2.1 Cell culture
The BHK-21 cell line (CL-0034, Procell, China) was cultured in Dulbecco’s modified Eagle’s medium (CORNING, USA) supplemented with 10% (v/v) fetal bovine serum (Cell-Box, China) and 1% (v/v) penicillin‒streptomycin (Cytiva, USA) in a water-saturated atmosphere of 5% CO2 at 37°C. The B16 cell line (CL-0029, Procell, China) was cultured in high-sugar DMEM (Procell, China) containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin‒streptomycin in a water-saturated atmosphere of 5% CO2 at 37°C.
2.2 VSV-CHIKV construction, propagation, purification and observation
VSV-CHIKV was constructed following the methodology described in the literature [12]. To reduce the neurotoxicity of VSV, the glycoprotein G gene was replaced with the E3-E2-6K-E1 gene of CHIKV (Fig. 1A). VSV-CHIKV was propagated in BHK cells. Briefly, at 80% confluence, BHK cells were inoculated with VSV-CHIKV recombinants for 2 h at 37°C in FBS-free medium, after which complete culture medium was added for further culture. Cell supernatants showing cytopathic effects (CPEs) were collected. The released viruses were then purified at 4°C and at 130,000 × g for 2 hours. The virus pellet was dissolved in Tris-EDTA (TE) buffer overnight at 4°C and stored separately at -80°C.
2.3 Cell pyroptosis analysis
A total of 2 × 105 B16 cells were seeded into 6-well plates with high-sugar DMEM containing 10% (v/v) FBS at 37°C for 24 h. Next, the culture medium in each well was washed with PBS, and fresh medium was added. The experiment involved four different groups: mock, VSV-CHIKV (MOI = 1), VSV-CHIKV (MOI = 1) + vehicle and VSV-CHIKV (MOI = 1) + the GSDMD inhibitor LDC7559 (10 µM). LDC7559 (MedChemExpress, USA) was diluted in 10% DMSO and 90% corn oil before use. Among them, VSV-CHIKV, VSV-CHIKV + vehicle and VSV-CHIKV + LDC7559 were filtered through a 0.22 µm sterile filter membrane and added to B16 cells for 24 h. Images of pyroptotic cells with air bubbles were observed via optical microscopy. LDH release was used to detect the integrity of the cell membranes and was assessed according to the manufacturer's instructions (Beyotime, China). Culture supernatants were collected, and the levels of the inflammatory factors IL-1β and IL-18 were detected via ELISA according to the manufacturer's instructions (Servicebio, China).
2.4 Animal tests
C57BL/6 mice (6 weeks of age and weighing 20–22 g) were provided by the Animal Center of Fourth Military Medical University. All procedures were examined and approved by the Institutional Ethics Committee of the School of Stomatology, Fourth Military Medical University (kq-2022-020). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. A total of 3 × 106 B16 cells were implanted subcutaneously into the right dorsal side of C57BL/6 mice. When the tumors were visible, the length (L) and width (W) of each tumor were measured daily with calipers, and the tumor volume was calculated using the following formula: (L × W2)/2. Twelve days after the injection of tumor cells, VSV-CHIKV (1 × 106 PFU/mL) or VSV-CHIKV (1 × 106 PFU/mL) + LDC7559 (10 mg/kg) was injected within the tumor every two days for a total of five injections (Fig. 3A, 4A). Tumor tissues were taken from each rat for ELISA, immunohistochemistry, Western blotting, PCR and flow cytometry. The heart, liver, spleen, lungs and kidneys were removed for hematoxylin and eosin (H&E) staining. For HE and immunohistochemical analyses, tissue blocks were extracted, fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Then, the tissue samples were embedded in paraffin blocks and sectioned at 5 µm thickness.
To investigate the efficacy of the treatment of VSV-CHIKV in combination with PD-1 blockade, 12 days after the injection of tumor cells, PBS or VSV-CHIKV (1 × 106 PFU/mL) was injected into the tumor first, and lgG or aPD-1 (5 mg/kg) was injected intraperitoneally every other day for a total of 5 injections (Fig. 6A). In VivoMab-conjugated anti-mouse PD-1 (CD279) (BE0146) was purchased from BioXCell.
2.5 Histology and immunohistochemical staining
The serial sections were stained with HE as reported previously [18]. IHC staining was performed by avidin-biotin complex (ABC) staining. In brief, after deparaffinization, hydration and blockage of endogenous peroxidase, the sections were incubated with 5% goat serum to block specific sites and then incubated overnight with the following primary antibodies: anti-NLRP3 antibody (68102-1-Ig, Proteintech, USA), anti-Caspase1 antibody (sc-56036, Santa Cruz Biotechnology, USA), anti-cleaved caspase 1 antibody (AF4022, Affinity, USA), anti-GSDMD antibody (ab219800, Abcam, UK) and anti-GSDMD-N antibody (DF13758, Affinity, USA). After rinsing, the sections were incubated with biotinylated conjugated goat anti-rabbit or donkey anti-goat secondary antibody and then incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate for 30 s to 2 min for different antibodies. Sections were mounted with balsam after being dehydrated in serial alcohol solutions. Images were acquired using a Leica DFC490 system under a light microscope (DM 2500, Wetzlar, Germany).
