Mebendazole

Pairwise drug combinations were tested for synergistic effects using MTT-based cell viability data of tumor cell lines exposed for 48 h to four different concentrations of either cisplatin and doxorubicin or cisplatin and mebendazole (all from Selleckchem). Maximum synergy scores and the corresponding concentrations were determined using the HSA statistical reference model of the Synergyfinder 2.0 software [24 (link)].

The click-iT EdU cell proliferation kit (Thermo Fisher) was applied according to manufacturer’s instructions for the detection of proliferating cell portions. In brief, 1 × 10⁵ cells/well were seeded in 24-well plates and cultured overnight before treatment. After refreshing media on the next day, cells were labelled with 100 µM ethynyl deoxyuridine (EdU) and subsequently exposed to mebendazole (Selleckchem) or dimethyl sulfoxide (DMSO) for 24 h at 37 °C. After 3.7% paraformaldehyde/PBS fixation and 0.5% Triton-X permeabilization, cells were stained with Alexa Fluor 555 azide for 30 min. Hoechst 33342 was used for staining of nuclei. Images were captured with the 10× objective of the EVOS M7000 microscope (Invitrogen) and EdU-positive nuclei counted in relation to the total number of Hoechst 33342-positive nuclei using the EVOS software.

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis MO, USA) viability assays were performed for determining drug responses of the cells. 1 × 10⁴ cells/well were seeded in 96-well plates 24 h prior to drug exposure. All cell lines and PDXs were exposed to DMSO as a control or seven increasing concentrations of mebendazole (Selleckchem) ranging from 0.128 nM to 20 µM as 1:5 serial dilutions. Absorbance values were measured with the Sunrise plate reader (Tecan, Männedorf, Switzerland) after 48 h incubation, and half-maximal inhibitory concentrations (IC₅₀) were calculated using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA).