Evaluating mTOR Inhibitor Everolimus on Neuroendocrine Tumor Cells

We tested the effect of the mTOR inhibitor everolimus (known as RAD001; LC Laboratories, Woburn, MA, USA) on the proliferation of both BON-1 and QGP-1. RAD001 was dissolved in 100% dimethylsulfoxide (DMSO) to a 1 mM concentration and stored at −20 °C before dilution to intermediate concentrations in 40% DMSO and use at 100×. In all the experiments, controls were treated with an equivalent 0.4% vehicle DMSO concentration equivalent to the 0.4% final DMSO concentration in the treatment dilution. For BON-1 and QGP-1 cells, we used the IC₅₀ concentrations of RAD001 of 1 nM and 5 nM, respectively, as previously described [25 (link)]. HPF cells were also treated with RAD001 at the concentration of 10 nM. The protocol followed the same steps as the ones described in the conditioned media and co-culture experiments. A dose curve response to RAD001 (0.001 to 100 nM) was performed for both cell lines after a total of seven days of incubation with DMEM/F12 + BSA 0.2% (control conditions) or HPFcm and DMEM/F12 + 0.2% BSA in a 1:1 ratio (1 mL total volume).