Cell lines and culture
The B16-F10 Mus musculus skin melanoma cell line of C57BL/6 origin was obtained from Dr. Michael Ostrowski’s laboratory at Ohio State University and was cultured in RPMI-1640 Medium (Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, USA), 100 ug/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA). All cells were maintained in a humidified environment with 5% (v/v) CO2 at 37°C and were in the logarithmic growth phase when harvested for injection with ~50% confluence in B16-F10 cell line.
B16-F10 cell Sirt2 knockout with CRISPR/Cas9 gene editing and Sirt2 overexpression
Mouse SIRT2 CRISPR KO lentivirus vector targeting 5’- GTCATCTGTTTGGTGGGAGC-3’ was purchased from Applied Biological Materials Inc. (Richmond, BC, Canada). Third generation lentivirus was packed as previously described42. B16-F10 cells were infected by lentivirus and selected in the medium containing 2.0 µg/ml puromycin to obtain stable B16-F10 Sirt2-KO cell line. B16-F10 cells were also transfected with Flag-tagged WT-Sirt2 using FuGENE HD (Promega, Madison, WI) as before  to get SIRT2- overexpressing stable B16-F10 cell line after selection with 1,000 µg/ml neomycin.
SIRT2+/+ (C57BL/6), Sirt2 transgenic (Sirt2-KI or SIRT2tg ) and wild-type littermates with a C57BL/6 background were obtained from the Sinclair Laboratory (David A. Sinclair, PhD) in the Department of Genetics at Harvard Medical School. SIRT2−/− C57BL/6 (Sirt2-KO) mice were obtained from Tiago F. Outeiro (Department of Neurodegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen). All mice were bred in the facilities of the Department of Laboratory Animal Medicine (DLAM) at the University of Arkansas for Medical Sciences. All procedures were approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee (IACUC). All mouse experiments were performed in accordance with NIH regulations about the use and care of experimental animals. Appropriate numbers of tumor cells were suspended in ice-cold phosphate-buffered saline (PBS) and checked for viability using trypan blue staining. Only when cell viability was greater than 90% was the cell batch considered for injection.
Mouse subcutaneous melanoma xenograft model and treatment
For the subcutaneous melanoma model, 6-8-week-old Sirt2-KI and wild-type littermates were inoculated subcutaneously with 1×105 B16-F10 melanoma cells resuspended in 100μl ice-cold PBS per injection into the lower flank. The day of tumor cell inoculation was designated as day 0. Measurement of tumor size began on the 6th day and continued up to 20 days post injection. For each tumor measurement, the tumor’s longest dimension (a) and the one perpendicular to it (b) were measured using digital caliper once every 2 to 3 days. Tumor volume (V) was calculated according to the following formula: V = π/6 x ab2. The volumes calculated with this formula were closely related to the weight of the tumors isolated after sacrifice (data not shown). For tumor treatment, SIRT2 was pharmacologically inhibited with the SIRT2 specific inhibitor, SirReal2 (Cat #S7845), ordered from Selleck Chemicals (Houston, TX, USA). It was dissolved in DMSO as an 84 mg/ml stock solution and stored at -20ºC. Daily vehicle or SirReal2 treatment (50 mg/kg in PBS containing 20% PEG400 and 10% Tween-20) was given 5 days before B16-F10 tumor inoculation and repeated once every 3 days until the end of the experiment.
