Mouse xenograft tumor model

Human colon cancer cell line, HCT116 (10⁷ cells in 100 μL of sterile PBS and matrigel), were injected subcutaneously into the right flank region of female athymic nude mice (4 to 6 weeks of age, 18–22 grams). After tumor development, mice were divided into two treatment groups (n = 6 each): (I) dimethyl sulfoxide (DMSO) as vehicle control and (II) 5 mg/kg of LY5 (dissolved in 10% DMSO, 18% Cremophor EL and 72% sterile 5% dextrose). Vehicle and LY5 were administered via intraperitoneal injection once daily for 11 days. Tumor growth was determined by measuring the length (L) and width (W) of the tumor every other day with a caliper. And tumor volume was calculated on the basis of the following formula: volume = (π/6)*L*W². The tumors were harvested after mice was killed, snap-frozen in liquid nitrogen, and stored at −80°C. Tumors tissue homogenates were lysed and separated by SDS-PAGE. A portion of tumor tissues were fixed by using formalin and embedded in paraffin. The expression of STAT3, its downstream target genes Bcl-2 and cleaved caspase 3 in xenograft tumors was examined by Western Blot assay. The expression of P-STAT3 (Y705) and Bcl-2 was also examined by immunohistochemistry (IHC) staining. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was used to detect the percentage of apoptosis in xenograft tumors.