To the best of our knowledge, the cytotoxicity of ATV against glioblastoma stem cells or cancer stem cells in general has not been investigated before. However, for another statin, pitavastatin, autophagy induction in patient-derived stem cell-like primary GBM (as well as delayed U87 GBM tumor growth in vivo) was observed. Similar to CT-2A, the bRiTS-G2 brain tumor initiating cells rapidly formed spheroids when placed in U-bottom shaped 96 wells. Surprisingly, A-pBuOzi-A/ATV and to a lesser extent DMSO/ATV were highly active against the tumor spheres after 48 h and 72 h incubation (Figure ). Interestingly, a recently reported micellar mitotane nanoformulation of A-pBuOx-A was also slightly more active than mitotane simply dissolved in EtOH against adrenocortical NCI-H295R cells in a 3D tumor spheroid model (IC 50,48h = 43 µM & 47 µM for nanoformulated & EtOH-dissolved mitotane, respectively). Important to note, the pronounced cytotoxicity of A-pBuOzi-A/ATV after 72 h treatment determined by CellTiterGlo®-3D luminescence assay was confirmed when quantifying the cell-viability by the intrinsic fluorescence of the GFP expressing bRiTS-G2 cells (inset Figure ). Although the absolute values slightly differed, both measurement techniques gave comparable results.