Everolimus suppresses mTOR signaling and improves survival in mice transplanted with tgRhoA; Tet2-deleted tumor cells. (A) Spleen weight as percentage body weight in Nod.SCID.IL2rɣ^−/− mice. Control (Cont) mice were euthanized 14 days after injection with 500 000 cells from tumor 1. The remainder of the cohort was dosed with daily gavage of 10 mg/kg Everolimus (Ever) or vehicle (Veh) daily for 5 consecutive days until they were euthanized for analysis 2 hours after the final dose. Quantification of the percentage (B) or absolute number (C) of CD4⁺ cells present in the spleens of mice. (D) Intracellular flow cytometric analysis of pAkt and mTORc1 signaling components pS6 and p4EBP1 in splenic CD4⁺ cells from everolimus-treated (red) or vehicle-treated (blue) mice. Control peaks are from unstained vehicle CD4⁺ cells. All histograms display frequency of events as percentage of cells within the population indicated. (E-G) Mean fluorescence intensity from replicates of indicated phosphoproteins. All P values from t test with Welch’s correction. (H) Overall survival of recipients of tgRhoA; Tet2^fl/fl; Vav-Cre⁺; OT-II tumor cells treated with either daily oral gavage of 10 mg/kg everolimus or vehicle from days 14 to 34 (indicated by black bar) after transfer. Each group consisted of 4 mice. P value from Mantel-Cox test.