Figure 7.

Validation of BRC treatment in vitro and in vivo

Dose- and time-dependent inhibition of proliferation in response to RAD001 and CPI-613 in MKN74 (A, C) and KATO-III cells (B, D).

Proliferation inhibition of MKN74 cells in response to either RAD001 (25 μM), CPI-613 (200 μM or 250 μM), or combinations at 24–48 h (E-F) with BLISS synergy score for each combination (G).

BLISS score >10 indicates synergistic effect. Mean of n = 3–12 replicates/treatment with SD. Two-way ANOVA between treatments (time x dose). Proliferation was measured using CCK-8 kit at 450 nm. See also Figures S2 and S4. Timeline for in vivo treatment of BoNT-A, RAD001, CPI-613 with and without FUOX over a period of two months (H).

Kaplan-Meier survival curves (I) and body weight change (J) in mouse GC (age 9–15 months) that received more than 1 cycle of treatments of BoNT-A (0.1 U/month) + RAD001 (1.5 mg/kg/day) + CPI-613 (20 mg/kg/week) (BRC), 5-fluorouracil (5 mg/kg/week) + Oxaliplatin (25 mg/kg/week) (FUOX), or BRC + FUOX or no treatment (age-matched control, AMC). Log rank (Mantel-Cox) post hoc test between groups (two-tailed). GraphPad Prism v6.

Tumor size (expressed as volume density in % of glandular area of stomach occupied by tumor) of mouse GC after 2 months treatment with indicated drugs (K). Mean ± SEM with paired t test (AMC: two-tailed, treatments: one-tailed) between anterior (denervated) and posterior (innervated) side of the stomach or non-parametric test as appropriate. Sham: Laparotomy procedure without denervation surgery.