Tumor xenograft study

Balb/c-nu mouse (male, 4-weeks-old; OrientBio, Seoul, Korea) were allowed one-week acclimation prior to the experiment. HCC827 (2 × 10⁶ cells), HCC827-gef (4 × 10⁶ cells), or HCC827-osi (4 × 10⁶ cells) cells were prepared in 100 µl PBS and mixed with the equal amount of Matrigel (Corning, Bedford, MA, USA) right before injecting subcutaneously into the flanks of the mice. When the tumor volume reached 50 mm³ (HCC827) and 100 mm³ (HCC827-Gef, HCC827-osi) on average, the mice were randomized into the vehicle control and treatment groups (n = 5). Drugs were mixed with vehicle (EtOH:Tween80:Saline solution 1:1:98). Each drug was administrated orally once a day and 6 times per week for 22 days (HCC827-gef, HCC827-osi) and 90 days (HCC827). The body weight and tumor size were measured every 3–7 days. The tumor size was measured using a digital slide caliper and volumes (mm³) were calculated as follows: (width × length × height) × π/6. The normalized tumor volume as follows: (TV_j,treated/TV_i,control), where TV_i is the initial tumor volume of first administration, and TV_j is the tumor volume of day j. Animals were sacrificed after the final drug administration and tumors were collected for ex vivo analysis.