Female balb c nude mice

First, 1 × 10⁶ CRC cells with different treatments were subcutaneously injected into the female BALB/c nude mice (Beijing HFK Bioscience Co., LTD, Beijing China) aged 5 weeks for 4 weeks to generate subcutaneous tumors and then the mice were sacrificed. The schematic representation for positions of subcutaneous tumor after injection with CRC cells were shown in Additional file 1: Figure S1c. Second, the subcutaneous tumors were immediately isolated from the sacrificed mice and maintained in normal saline. Other cohort of female BALB/c nude mice (Beijing HFK Bioscience Co., LTD, Beijing China) aged 5 weeks were anesthetized and their cecum were exteriorized by laparotomy. The subcutaneous tumors were cut into the same size (about 30 mm³) to be embedded into the mesentery at the distal end of cecum, and then the cecum was restored prior to surgical sutures. Six weeks later, the mice were sacrificed and their intestines, livers and lungs were removed for biopsy. LEICA DM600 upright microscope (Germany) was used to observe metastatic lesions after HE staining. The nude mice were maintained under specific pathogen-free (SPF) conditions in the experimental animal center of Chongqing medical university. All animal experiments were performed according to protocols approved by the institutional animal care and use committee at the Chongqing medical university.

All reagents were purchased from commercial companies and directly used unless stated otherwise. If necessary, the reactions were carried out in dry solvents and under an argon atmosphere. The human colorectal tumor cell lines HCT116, DLD1, and HT29 were purchased from ATCC. The cells were propagated in RPMI 1640 or DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, United States) and 1% antibiotics (penicillin and streptomycin) in 5% CO₂ at 37°C. MTT, DMSO, 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol -4-yl)amino)-2-deoxyglucose (2-NBDG) and PI were from Sigma Chemical Co. (St Louis, MO, United States). The primary antibodies against Ki-67 and cleaved-caspase-3 (CC-3) were purchased from Cell Signaling Technology (Beverly, MA, United States). The Annexin V-FITC Apoptosis Detection Kit was purchased from KeyGen Biotech (Nan-jing, China). Female BALB/c nude mice (5–6 weeks old) were purchased from Beijing HFK Bioscience. Animal experiments were approved by the Institutional Animal Care and Treatment Committee of the State Key Laboratory of Biotherapy at Sichuan University.