Tumor xenograft model

Four-week-old male Balb/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). Hep3B spheres were treated with or without 10 μmol/L WM130 for 4 days and then allowed to recover and expand for another 4 days without treatment prior to injection. The WM130-treated cells (1×10⁵) were mixed with Matrigel (BD Biosciences) and subcutaneously injected into the flank of each mouse. To avoid individual differences, an equal number of vehicle-treated control cells were subcutaneously injected into the opposite flank of the same mouse. Tumor growth was monitored for 45 days after cell injection.

For in vivo drug treatments, 1×10⁶ MHCC-LM3 cells were subcutaneously injected into nude mice. Drug treatments were initiated at 24 h after cell injection. Animals were administered either saline, WM130 (20 mg/kg, orally by gavage daily), DOX (6 mg/kg, intraperitoneal injection, once a week), or WM130 in combination with DOX for 3 weeks. The tumor size was measured with a caliper as the longest surface length (mm; L) and width (mm; W). Tumor volume (mm³; V) was calculated as V=1/2×LW². All mice were sacrificed, and tumor tissues were excised and weighted at the end of experiments.

A portion of the tumor tissues was dissociated enzymatically to obtain a single cell suspension for spheroid formation assay, proliferation assay and colony formation assay, and the remaining tumor tissues were used for real-time RT-PCR analysis, western blotting and immunostaining. All procedures involving animals and their care were approved by our institutional Animal Care Committee in accordance with the institutional guidelines for animal experiments.