17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) and PI-103 concentration-dependent inhibition of tumor spheroid growth. (a) Schematic illustration of tumor spheroid growth kinetics and compound treatment procedures. Spheroids were initiated on day 0, treated with compound or vehicle on day 4, and 50% medium replenishment performed on days 7, 10 and 12. A Celigo cytometer was used for automated imaging and analysis. (b-g) Day 4 U-87 MG spheroids were treated with 17-AAG (heat shock protein 90 (HSP90) inhibitor) or PI-103 (phosphatidylinositol-3-kinase (PI3K)- mammalian target of rapamycin (mTOR) inhibitor) with 1:2 serial dilutions (final concentrations 0 to 4 μM for 17-AAG and 0 to 2 μM for PI-103). Control spheroids were treated with vehicle. Values are means ± SD (n = 6) and a representative of three separate experiments for each agent is shown. Student t test with Welch's correction was performed relative to the 14-day values: (b) vehicle controls vs 0.015 and 0.031 μM not significant; vs 0.062 μM P < 0.01; vs all higher concentrations P < 0.001; (e) vehicle controls vs 0.007 to 0.031 μM not significant; vs all higher concentrations P < 0.001. Screen prints of plates for (c) 17-AAG or (f) PI-103, corresponding to day 14 of the above graphs are also shown. Concentration response curves at day 14 for 17-AAG (d) and PI-103 (g) are shown. Values are means ± SD (n = 6).