Human Tumor Tissue Preparation
Fresh tumor tissue samples (6 cases) diagnosed as OSSN from June 2021 to June 2022 in Union Hospital were collected and healthy conjunctival tissues from donors in the same period were taken as control group (3 cases). All tissues were preserved in 4% paraformaldehyde or liquid nitrogen frozen after comprehensive irrigating with sterile saline solution several times. Detailed ophthalmology examinations of OSSN cases were performed and anterior segment photographs were obtained before surgery.
Histology and Immunofluorescence Staining
Tissue samples were fixed in 4% paraformaldehyde for 24 hour and dehydrated with a series of sucrose before embedded in optimum cutting temperature compound (Sakura Finetek) for sectioning at a thickness of 6μm. Then morphology of the sections was observed after hematoxylin and eosin (H&E) stained.
The sections were blocked in 10% donkey serum (AntGene) for 30 minutes at room temperature, and incubated with rabbit polyclonal antibodies against IFNAR1 (ABclonal), IFNAR2 (ABclonal), TP (Proteintech), TS(Proteintech), and DPD (Affinity Biosciences) at 4°C overnight. After washing with TBST, samples were stained by donkey anti-rabbit IgG (Thermo Fisher Scientific) for detection. Then 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI; Beijing Solarbio Biotech Co.) was used for nucleus staining. Images were obtained with laser scanningconfocal microscope (Nikon).
Cell Culture and Cell Viability Assay
Human oral squamous cell carcinoma Cal27 cell line was purchased from FuHeng BioLogy (shanghai,China) and cultivated in DMEM high glucose medium containing fetal boving serum (HYCEZMBIO) at 37°C with 5% CO2/95% air. Cal27 cells were passaged upon 80% confluence.
Cell viability was performed via the Cell Counting Kit-8 assay (CCK8; Abbkine, Wuhan, China). Cal27 cells were seeded in 96-well plates, and negative blank wells with medium and CCK8 only and positive control wells with untreated cells, medium and CCK8 were set. After cultivated for 24 hours, cells were simultaneously treated with IFNα2b ,5-FU or a combination of IFNα2b and 5-FU of different concentrations for 48 h. The Concentration gradients of IFNα2b are 2.56*10IU/mL, 1.28*102IU/mL, 6.4*102IU/mL, 3.2*103IU/mL, 1.6*104IU/mL, 8*104IU/mL, 4*105IU/mL, 2*106IU/mL, and the set gradients of 5-FU are 0.064μg/ml, 0.32μg/ml, 1.6μg/ml, 8μg/ml, 40μg/ml, 200μg/ml, 1000μg/ml, 5000μg/ml. Then, 20μL CCK8 solution per well was added and incubated at 37°C for another 2 hours. The optical density (OD) value was measured at 450 nm using a microplate reader (PerkinElmer). The effect-dose curve was drawn, and the fraction affected and combination index of combination drugs was calculated by CompuSyn.
Western Blotting
Different concentrations of IFNα2b were added to the cal27 cell line at 1*105 IU/L,1*107 IU/L,1*109 IU/L for 48h and he expressions of key enzymes TP, TS and DPD of 5-FU were analyzed. Proteins were extracted using RIPA lysis buffer adding PMSF and the concentrations were measured. They were separated by electrophoresis on 12% SDS-PAGE gels and transferred to PVDF membranes. The samples were blocked by 5% fat-free milk for 1 hours, followed by incubation with primary antibody (TP,1:2000, Proteintech; TS,1:5000, Proteintech; DPD,1:1000, Affinity; GAPDH,1:50000, 60004-1-IG) and HRP-conjugated goat anti-rabbit IgG antibody (1:5000, Proteintech). The products were visualized with gel imaging system (Bio-rad).
Flow cytometry
For analysis of cell apoptosis and cycle, Cal27 cells were treated with different drugs of 1*105 IU/L IFNα2b,1*107 IU/L IFNα2b,1*109 IU/L IFNα2b, 0.2ug/ml 5-FU,2ug/ml 5-FU,20ug/ml 5-FU and 1*105 IU/L IFNα2b+0.2ug/ml 5-FU, respectively for 48 h. Untreated Cal27 cells served as control.
After two washes with PBS, adding 100 μL of 1×Binding Buffer to resuspend Cells. 5μL Annexin FITC and 2μL PI were added and mixed gently. Then 400 μL of 1×Binding Buffer was added, mixed and samples were placed on ice. Flow cytometry was performed within 1 hour.
Cell suspension at a density of 5x105 to 1x106 was slowly added into precooled 70% Ethanol, fully mixed, and incubated overnight at 4°C. Monitor the fluorescence intensity with a flow cytometer to detect cell cycle.
TUNEL Assay
Single cells were resuspended and seeded to 24-well plates. After incubated at 37°C with 5% CO2 overnight, cells were treated with multiple drugs of blank, 1*105 IU/L IFNα2b,1*107 IU/L IFNα2b,1*109 IU/L IFNα2b,0.2ug/ml 5-FU,2ug/ml 5-FU,20ug/ml 5-FU,1*105 IU/L IFNα2b+0.2ug/ml 5-FU, cultivated for 48 h. Samples were fixed in 4% paraformaldehyde for 30min, followed by immersion in 0.3% Triton X-100 /PBS at 37 °C for 5min and two washes with PBS . Sections were stained in 50μL of TUNEL reaction mixture for 1 hour at 37°C in the dark. After washing the specimens three times with PBS, nuclei were revealed by DAPI staining and then images were acquired.
Cell Migration
For tumor cell migration assays, Cal27 cells were seeded at 3.5x105 cells/well in 6-well plates and cultivated at 37°C with 5% CO2 overnight. The cells were stimulated with different drugs of 1*105 IU/L IFNα2b,1*107 IU/L IFNα2b,1*109 IU/L IFNα2b,0.2ug/ml 5-FU,2ug/ml 5-FU,20ug/ml 5-FU,1*105 IU/L IFNα2b+0.2ug/ml 5-FU, respectively. Untreated Cal27 cells served as control. The cells were then scratched with a 200 ul pipette tip, rinsed three times using PBS and added serum-free medium. Images of the cells were collected consecutively at 0h,6h,24h,48h and analyzed.
Statistical Analysis
All results were expressed as mean ± S.D. from at least three independent experiments. Original experiment data were analyzed using GraphPad Prism 8 software. Statistical significance for multiple groups was assessed using one-way analysis of variance and independent sample t-test was used for two groups. Differences were considered statistically significant when p < 0.05.