2.6 Immunofluorescence
B16 cells were stained with immunofluorescence to evaluate the expression of GSDMD-N. Cells were incubated with primary antibody against GSDMD-N on round, sterilized glass cover slips (14 mm) and then incubated with Cy3-labeled secondary antibody diluted with DAPI for 2 h. Cover slips were placed on clean slides, sealed with antifluorescence quenching sealing liquid, stored at 4°C and then observed by confocal laser scanning microscopy (Olympus, FV1000, Tokyo, Japan).
2.6 Flow cytometry
Tumor tissue samples were collected, minced and separated into homogenates with type I collagenase (1 mg ml− 1; Worthington, China) on a shaker at 37°C and 220 rpm for 1 h. The homogenate was filtered through a nylon mesh filter and washed with PBS. Erythrocyte lysate was added, and the samples were lysed at 4°C for 15 min and then centrifuged and washed with PBS. To analyze the presence of active T cells in tumors, single-cell suspensions were incubated with PE-conjugated anti-CD4 (100408, Biolegend, USA), APC-conjugated anti-CD8 (100765, Biolegend, USA) or FITC-conjugated anti-CD3 (100306, Biolegend, USA) for 30 min at four°C. The data were analyzed with a Beckman CytoFLEX (Beckman Coulter) instrument, and the data were analyzed using FlowJo V10 software.
2.7 RT-qPCR analysis
Total RNA was extracted from tumor tissues and cells with TRIzol (Invitrogen, China). All the genes were analyzed using CFX96 real-time PCR (Bio-Rad, USA). The sequences of primers used were designed and synthesized based on the mRNA sequences obtained from the NCBI database, as shown in Table 1. The expression of each target gene was analyzed three times relative to that of GAPDH, and the mean values were calculated using the 2−ΔΔCt method.
Table 1
Gene | Forward | Reverse |
IFN-γ | 5’-ATGAACGCTACACACTGCATC-3’ | 5’-CCATCCTTTTGCCAGTTCCTC-3’ |
IL-2 | 5’-TGAGCAGGATGGAGAATTACAGG-3’ | 5’-GTCCAAGTTCATCTTCTAGGCAC-3’ |
IL-12 | 5’-GTCCTCAGAAGCTAACCATCTCC-3’ | 5’-CCAGAGCCTATGACTCCATGTC-3’ |
TGF-β | 5’-TCTGCATTGCACTTATGCTGA-3’ | 5’-AAAGGGCGATCTAGTGATGGA-3’ |
Foxp3 | 5’-CACCTATGCCACCCTTATCCG-3’ | 5’-CATGCGAGTAAACCAATGGTAGA-3’ |
GAPDH | 5’-TGTGTCCGTCGTGGATCTGA-3’ | 5’-TTGCTGTTGAAGTCGCAGGAG-3’ |
2.8 Western blotting
Isolated cells and tissues were subjected to RIPA lysis buffer (NCM Biotech, China) supplemented with ProtLytic Protease and Phosphatase Inhibitor Cocktail (NCM Biotech, China), and the protein concentration was detected with a BCA protein assay kit. Equal amounts of proteins from each group were separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in BSA for 1 h and then incubated overnight with primary antibodies against NLRP3, Caspase 1, Cleaved-caspase 1, GSDMD, GSDMD N-terminal and β-tubulin (AF7011, Affinity, USA) at 4°C. The membranes were then incubated with a 1:2000 dilution of HRP-conjugated Affinipure goat anti-rabbit IgG (H + L) (SA00001-2, Proteintech, USA) or HRP-conjugated Affinipure goat anti-mouse IgG (H + L) (SA00001-1, Proteintech, USA) for 1 h at RT. A SuperSignal West Pico chemiluminescent substrate kit (Thermo Scientific, Rockford, IL) was used to visualize the blots according to the manufacturer’s instructions. Then, the membranes were scanned with a ChemiDoc XRS + WB luminous imaging system. Image Lab 5.2.1 software was used for analysis after image acquisition.
2.9 Statistical analysis
All the data are expressed as the mean ± standard deviation (SD). The results presented are representative of at least three independent experiments and are expressed as the mean ± SD. Student’s t test was used to compare the differences between two groups. A one-way analysis of variance model was used to compare multiple groups, and Tukey’s multiple comparisons test was used to compare 2 groups. P < 0.05 was considered to indicate statistical significance. All the statistical analyses were conducted with GraphPad Prism 8.0.