br Mice and murine tumor model br Female
2.2. Mice and murine tumor model
Female BALB/c mice, aged 6–8 weeks, were purchased from Bei-jing Huafukang Biology Technology Co. Ltd. (Beijing, China) and kept in micro-isolator cages under pathogen-free conditions. All animal procedures were conducted according to the National Insti-tutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Murine 4T1 breast can-cer AMG 925 were provided by the National Engineering Laboratory for AIDS Vaccine, Jilin University. To obtain a model of breast cancer in situ, BALB/c mice (eight per group) were challenged with 2 104 4T1 cells subcutaneously on the right lower flank. All mice were weighed every two days after tumor challenge. When the tumor mass was detectable, the tumor volume was measured every 2 days using calipers and calculated using the following for-mula: (length width2)/2 (mm3). All experimental animals were euthanized within 30 days after tumor challenge for the tumor vol-ume of mice in the Vec group was too large. For survival studies, the animals were monitored for approximately 45 days and ani-mals were sacrificed when the humane endpoints were reached, including tumor ulceration, tumor size reaching 2000 mm3, or decline in health (mice did not eat or had a severe weight loss).
2.3. Immunization of mice
For the immunogenicity assays, BALB/c mice (five per group) were immunized with plasmids (100 lg) three times into the skeletal muscle of both hind limbs every 2 weeks. In a prophylactic setting, BALB/c mice (eight per group) were immunized as the immunogenicity strategy, two weeks after the final immunization, the mice were challenged subcutaneously with 2 104 4T1 cells. There were two therapeutic settings, i.e., BALB/c mice (eight per group) were injected with 2 104 4T1 cells subcutaneously on day and immunized with plasmids (100 lg) three times on days 2, 9, and 16 [Therapeutic setting (a)] or on days 7, 14, and 21 [Ther-apeutic setting (b)].
2.4. Cytotoxicity assay
The cytotoxicity assay was conducted in accordance with previ-ously published procedures . The three H-2Db-restricted FAPa peptides (LSPDRQFVY, VGPQEVPVP, and GGPCSQSVR) and unre-lated peptides from human MUC1 (HGVTSAPDT, VTSAPDTRP, and APDTRPAPG) were incubated with target P815 cells for 2 h at 37 LC, and the target cells were labeled with different concentra-tions of CFSE fluorescent dye. Splenocytes from vaccinated mice were then incubated with the target cells for 6–8 h at 37 LC. Cyto-lytic activity was detected by flow cytometry and specific killing was calculated as follows: % killing = [1 (peptide-loaded cells/unloaded cells from the immunized group)/(peptide-loaded cells/unloaded cells from the naïve group)] 100. All of the peptides were synthesized by Shanghai GL Peptide Ltd. (Shanghai, China) at 95% purity.
The release of IFN-c was detected using the BDTM ELISPOT Kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, 1 106 spleno-cytes were incubated with the peptides, as described for the cyto-toxicity assay, and the procedure was performed according to
previously described methods . IFN-c secretion spots were counted and analyzed.
2.6. Cytokine IL-2 detection by ELISA
Splenocytes (1 107) from vaccinated mice were stimulated with FAPa (99–499 aa) at a concentration of 5 lg/mL for 5 days in a 12-well plate. The cell culture supernatants were then col-lected to detect the expression of secreted IL-2 using the Mouse IL-2 ELISA MAXTM Standard Set (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. The culture medium was diluted 1:3 in PBS with 1% FBS for detection.
2.7. Quantitative real-time PCR (qRT-PCR)
The procedure for qRT-PCR was published previously . qRT-PCR primers for IL-2, IL-4, IL-6, IL-10, IFN-c, TNF-a, TGF-b, GzmB, GM-CSF, PD-L1, FoxP3, FAPa, collagen I, SDF-1 (CXCL12), CCL2, VEGFa and PDGFwere used to detect mRNA expression levels in spleno-cytes or tumor tissues. GAPDH was used as an internal reference.