Tumor xenograft model

To generate subcutaneous tumor, 5 × 10⁶ AGS or MKN45 cells were suspended in 100 mL of PBS and injected subcutaneously into the right flank of male nude mice (BALB/c, 4 to 6 weeks of age). When tumors reached a size measuring 40 to 70 mm³, calculated as V = L × W²/2(V = Tumor volume, L = Length, W = Width), mice were treated with hydroxypropyl-cyclodextrin (vehicle control), itraconazole (75 mg/kg twice daily by oral administration), 5-FU (20 mg/kg every 2 days by intraperitoneal injection), or a combination of itraconazole and 5-FU. All treatments were administered in 10 mL/kg volumes with 12-h separation between bi-daily treatments. Tumors were measured three times per week for 2 to 3 weeks. Then mice were sacrificed and tumor tissues were isolated and frozen in liquid nitrogen or fixed in formalin immediately.