Balb c nu nu nude mice

Cultured 5 × 10⁶ exponentially growing cells were subcutaneously injected into the right flanks of 6-week-old female BALB/c (nu/nu) nude mice (GemPharmatech Co., Ltd, Nanjing, China), and 0.1 ml of PBS was injected into the left flanks as the control. Tumor size was measured by calipers every 2 days. The approximate tumor volume was calculated using the equation V = (length × width × width)/2. All animals were ear-tagged and monitored individually throughout the experiment. All animal care was in compliance with the guidelines established by the internal Institutional Animal Care and Use Committee and Ethics Committee of Sichuan University. Mice were euthanized by cervical dislocation after anesthetizing with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine via intraperitoneal injection. Then, the tumor mass was excised, fixed in 10% formalin, and routinely processed for paraffin embedding. Five-millimeter-thick sections were obtained and prepared for standard histopathological examination.

All animal studies were approved by the Animal Experimental Ethical Committee of the Fudan University, Shanghai, China (No. 20200306-071). Male C57BL/6, female BALB/c, and male BALB/c-nu/nu nude mice at the age between 6- and 8-week-old were purchased from GemPharmatech, China, and maintained under a 12-h dark/12-h light cycle with food and water provided ad libitum. Humanized NSG mice were constructed by Shanghai Model Organisms, China. In brief, human CD34⁺ cells were isolated from cord blood and were transplanted into each 1.4 Gy-irradiated NSG mouse. After 16 weeks, the humanization of each mouse is analyzed by detecting the percentage of human CD45⁺ cells in all CD45⁺ cells in peripheral blood. Human CD45⁺ cell percentage greater than 25% was considered successful in modeling. All animals were randomly assigned to groups before experiments. No statistical methods were used to predetermine the sample size. The experimenter was not blind to the assignment of the groups and the evaluation of the results. No samples, animals, or data were excluded.