In vivo investigation of effects of BAG956 and RAD001, alone and combined. For BAG956+RAD001 combination studies, BAG956 was prepared as described in Figure 6, and gavage volumes were fixed to administer a final dose of 75 mg/kg BAG956. RAD001 was synthesized by Novartis Pharma, and was supplied as a 2% RAD microemulsion preconcentrate (active ingredient concentration 2% (w/w); formulation contained 20 mg RAD001/g; density of the formulation was 0.995 g/cm³). For a final concentration of 4 mg/kg for RAD001-treated mice, 0.049 g 2% RAD microemulsion was diluted in 1.4 mL water. For vehicle-treated mice, 0.049 g placebo microemulsion (same vehicle as used for RAD001) was diluted in 1.4 mL water. Gavage volumes were fixed according to the average weight of mice to achieve a final dose of 4 mg/kg RAD001 per mouse. In vivo imaging studies were performed that included treatment of mice with vehicle (both the vehicle used for BAG956 and the vehicle used for RAD001), BAG956 alone (75 mg/kg), RAD001 (4 mg/kg), or a combination of BAG956 (75 mg/kg) and RAD001 (4 mg/kg). Baseline imaging was performed for BAG956+RAD001 combination studies on the day after intravenous injection of cells, and drug treatments were started on that day as well. Two independent studies involving BAG956 and RAD001 treatments were performed, and data from both experiments were pooled. Histopathologic analysis and confirmation of tumor burden in vital organ tissues was carried out. (A) Raw photo images: experiment 1, 6-day treatment (1 times daily) with BAG (75 mg/kg) and RAD001 (4 mg/kg) alone, and together. First imaging day was 3 days after intravenous injection of cells. First treatment day was 4 days after intravenous injection of 32D.p210-luc⁺ cells. Last imaging day: 10 days after intravenous injection of cells. (B) Raw photo images: experiment 2, 7-day treatment with BAG (75 mg/kg) and RAD001 (4 mg/kg) alone, and together. First imaging day and first drug treatments were 1 day after intravenous injection cells. Last imaging day: 8 days after intravenous injection of cells. Mice were killed on day 8 after intravenous injection of cells, and total and spleen weights were obtained on the same day. (C) Bioluminescence results shown in panels A and B, pooled together and graphed as percent baseline (left panel) and as total flux (right panel). (D) Percent spleen/total weights assessed for mice shown in panels A and B. Additional mice that died within the last imaging day (but that died before imaging and therefore could not be imaged) were included for calculation of percent spleen weights for the BAG only, RAD001 only, and drug combination groups.