NK cell detection
Mice tumor tissues were harvested from the tumor-bearing mice on days 14-15 after tumor transplantation. The tumor infiltrating lymphocytes (TIL) were isolated via Ficoll-Paque Premium (GE Healthcare, Pittsburgh, PA, USA) density gradient centrifugation after mincing in cell culture medium containing cell culture dish, grinding with a 5 ml syringe plunger on and passing through the 70 µm cell strainer. Next, 106 TILs were pelleted, re-suspended in 50 µL blocking buffer (HBSS containing 5% normal mouse serum, 1:100 Fc blocker 24G.2 antibody, cat#553141, BD Biosciences, San Jose, CA), and incubated on ice for 30 min. Subsequently, the cells were re-suspended and incubated in the antibody cocktail containing 1:100-1:400 of anti-mouse CD45 (clone 30-F11, eBioscience, San Diego, CA, USA), CD19 (1D3), CD3e (145-2C11), NKp46 (29A1.4), and Fixable Viability Dye 780 (BD Biosciences) on ice for another 30 min protected from light. Finally, the cells were washed, fixed with 4% paraformaldehyde, washed again, passed through the 70-µm cell strainer lid of flow cytometry sample tubes in HBSS with 2% FBS. The samples were wrapped with aluminum foil and stored in 4°C refrigerator until flow cytometry analysis. For NK cell detection, the stained TIL samples were tested with LSRII flow cytometer (BD Biosciences), with live cells gated first, then CD45+ cells, CD19-cells from CD45+ cells, CD3e-cells from CD45+CD19- cells, and NKp46+ cells from CD45+CD19-CD3e- cells. Data analysis was performed via FlowJo software (Tree Star, Ashland, OR, USA).
Depletion of NK cells in vivo
To deplete the NK cells in subcutaneous melanoma bearing mice, the C57BL/6 and C57BL/6 Sirt2-KI mice were subcutaneously injected with B16-F10 cells on day 0 as described above. All mice in the NK-cell depletion groups were intraperitoneally injected with 50 mL of antibodies against Asialo-GM1 (AsGM1, Wako, Osaka, Japan) combined with 50 mL of distilled water before the injection of the tumor cells and then once a week thereafter (on days -3, 4, 11, and 18). The mice in control groups were intraperitoneally injected with 100 mL distilled water. On days 14- 15, mice in the NK-cell depletion groups and the control groups were euthanized for harvesting of samples. The mice were monitored daily and euthanized when moribund.
4 µm mouse melanoma tissue sections were deparaffinized and rehydrated before the citrate buffer antigen retrieval method was performed to unmask the antigenic epitopes. Briefly, slides were arranged in a single layer in a staining holder, submerged in a 500 ml glass beaker with 300 ml of 10 mM citrate buffer at pH 6.0, and incubated at 95-100°C in microwave oven for 10 min. The beaker was then removed from the microwave oven to room temperature and slides were allowed to cool for 1 hour. Next, slides were incubated in 3% hydrogen peroxide for 20 minutes to quench endogenous peroxidase activity. The sections were washed with a solution of phosphate-buffered saline (PBS) and 0.1% Triton three times. They were incubated with 10% normal goat serum in PBS + 0.1% Triton for 30 min to block nonspecific binding sites in the tissue. The tumor sections were incubated with 1:200 (in blocking buffer) rabbit anti-Granzyme B (Abcam, cat#4059) or mouse anti-Ki67 (Cell signaling, Danvers, MA, cat#9499) antibody at 4°C overnight in a wet box in the refrigerator. The secondary antibody used was anti-rabbit AP and anti-mouse HRP antibody (ENZO Biochem, Farmingdale, NY). After staining with the HIGHDEF IHC chromogen substrate DAB and AP (ENZO Biochem), the tissue sections were dehydrated by subsequent alcohol washings using 70%, 95%, and 100% EtOH solutions for 5 minutes each. Total cells were counted under microscope (original magnification, 20x; Carl Zeiss, Thornwood, NY). Cell counts were performed on at least three consecutive slides with three mice in each group.
All data are represented as a mean with standard error of the mean (SEM). Unpaired two-tailed student’s t-tests or one-way ANOVA followed by Tukey multiple comparison test or two-way ANOVA followed by Bonferroni correction when more than two groups were compared (GraphPad Prism, version 6.05, GraphPad Software, Inc.) were used for statistical analysis. P-values less than 0.05 were considered statistically significant.