Orthotopic mouse xenograft model

Two million A4573 or SKES cells in 0.1 ml were injected into an orthotopic paraosseous location, adjacent to the left proximal tibia, in 5-week-old female severe combined immunodeficient/beige (SCID/bg) mice (Harlan Laboratories Inc.). After primary tumors reached 250 to 300 mm³, mice were randomized and received intraperitoneal injection with vehicle control of DMSO in 20 ml of saline once daily, VCR at a dose of 1 mg/kg once weekly, racemic YK-4-279 at different concentrations (10 to 150 mg/kg in A4573 and 75 to 400 mg/kg in SKES) once daily for 5 days on/2 days off or 7 days, or combined treatment with VCR injected 1 hour before racemic YK-4-279 at the indicated concentrations once daily for 5 days on/2 days off or 7 days. The tumor volume was determined by the formula (D× d²/6) × π, where D is the longer diameter and d is the shorter diameter. Tumor volume was monitored every day by caliper until the tumor size reached 1 cm³. Mice were euthanized, and primary tumors were collected. An Institutional Animal Care and Use Committee of the Georgetown University approved the animal studies.