In vivo investigation of effects of BAG956 and nilotinib, alone and combined. For BAG956⁺ nilotinib combination studies, solutions of BAG956 and nilotinib, respectively, were prepared just before administration by dissolving 100 mg in 1.0 mL of NMP and diluting with 9.0 mL PEG300. Gavage volumes were fixed according to the average weight of the mice to achieve 20 mg/kg nilotinib, and 75 mg/kg or 100 mg/kg BAG956, respectively. For these studies, cohorts of mice were treated with oral administration of vehicle (10% NMP-90% PEG300), oral administration of 20 mg/kg per day nilotinib (formulated as above), oral administration of BAG956 (at 75 or 100 mg/kg), or an orally administered combination of BAG956 and nilotinib at the respective doses. (A) Raw photo images: experiment 1, 6-day treatment with BAG956 (100 mg/kg) and nilotinib (20 mg/kg) alone, and together. Mice injected with 800,000 32D.p210-luc⁺ cells each. For this study, the first imaging day and initial drug treatments were on day 3 after intravenous injection of cells. The final imaging day was on day 9 after intravenous injection (6-day treatment). Mice were monitored for a period of time after the last imaging day and before death. One BAG956⁺ nilotinib mouse died 1 week before the day when the remaining mice were killed (17 days after intravenous injection). Body and spleen weights for all mice were recorded, and tissues were preserved in 10% formalin for histopathologic analysis and confirmation of tumor burden in vital organs. (B) Bioluminescence results shown in A, graphed as total flux. (C) Raw photo images: experiment 2, 8-day treatment with BAG956 (75 mg/kg) and nilotinib (20 mg/kg) alone, and together. For this study, drug treatments were started 2 days after intravenous injection of cells (the first imaging day was on day 2 after intravenous injection of cells, as well). The last imaging day was performed on day 10 after intravenous injection of cells, and the last treatment day was on day 9 after intravenous injection of cells (mice were treated with drug or vehicle for a total of 8 days). At the planned end of this study (on day 11 after intravenous injection of cells), any remaining mice were killed. Body and spleen weights for all mice were recorded, and tissues were preserved in 10% formalin for histopathologic analysis and confirmation of tumor burden in vital organs. (D) Bioluminescence results shown in panel C, graphed as total flux. (E) Percent spleen/total weights assessed for mice shown in panel C.