2.8. Mouse liver orthotopic transplantation model and tumorigenicity assay

Four‐ to five‐week‐old male BALB/c nude mice (Institute of Zoology Chinese Academy of Sciences) were housed in the Animal Laboratory Unit, Shanghai Jiao Tong University School of Medicine, China, within the pathogen‐free environment. All experiments were performed in accordance with the guidelines of the Shanghai Medical Experimental Animal Care Commission and also refer to the relative recommended protocols. 22 HCC cells were suspended in 25 µl serum‐free DMEM mixed with 25 µl Matrigel (1:1, v/v) every 1 × 10⁶ cell. Cells above were orthotopically injected into hepatic lobes of each mouse. All mice were killed in 6 weeks post‐injection. And the diameter and weight of all the xenografted livers were measured. For histological analysis, liver and lung from mice were collected, stained with haematoxylin and eosin (HE), and detected.