So far, we could show that ATV was able to efficiently decrease cell-viability of various glioblastoma cell-lines in conventional 2D cell culture. However, cancer cells grown in 3D cell-clusters such as spheroids can behave significantly different from their monolayer counterparts and we have recently reported differences in tumor spheroids depending whether the API was administered in polymer micelles or added as a ethanolic solution. The multi-cellular structures resemble actual tissues better in terms of structural and functional properties and often exhibit an enhanced resistance against therapeutic compounds. CT-2A cells were chosen due to their ability to form spheroids without the need of specialized devices or additives. Furthermore, the difference in cytotoxicity between A-pBuOzi-A (Figure ) and DMSO (Figure ) formulated ATV was most pronounced in this cell line. Spheroids were obtained by simply seeding the cells (5 x 10 3 cells/well) in Ubottom shaped 96 well-plates. Similar to conventional 2D-cell culture, both ATV formulations strongly decreased CT-2A cell viability in a concentration-dependent manner (Figure ). Here, A-pBuOzi-A/ATV again was clearly more effective than DMSO/ATV. Similar to conventional 2D cell culture (Figure ), cell viability in the spheroid model increased from 48 h (50 %) to 72 h (55 %) treatment with 8 µM DMSO/ATV. Unexpectedly, cytotoxicity of A-pBuOzi-A/ATV was higher in the spheroid model (Figure ,b) than in conventional 2D cell-culture (Figure ). A similarly high activity of ATV in 3D cell-culture was also observed for U87 glioma spheroids incorporated into a fibrin gel with IC 50 (48 h) ≈ 10 µM. Besides induction of apoptosis, migration and invasion of the U87 cells were inhibited in a dose-dependent manner in this case. To further challenge ATV as potential chemotherapeutic agent against glioblastoma, its activity against spheroids composed of mouse-derived, glioblastoma cancer-stem cells (CSCs) was evaluated (Figure ). CSCs are cancer cells found within tumors with the ability of self-renewal, generation of daughter cells of various phenotypes and differentiation into phenotypically diverse populations of cells. Therefore, small populations of CSCs present after treatment can cause rapid cancer relapse as well as metastasis. Unfortunately this is favored by an increased resistance of CSCs to various otherwise unfavorable conditions including increased resistance against radiotherapy and chemotherapy .