C57bl 6 mice

BMSCs derived from C57BL/6 mice were obtained from Cyagen Biosciences, Inc. (Santa Clara, CA, USA). Lentivirus vector-mediated transfection of BMSCs was performed as previously described in our study [11 (link)]. After successful and stable transfection, BMSCs carrying enhanced green fluorescence protein (eGFP) and BMSCs carrying the Lats2-shRNA gene or the empty vector were cultured and passaged in a 1 : 1 mix of Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12) culture medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich, St Louis, MO, USA) and 1% streptomycin and penicillin at 37°C in a humidified atmosphere of 5% CO₂. Passage 6 or 7 cells were used for subsequent experiments.

Rbck1^+/− (C57BL/6) mice were purchased from the Cyagen Biosciences Inc. (Guangzhou). Stat1^–/– (129S6) mice were gifts from Dr. Y. Eugene Chin (Soochow University). Wild-type (WT) C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animals. All mice were maintained under specific pathogen-free (SPF) conditions in the animal facility of Soochow University. Six- to eight-week-old mice were used in all experiments. Animal care and use protocol adhered to the National Regulations for the Administration of Affairs Concerning Experimental Animals. All animal experiments have received ethical approval by the Ethics Committee of the Soochow University, and were carried out in accordance with the Laboratory Animal Management Regulations with approval of the Scientific Investigation Board of Soochow University, Suzhou.

mMSCs derived from the bone marrow of C57BL/6 mice was obtained from Cyagen Biosciences, Inc. (Guangzhou, China). The details of the transfection of MSCs by lentivirus vectors were described in our previous work [12 (link)]. After transfection, mMSCs carrying an empty vector and enhanced green fluorescence protein (eGFP) (control mMSCs) or mMSCs carrying both the β-catenin gene and eGFP (mMSC-Ctnnb1) were cultured in a 1:1 mix of Dulbecco’s modified Eagle medium/nutrient mixture F-12 (Wisent, Inc., St-Bruno, Quebec, Canada) containing 10% foetal bovine serum (Wisent, Inc.) and 1% antibiotics (streptomycin and penicillin) and were incubated at 37°C in a humidified atmosphere of 5% CO₂. The cells were used at passages 6 to 10 for the experiments.

Six to eight weeks old female BALB/c mice (Guangdong Medical Laboratory Animal Center) and TLR4 knockout C57BL/6 mice generated using CRISPR/Cas9 technique (Cyagen Biosciences) were bred under standard pathogen-free conditions. For DNA vaccination, each mouse was immunized with 20 μg of endotoxin-free DNA vaccines or pVAX1 vector twice at four-week intervals intramuscularly plus electroporation as we described previously [13 (link)]. Two weeks after the last immunization, mice were euthanized for immunogenicity analysis. For tumor challenge, two weeks after the 2nd immunization, 5 × 10⁵ AB1-Gag cells were inoculated subcutaneously in the right flank of mice. The tumor size was measured each two-four days. Tumor size based on caliper measurement was calculated by the modified ellipsoidal formula, tumor size = (length × width²)/2. Mice were sacrificed at the endpoint (21 days post-challenge). The mice were raised under specific pathogen-free (SPF) conditions and were fed a normal diet.