Alisertib

GB9, GB30 and GB169 neurosphere cells were derived from patient surgical samples at the Ohio State University Cancer Center under an IRB-approved protocol. Minced tissue was passed through 18- and 21-gauge needles to achieve single cell suspensions. Cells were cultured in DMEM/F12 containing N2 supplement (Invitrogen) and 20 ng/ml each of epidermal growth factor and basic fibroblast growth factor (R&D). Neurospheres were passaged by trituration with a 21-gauge needle. U87 cells obtained from the American Type Culture Collection were grown in DMEM/10% FCS. No additional authentication was performed on these cells.
The investigational Aurora-A inhibitor alisertib was provided by Takeda Pharmaceuticals. TC-A2317 (11 (link)) was from Tocris Bioscience. For growth curves, cells were plated at 10⁴ cells/well in 24-well plates in the presence of drug or DMSO vehicle (0.0032 to 0.02%). Every 2 days 3 wells per condition were triturated and cells counted (Coulter model Z2). Metabolic activity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide conversion to formazan (MTT assay). Cells were plated in 96-well plates at 10⁴ cells/well in 100 µl of medium containing alisertib or DMSO. After 4 days at 37°C, 10 µl of MTT reagent (Roche) was added/well and plates processed per the manufacturer’s instructions.

Inhibitory concentration 50% (IC₅₀) of alisertib (Takeda Pharmaceuticals) against breast cancer cells in vitro was determined using MTT cell viability assay (ATCC). Briefly, breast cancer cells (5,000/well) were transferred to 96-well plates (Falcon) and treated with increasing concentrations of alisertib or vehicle control solution. At different time points, plates were incubated with MTT for 4 h and the assay was performed according to the manufacturer’s instructions (ATCC). IC₅₀ was determined using GraphPad Prism 5.0 computer software (GraphPad Software).
For combination treatment, cells were treated with IC₅₀ concentration of alisertib 2 h or 48 h prior to inoculation with MV strains at a multiplicity of infection (MOI) of 0.1 and 1. The anti-tumor effect of combination treatment was compared to those of samples treated with MV or alisertib alone.
Changes in cell morphology upon alisertib treatment and MV induced cytopathic effect in breast cancer cells were evaluated by light and fluorescent microscopy.