Cells
Human natural killer cell line NK92 was purchased from the American Type Culture Collection and grown in α-MEM medium (Cat. No.12571089; Life Technologies, Carlsbad, CA, USA) supplemented with 12.5% fetal bovine serum (FBS; Cat. No.1614007; Gibco, Grand Island, NY, USA), 12.5% horse serum (Cat. No. 26050070; Gibco), 1.5 g/L sodium bicarbonate, 2 mM L-glutamine (Cat. No. 25030149; Gibco), 100 to 200 U/ml recombinant IL-2 (Cat. No. 200-02; PeproTech, Rocky Hill, NJ, USA,), 0.1 mM 2-mercaptoethanol, 0.2 mM inositol (Cat. No. I5125; Sigma-Aldrich, St. Louis, MO, USA), 0.02 mM folic acid, and 1% penicillin-streptomycin solution (Cat. No. SV30010; Solarbio, Beijing, China). Mouse pancreatic carcinoma cell line PAN02 and human pancreatic carcinoma cell line PANC28, PANC1, SW1990 were purchased from the American Type Culture Collection and cultured in DMEM medium contained 10% FBS (Gibco, Gaithersburg, MD, USA) at 37°C in an atmosphere of 5% CO2.
Mouse Nk Cell Isolation
Mouse NK cells were isolated from fresh spleens of wild-type (WT) or GPR116−/− mice on C57BL/6 background. NK cells were purified with MagniSort Mouse NK cell Enrichment Kit (Invitrogen), followed by CD49b MACS (Miltenyi). This cell population was highly selected for NK cells with a purity of > 99%.
Quantitative Real-time Polymerase Chain Reaction (Qrt-pcr)
The NK92 cells, mouse NK cells from the spleens of WT or GPR116−/− mice were stimulated by IL-15. After 24 h, the total RNAs were collected using Trizol reagent (Takara, Japan), and then reversely transcribed through the Prime Script RT-PCR kit (Takara, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was conducted with SYBR Premix Ex Taq (Takara, Japan) on a 7500 Real-time PCR system (Applied Biosystems, Inc., USA).
Flow Cytometry
1×106 single suspension cells were stained with the proper antibodies diluted in cell staining buffer (420201, BioLegend, USA) for 30 min at 4°C. For surface staining, anti-mouse CD45- Alexa Fluor® 700 (Biolegend, 109821), anti-mouse CD11b-PercpCy5.5 (Biolegend, 101227), anti-mouse CD3-PE (Biolegend, 100206), anti-mouse CD3-FITC (Biolegend, 100203), anti-mouse CD4-APC (Biolegend, 100412), anti-mouse CD8-PercpCy5.5 (Biolegend, 100721) and anti-mouse NK1.1-APC (eBioscience, 17-55941-82) were used for extracellular staining. For intracellular staining, the cells were stimulated with or without various cytokines and GolgiPlug for 4 h and then fixed/permeabilized with BD Cytofix/Cytoperm kit as the recommendation of the manufacturer. After washing with wash buffer, anti-human IFN-γ-Percp-cy5.5 (Invitrogen, 45-7319-42), anti-human and Granzyme B-PE (Invitrogen, 12-8899-41) were used for intracellular staining. All samples determined using FACSalibur flow cytometer (Becton-Dickinson, FL, NJ, USA), and the percentages of cells within each phase of the cell cycle were analyzed using FACS Express 7 software (De Novo Software).
Western Blot
In detail, cells were collected and lysed in radio immunoprecipitation assay (RIPA) buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholine, and 1 mM NaF), and the protein concentration was measured using a BCA protein assay kit (Pierce Biotechnology). Proteins were separated on 10% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After 2 h, the membranes were blocked with 5% skim milk and incubated with the primary antibodies at 4°C overnight. After that, the membranes were washed with TBST for three times and incubated with HRP-labeled goat anti-rabbit antibody (Cell Signaling Technology (CST), USA) for 0.5 hour. Finally, the immunoreactive signals were detected using the ECL detection system (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, IL, USA). The following primary antibodies were applied: rabbit anti-HIF1α (36169S, CST, USA), rabbit anti-P-p65 (3033S, CST, USA), rabbit anti-GPR116 (ab136262, Abcam, Shanghai, China), rabbit anti-p65 (8242S, CST, USA), rabbit anti-β-actin (4970 L, CST, USA).
Mouse Models
WT or GPR116−/− mice on C57BL/6 background at the age of 6 to 8 weeks were used in this study. PAN02 cells were suspended at 5×106/100 µL PBS and subcutaneously injected into the right back flank of mice to establish subcutaneous tumor models. Tumor volumes were measured and calculated as length×width2×0.5. One week later, according to tumor size, they were divided into three groups and the mice was injected with PBS, WT-NK and GPR116−/−-NK through tail vein, respectively.
6 to 8-week-old female NSG were used to establish subcutaneous tumor models to analyze the therapeutic effect of CAR-NK cells in this study. For the cell line PANC28-luc subcutaneous xenograft models, 5×106 PANC28-luc cells in 100 µL PBS were subcutaneously inoculated into the right flanks of NSG mice (on day 0). When tumor volume reached approximately 80 mm3, the mice were randomly divided into four groups: PBS, NK92, NKG2D-CAR-NK92 and SH-NKG2D-CAR-NK92 (n = 4), and received PBS or 5×106 irradiated CAR-NK (NKG2D-CAR-NK92 or SH-NKG2D-CAR-NK92) cells intravenously (every 7 days, 3 times in total). All animal studies were carried out under protocols approved by the Institutional Animal Care and Use Committee of East China Normal University.
Analysis of tumor-infiltrating immune cell subsets.
Tumors were excised and digested postmortem using a cocktail of 1 mg/ml collagenase type IV (Sigma-Aldrich) and 0.02 mg/mL DNaseІ (Sigma-Aldrich). After digestion at 37°C for 30 minutes, cells were passed through a 70-µm filter twice. Cells were then analyzed for various functional parameters including cytokine production by flow cytometry directly ex vivo as previously described [41].
Statistics
Data were analyzed using Prism v7.0 (GraphPad Software). Survival curves were analyzed using the log-rank test. Statistically significant differences of P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 are noted with *, ** and ***.