Mice and tumor cell line
All mouse procedures and protocols were approved by the Arizona State University Institutional Animal Care and Use Committee (protocol #1568R). Animals were purchased from The Jackson Laboratory and housed at ASU specific pathogen free (SPF) at the Interdisciplinary Science and Technology Building 1 (ISTB1) administrated by the Department of Animal Care and Technologies (DACT). Mice were caging in a ventilated Thoren with 250 cages or less, room temperature of 74 ± 2°F (23 ± 1°C), light cycle of 12 hours of light /12 hours of dark and 10‐15 air changes per hour. The 4T1 cells were purchased from ATCC (ATCC® CRL-2539™) in 2006, cultured as specified and aliquots stored at -180 ºC until use. For the experiments, cells between 2 and 5 passages were used and the cell line was not re-authenticated. Serum samples from the KPC pancreatic tumor mouse model were obtained from C57/BL6 mice challenged by Dr Haiyong Han’s team, at TGen.
4T1 BALB/c breast cancer mouse model and vaccination regimen
4T1 tumor cells were grown in RPMI-1640 culture medium supplemented with 10 units/ml penicillin-streptomycin (Sigma Aldrich) and 10 % fetal bovine serum (FBS) at 37 ºC in 5 % CO2 until 80 % confluence. Cells were detached with trypsin (TrypLE, Thermo fisher Scientific), washed 3 times and suspended in sterile PBS 1X at 5 x 103 cells/ml. 4T1 cell suspension (500 cells in 100 µl PBS 1X) was inoculated subcutaneously (s.c.) into the right flank, near the mammary glands, of each female BALB/c mice (6-8 weeks-old) [19, 20]. Mice were randomized into the treatment groups (n= 10 mice/group) (5 mice/cage). Tumors were measured twice per week. For the FAST vaccine experiments, 4T1-tumor bearing mice were immunized at day 7 and boosted at day 14. For the PCV experiments, 4T1-tumor bearing mice were immunized at day 12 and boosted at day 19. Both vaccines were administrated subcutaneously in the left flank (opposite to the tumor injection). Each immunization was composed of 50 µg vaccine peptide pool (5 µg/FS peptide) and 50 µg Poly (I:C) (Polyinosinic–polycytidylic acid potassium salt, Sigma Aldrich) in 100 µl sterile PBS 1X. PCV peptides were conjugated to KLH according to the manufacturer’s protocol (Imject Maleimide-Activated mcKLH™, Thermo Scientific) and purified by dialysis. For the immunotherapeutic treatment, mice were treated with anti-mouse PD-L1 (200 µg/mouse, clone: 10F.9G2, BioXCell, West Lebanon, NH) and anti-mouse CTLA-4 (100 µg per mouse, clone: UC10-4F10-11, BioXCell, West Lebanon, NH) on days 8, 15 and 21 (FAST) or days: 13, 16, 20 and 23 (PCV). The control group was immunized with PBS 1X at the same schedule and injection volumes as the vaccine groups. All tumor challenges, immunizations and tumor measurements were performed in the morning. Tumor growth was monitored and the volume was calculated as: tumor volume = (length × width2)/2. Animals were euthanized with carbon dioxide overdose (CO2) followed by confirmation with cervical dislocation when tumor volume reached 2,000 mm3 or when animals became moribund with severe weight loss or ulceration. Values were reported as means ± S.E., with statistical analysis performed using Prism 8.0 (GraphPad Software). All statistical analysis were performed in: 8/10 mice in mock group, since 2 animals did not develop tumor as described before by [19]; 10/10 mice in all other treated groups.
FS peptide array and vaccine peptide selection
To identify FSP for both vaccine approaches, we collected pre-immune sera and sera from 7-days post 4T1 tumor challenge (n=24 mice) or KPC tumor challenge (n=18). A FS peptide microarray similar to our previous FS peptide microarray[10] with 788 peptides representing 200 Fs antigens as 20mer peptides was used for these studies. The FS antigens were from FS transcripts that could be generated by the errors of the RNA processing: insertion and deletion (INDEL) of the microsatellite (MS) region during the transcription and mis-splicing of the exons. The majority of these FS antigens were predicted MS INDEL FS antigens from mono-repeat MS regions (minimum repeat length was 7 nt). MS FS antigens were selected based on the length of the homopolymer and the length of the predicted FSP length: the FS antigens with longest microsatellite repeats were selected, since they have higher INDEL rates during the transcription [14] and FSPs length needs to be longer than 17 amino acids. The remaining FS antigens were selected from human EST library analysis where FS with high frequencies in tumor EST libraries and low frequencies in normal EST libraries were selected[21]. Peptides were synthesized (Sigma-Aldrich, St. Louis, MO and WatsonBio, Houston, TX) without purification and spotted (Applied Microarrays, Tempe, USA) on NSB-9 amine slides (NSB Postech, Seoul, South Korea) using our previously developed methods[11]. Dilute sera was spotted on filter paper (903, Whatman) and dried overnight at room temperature. A 6 mm spot was punched and the filter paper was added to 150 μl blocking buffer (PBS 1X, 0.05 % Tween-20, 3 % BSA (bovine serum albumin)) with E. coli extract (1mg/ml) (dilution 1:200) and probed on peptide array for 1.5 h at room temperature (R.T). Arrays were washed 3 times with PBST (PBS 1X, 0.05 % Tween-20), incubated with 200 μl of 3-5 nM goat anti-mouse IgG-AlexaFluor 647 (Thermo Scientific, Waltham, MA), washed and scanned on an Innoscan 910 (Innopsys, Carbonne, France) at 80 % gain at 647 nm excitation and 20 % gain at 532 nm excitation. Microarray data was image-processed with GenePix Pro-6.0 (Molecular Devices, Sunnyvale, CA) and exported to Excel prior to analysis with JMP 12 (Statistical Discovery Software). Raw intensities were median normalized by slide and reactive peptides were defined as post-challenge signal two times higher than the standard deviation of the naïve mice (average). FSP positive rates were calculated for each positive peptide and the top 10 peptides with highest incidence chosen to compose the FAST vaccine. For PCVs, from the positives peptides for each mouse we selected 10 peptides with higher fluorescence intensities and that represented different frameshifts antigens. For the non-reactive Fs vaccines, we selected 10 non-reactive peptides for each mouse.
