Tumor xenograft assay in nude mice

BALB/c nude mice (nu/nu, n = 24, 4 weeks old, 18.81±0.8 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and housed under specific pathogen-free(SPF) conditions in the animal care facilities of the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Animal welfare and experimental procedures were in compliance with the guide for the Care and Use of Laboratory Animals (the Ministry of Science and Technology of China, 2006) and the related ethical regulations of the hospital. The Laboratory Animal Care and Use Committees of the hospital approved all experimental procedures. In brief, a total of 2 × 10⁶ Cal27 cells were washed three times in PBS and suspended in 50 μl DMEM mixed with 50μl Matrigel (Becton Dickson, Bedford, MA, USA) for a total volume of 100 μl per injection site. Subcutaneous injection was performed in the right side of the shoulder region. When the average tumor volume reached 100–150 mm [3], the mice were divided randomly into the control group and carrimycin group (n = 6 for each group). In the carrimyicn group, the mice were orally administered carrimycin solution (52 mg/kg) each day, while the control group were orally administered saline. The tumor volume and mice weight were measured every 2 days until the length of the tumor was greater than 1.5 cm, after which the mice were humanely sacrificed by euthanasia. The tumor volume (V) was calculated using tumor length (L) and width (W) as V=LxW²/2. Xenograft samples were collected and analyzed by immunohistochemistry, western blot assay and TUNEL assay.