Xenograft studies

The animal study protocol was approved by the Institutional Animal Use and Care Committee (IACUC). Female athymic pathogen-free nude mice (nu/nu, 4-6 weeks) were purchased from Charles River Laboratories (Wilmington, MA, USA). To establish HT29 xenografts, a total of 5 × 10⁶ cells (in 0.1 ml) were subcutaneously injected into the left inguinal area of the mice. All animals were monitored for activity, physical condition, body weight, and tumor growth. When the tumor volume reached ~100 mm³, the mice bearing HT29 xenografts were randomly divided into multiple treatment and control groups (6-8 mice/group). Test compounds were dissolved in a vehicle of composition, PEG-400: EtOH: saline (57.1:14.3:28.6, v/v/v). CA4 (100 mg/kg/d) and KSS19 (25 mg/kg/d) were administered by i.p. injections five times a week for 3 weeks. The control group received the vehicle only and tumor volume was measured using the calipers. At the end of the experiments, the xenograft tumors, hearts, lungs, livers, kidneys, spleens, and brains were removed, weighed, and snap frozen for western blotting, immunohistochemistry and immunofluorescence studies.