4T1 tumor metastasis
To evaluate the 4T1 clonogenic spontaneous metastasis, at the endpoint, lungs were aseptically removed, minced and digested with a solution of 10 mg/ml collagenase type I and 10 mg/ml hyaluronidase for 20–30 min at 37 °C under slow rotation. The suspension was filtered through 70 μm cell strainers and washed two times with complete RPMI-160 culture medium. Cells were suspended in the same medium supplemented with 60 µM 6-thioguanine (Sigma Aldrich) (10 ml/plate) and cultured in petri dishes for 14 days at 37 ºC and 5 % CO2. Plates were fixed with methanol for 5 minutes, carefully washed with water and stained with 0.03 % methylene blue and counted. Data are expressed as total number of metastatic colonies per mouse[20].
Peptide ELISAs
Specific IgG antibody responses in the sera of immunized mice were determined by ELISA in 96-well MaxiSorp plates (Nunc) coated with each vaccine peptide. Peptides were coated (10 µg/ml peptide/well) in carbonate-bicarbonate buffer (pH 9.6), overnight at 4 ºC. Peptide-coated plates were blocked with blocking solution (PBS 1X, 0.05 % Tween-20, 3 % BSA) for 1.5 h a 37 ºC, and washed thrice with PBS-T 1X (PBS 1X, 0.05 % Tween-20). Mouse sera, either pooled sera by group or from individual mice, was diluted 1:200 in blocking solution, added to the plates, and incubated for 1.5 hr at room temperature. Plates were washed and bound IgG was detected with horseradish peroxidase-conjugated anti-mouse IgG (Bethyl Laboratories Inc.) followed by TMB Substrate Solution (Thermo Fisher Scientific). The reaction was stopped with 0.5 M HCl and the final absorbance at 450 nm was measured in a plate reader (SpectraMax 190, Molecular devices). For the final absorbance, the pre-immune response was subtracted.
IFN-γ ELISPOT
At endpoint, vaccinated mice were euthanized and spleens were aseptically removed, minced and filtered with 100 µm screens in complete RPMI culture medium (10 % FBS, HEPES, L-glutamine, ß-mercatoethanol, sodium pyruvate and penicillin/streptomycin). Red blood cells were removed by lysis with BD Pharm Lyse ™ (BD biosciences), splenocytes were suspended and then counted. Cells were diluted in freeze medium (complete RPMI with 10 % DMSO) and stored in liquid nitrogen until use. ELISPOT plates (BD biosciences) were coated with anti-mouse IFNγ (10 µg/ml) as described by manufacturer and incubated overnight at 4 ºC. Plates were washed and blocked with complete RPMI for 2 hours at 37 ºC with 5 % CO2. Splenocytes were thawed and diluted to 5 x 106 cells/ml in complete RPMI medium, and 100 µl cells added to each well and stimulated with FS peptide pool (3 – 4 FS antigens/well) (1 µg/well each FS peptide) or 4T1 tumor cells (1x105 cells/well). As a negative control, splenocytes were stimulated with medium only. Concanavalin A (Sigma Aldrich) was used as positive control. Plates were incubated 20-24 hours for the peptide stimulation and 72-96 hours for tumor cell stimulation, at 37 ºC with 5 % CO2. Plates were washed, incubated with biotinylated anti-IFN-γ according to manufacturer’s protocol, developed with AEC substrate set (BD biosciences) and spots counted by the AID EliSpot Reader System (Autoimmun Diagnostika GmbH, Germany). Final numbers are represented as sum of the spots for the all vaccine peptides.
Flow cytometry
Frozen splenocytes prepare as described before were thaw and washed twice with complete RPMI culture medium. Cells were counted and prepare to 1 x 107 cells/ml, and one million cells were re-stimulated in vitro as follow. Lymphocytes were identified by forward and side scatter, and dead cells and doublets were excluded. Cells were stained extracellularly with amine reactive viability dye (Ghost Dye™ Red 780) (TonBio Biosciences) and fluorochrome-conjugated Abs specific for: CD8a (PerCP-Cy5.5, clone 53-6.7), CD4 (PE, clone GK1.5), IFN-γ (APC, XMG1.2), Granzyme B (FITC, clone NGZB), IL-2 (PE-CF594, clone JES6-5H4), TNF-α (BV421, Clone MP6-XT22 ), and PD-1 (PE-Cyanine7, clone J43). Cells were surface stained ex vivo, then fixed and permeabilized for intracellular staining (Fixation/Permeabilization Solution Kit with BD GolgiStop; BD Bioscience). For the intracellular cytokine analysis, cells were stimulated for 3-4 h with: FS peptide pools (3-4 FS antigens per pool), or PMA (50 ng/ml) and ionomycin (500 ng/ml) or medium only in the presence of GolgiStop (BD Bioscience), as recommended by the manufacturer. Data were acquired on an Attune NxT Flow cytometer (Thermo Fischer Scientific) and analyzed with FlowJo™ v10.6.1 software. Gating strategy were confirmed on unstimulated control samples or fluorescence minus one controls, as appropriate. Presentation of cytokine production was performed using SPICE version 6.0 software (National Institute of Allergy and Infectious Diseases, National Institutes of Health)[22].