Everolimus

Everolimus (Novartis Pharma AG, Basel, Switzerland) was dissolved in DMSO as a 10 mM stock solution and stored in aliquots at −20°C. Prior to experiments, Everolimus was diluted in cell culture medium. Cell growth experiments were carried out in the presence of 1 – 1000 nM Everolimus. Resistance towards Everolimus was induced by subjecting Caki-1, KTCTL-26, or A498 cells with stepwise ascending concentrations from 1 nM up to 1 μM. The tumor cells were further exposed to 1 μM Everolimus twice weekly for over a year. Tumor cells, resistant to Everolimus, were designated Caki-1^res, KTCTL-26^res, and A498^res. Control cells, sensitive to Everolimus, were designated Caki-1^par, KTCTL-26^par, and A498^par. L-Sulforaphane was provided by Biomol, Hamburg, Germany. Concentrations from 1.25 – 20 μM SFN were applied to cell cultures to evaluate effects on the growth of both Everolimus- resistant and Everolimus-sensitive tumor cells. Since 20 μM SFN showed the greatest growth inhibitory effect, all further experiments were carried out with 20 μM SFN. Controls remained untreated. To evaluate toxic effects of Everolimus and SFN, cell viability was determined by trypan blue (Gibco/Invitrogen, Darmstadt, Germany).

Everolimus (formerly known as RAD001) and placebo were kindly provided by Novartis Pharma GmbH (Nuremberg, Germany). We applied a weekly dose of 5 mg/kg body weight as suggested by previously published studies [18 (link)]. The pharmaceuticals were freshly prepared from the pre-concentrate once a week right before the oral gavage. Following the manufacturer’s manual, Everolimus pre-concentrate was diluted with 5% glucose solution to a concentration of 0.25 mg/mL corresponding to an administered volume of ~0.5 mL. Equivalent amounts of pre-concentrate and glucose solution were used for the preparation of the placebo solution.
No-carrier added 177Lu was obtained from Isotope Technologies Garching GmbH (Garching, Germany). DOTA0, TYR3-octreotate was purchased from ABX advanced biochemical compounds (Dresden, Germany). Radiolabeling was performed according to a previously described protocol [19 (link)]. The amount of 80 MBq was chosen according to data by Svensson et al. as a trade-off between moderate toxicity and anti-tumor activity [20 (link)]. Radiolabeling of [68Ga]Ga-DOTA-TATE was performed by a radiochemist of the department of nuclear medicine according to protocols described elsewhere labeled with 68Ga obtained from a 68Ge/68Ga generator system (GalliaPharm, Eckert & Ziegler AG, Berlin, Germany) [21 (link)]. All radiopharmaceuticals were administered via a tail vein.

7 week-old C57BL/6 mice were treated daily with solvent control (2.5% DMSO in H₂O) or the mTORC1 inhibitor Everolimus (5 mg/kg body weight) for 4 weeks by oral gavage (n = 6 for the solvent control, n = 7 for Everolimus treatment). Everolimus was a kind gift of Novartis. The animal study protocol was performed in accordance with national laws and guidelines and was approved by the Medical University of Vienna's Institutional Review Board (BMWF-66.009/0304-II/3b/2013). All animal sacrifice was performed under ketamine/rompun anesthesia, and all efforts were made to minimize suffering and the number of animals used.

Once the tumor volume had reached 100 mm³, the mice with the two cell lines were randomized to experimental groups for 28 days of treatment.
Consequently, for BxPC-3 cells, the mice were divided into 7 experimental treatment groups: placebo (P), Gemcitabin (G), Everolimus (E), Gemcitabin + Everolimus (G+E), Gemcitabin + Zoledronic Acid (G + AZ), Everolimus + Zoledronic Acid (E+AZ) and Gemcitabin + Everolimus + Zoledronic Acid (G+E+AZ). For Panc-1 cells, the mice were randomized into only 4 experimental treatment groups: placebo (P), Gemcitabin (G), Everolimus + Zoledronic Acid (E+AZ) and Gemcitabin + Everolimus + Zoledronic Acid (G+E+AZ).
Gemcitabine (GEMZAR, Eli Lilly, France) was administered by peritoneal injection (PI), twice a week at 100 mg/kg of body weight. Zoledronic Acid (Zometa®, Novartis Pharma, France) was administered once a week by PI at 100μg/kg of body weight, and Everolimus (Afinitor®, Novartis Pharma, France) was administered by gavage at 3 mg/kg of body weight. Treatments were provided and prepared by the pharmacy of Angers University Hospital. Control animals in placebo groups received an equal volume of saline solution to the active molecule [32 (link), 33 (link)].