Mice and cell
BALB/c mice were purchased from Orient Bio (Seongnam, South Korea) and were kept under pathogen-free conditions. Female mice at 6–8 weeks of age were used in the experiments, according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University.
A mouse breast cancer cell line, 4T-1, was incubated in RPMI-1640 medium (Biowest, France) supplemented with 10% fetal bovine serum (Biowest, France) and 50 U/mL penicillin streptomycin (Biowest, France) at 37°C and 5% CO2.
Total RNA isolation and cDNA synthesis
For isolation of RNA from 4T1 mouse breast cancer cells, the cells were collected and centrifuged at 1,600 rpm. Then, pellets were resuspended in 1 mL TRIzol Reagent (Invitrogen, USA) and incubated for 5 min. This was followed by addition of 0.2 mL chloroform (Sigma-Aldrich, Germany) and incubation for 3 min. After centrifugation at 12,000 rpm for 15 min, the top layer of the aqueous phase was collected. Then, RNA was precipitated by adding 0.5 mL isopropanol and incubating for 10 min followed by centrifugation at 12,000 rpm. Pellets were washed with 1 mL 75% ethanol and centrifuged at 12,000 rpm. After evaporating the ethanol, 20 μl RNase-free water (Qiagen) was added to the pellet and heated at 55°C for 5 min. Then, cDNA (Complementary DNA) was synthesized from total RNA using a real-time PCR (Polymerase Chain Reaction) kit (Promega, USA) and amplified by a PCR kit (NanoHelix, South Korea) based on neoantigen gene primers. (The detailed primer sequences are provided in Additional file 2).
Direct sequence analysis
Gel electrophoresis was performed using 1% agarose gel (DYNEBIO INC., South Korea) and a single band at 300–600 bp (base pair) was detected by Core Bio i-MAX™ gel image analysis system (Corebiosystem, South of Korea). DNA was extracted using a gel extraction kit (DYNEBIO INC, South Korea) and the mutation in the sequence was analyzed by Cosmogenetech (Seoul, South Korea). The proportion of mutation in base pairs was evaluated as follows: 100%, a single peak; 50%, double peaks including mutated and non-mutated in equal degrees; and 0%, a peak with no mutation. In addition, >50% indicated double peaks including high proportion of mutated sequences and <50% indicated double peaks including less proportion of mutated than non-mutated sequences.
Neoantigen peptide synthesis
All neoantigen peptides used in our experiments were custom-made by Anygen (Gwangju, South Korea). The genes and their peptide sequences are provided as follows[5]: Mutant Gen1 (IPHNPRVAVKTTNNLVMKNSVC LERDS), Mutant Polr2a (LAAQSLGEPATQITLNTFHYAGVSAKN), Wild type Zfr (AHIRGAKHQKVVKLH TKLGKPIPSTEP), Mutant Zfr (AHIRGAKHQKVVTHTKLGKPIPSTEP), Mutant Cep120 (ELAWEIDRKVL HQNRLQRTPIKLQCFA), Mutant Malt1 (FLKDRLLEDKKIAVLLDEVAEDMGKCH), Mutant Wdr11 (NDE PDLDPVQELIYDLRSQCDAIRVTKA), Mutant Kbtbd2 (DAAALQMIIAYAYRGNLAVNDSTVEQL), Mutant Gprc5a (FAICFSCLLAHALNLIKLVRGRKPLSW), Mutant Zzz3 (KELLQFKKLKKQNLQQMQAE SGFVQH V), Mutant Ilkap (RKGEREEMQDAHVSLNDITQECNPPSS), Mutant Cenpf (RVEKLQLESELNE SRTECITA TSQMTA).
In vivo tumor treatment experiments.
For tumor treatment experiments in vivo, 4T-1 cells (1x106 per mouse) were injected subcutaneously into BALB/c mice (6 per group). Doses of 5 mg/kg cisplatin (Sigma-Aldrich, Germany) were administered via intraperitoneal injection on days 12 and 15. Doses of 20 μg (per mouse) neoantigen peptide were injected intratumorally on days 13, 16, 19, 22, and 25. Doses of 100 μg (per mouse) anti-PD-L1 (clone 10 F.9G2) antibodies (BioXcell, USA) were injected intraperitoneally on days 18, 20, 22, 24, 27, and 29. Tumor sizes were measured twice a week and tumor masses were calculated using the formula (length × width2)/2. Euthanasia was performed when the size of the tumor exceeded 10% of the weight of the mouse or was 2 cm or more. Euthanasia proceeds with CO2 gas (injection at a rate of slowly filling the euthanasia chamber at a rate of 10-20% per minute).
Enzyme-linked immunosorbent assay (ELISA).
For in vivo cytokine analysis, splenocytes were harvested from the tumor-bearing BALB/c mice, one week after the last peptide or antibody injection. The cells were then treated with ACK(Ammonium-Chloride-Potassium) solution (Quality Biological, Gaithersburg, MD, USA) for red blood cell lysis. The splenocytes were then incubated with neoantigen peptide. After incubation for 24–48 h, the supernatants were harvested and assessed for IFN-γ cytokine levels using a mouse IFN gamma ELISA kit (Invitrogen, USA) following the manufacturer’s recommendations.
Statistical analysis
The t-tests used represent statistical significance as follows: *P<0.01; **P<0.05; ***P<0.001. All experiments were performed three times independently, and IBM (International Business Machines Co.) SPSS (Statistical Package for the Social Sciences) Statistics Base 22.0 was used as a statistical tool to analyze the differences between the groups in survival experiments.