In vivo lung metastasis model

Twenty-one nude mice were randomly divided into three groups as mentioned above. Cell suspensions of 5 × 10⁷ cells/ml were injected into the tail vein of 4-week-old female nude mice (Laboratory Animal Center, Xinqiao Hospital, TMMU) in a volume of 100 μl. The mice were evaluated every two days for weight and emaciation incapacitating tumor burden. Natural deaths of the nude mice were recorded to calculate the lifetime at 38 days after injection. The surviving nude mice were sacrificed at 38 days. All the lungs were resected and fixed in 10% neutral buffered formalin, and the fixed lungs were embedded in paraffin, sectioned, and stained with H&E. Microscopic lung metastases were counted under a light microscope after H&E staining. Five discontinuous and deep sections were used to define whether there was a metastasis nodule in the fixed lungs and the number of lung metastasis nodules was counted in one section with the most nodules.

All the animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Xinqiao Hospital, TMMU, and were performed according to the Guide for the Care Use of Laboratory Animals.