D and 2D cells were cultured for RNA extraction and hybridization on the Clariom D, human array. For 2D culture, cells were harvested at approximately 80% confluency. For 3D culture, 2,000 cells were seeded in 96-well ultra-low attachment microplates , spun at 1,200 rpm for 2 min for spheroid formation, then pooled and harvested 48 h post-seeding.