Figure 10. Inducible knocking down of SNAIL affects CTC and metastasis formation.

(A) Schematic of the 3-arm UM-UC3 SNAIL knockdown experiment with IHC staining for the “switch” group. After transduction in vitro, cells were orthotopically injected and primary tumors were grown for 2 weeks. Cells from primary tumors were isolated and inoculated in 50 mice. Mice were randomized into one of three arms (15 to 20 mice per arm). Primary tumors were established for 5 days at which point treatment commenced. The control arm received water and tumors expressed SNAIL (vehicle, no doxycycline). The mice in the treatment arm received doxycycline for 24 days continuously, knocking down SNAIL for the entirety. To assess the effect of delayed reactivation of SNAIL, mice in the “switch” group were given doxycycline until Day 12 at which point the doxycycline treatment was stopped. (B) Growth of primary tumors (p = 0.99) (C) Metastasis in the 3 treatment groups by photon count and percent of mice with metastasis (p < 0.0001). (D) Kaplan-Meier survival curves of the 3 different treatment groups demonstrates more favorable survival in those mice with inhibited SNAIL expression (log-rank test: p = 0.001). (E) Blood was procured for CTC quantification by tail vein aspiration (Treatment Days 7, 12, and 19) and by terminal cardiac puncture (Day 24). CTC counts were graphically compared (Mean ± SEM).