Cell lines and oncolytic virus.
Mouse Pan02 (RRID: CVCL_D627) and mouse SCC-VII (RRID: CVCL_V412) were kindly provided by Dr. Sho (Nara Medical University) and Dr. Masunaga (Kyoto University), respectively. African green monkey kidney cells (Vero cells; RRID: CVCL_0059) were obtained from the RIKEN cell bank (Tsukuba, Japan). All cell lines were cultured in Dulbecco’s modified eagle medium with high glucose (DMEM; Wako, Japan) and supplemented with 10% heat- inactivated fetal bovine serum (FBS; Biosera, France), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Wako, Osaka, Japan) at 37 °C in a humidified atmosphere containing 5% CO2. All cell lines were tested by PCR for mycoplasma infection and cultured consecutively for at most 4 weeks. All experiments were performed with mycoplasma-free cells.
C-REV is an attenuated mutant clone derived from HSV-1 strain HF. The virus was propagated in Vero cells and stored in aliquots at −80 °C. C-REV was diluted in PBS for in vivo and in vitro experiments. Viral titers were assayed in Vero cells and are expressed as plaque-forming units per milliliter (PFU/mL).
Metformin Hydrochloride (1, 1-Dimethylbiguanide Hydrochloride) (138-18661) was purchased from Wako Pure Chemical Corporation, Osaka, Japan. Metformin stored protected from light in 4°C until further usage.
Cell proliferation assay.
Cell proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction method. Pan02 and SCC-VII cells were seeded, grown in DMEM, and incubated for 24 hours at 37 °C with 5% CO2. After 24 hours, infected with C-REV at several multiplicities of infection (MOIs) or treated with metformin then cells were cultured in high or low glucose medium, incubated at 37 °C with 5% CO2. The first day of treatment was defined as day 0, and cells were grown for 3 days. Viable cells were quantified by colorimetric MTT assays.
Tumor challenge and treatments.
Six- to seven-week-old female C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). Mice were kept under constant temperature and humidity conditions and fed with a standard diet and water ad libitum. All mice were maintained under specific pathogen-free conditions. All experiments were reviewed and approved by the Animal Care University Committee following the Guidelines for Animal Experimentation at Nagoya University (Nos. 31322, 31323) (Nagoya, Japan). All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines.
A bilateral tumor model of Pan02 was used to evaluate antitumor effects. Tumors were cut into cubes (2 mm3). Pan02 tumors were inoculated into mice; one tumor cube was inoculated into each flank (right and left). When the average tumor size reached 100 mm3, and treatments were then started on day 0. Mice were randomly divided into four groups (n= 4 mice/group) with an equal average tumor volume among the groups. C-REV (1×106 PFU/100 μL PBS) was injected according to the experimental timeline on one side only (injected side). Metformin continuously supplied in the drinking water (5 mg/ml) when C-REV treatment started. Clinical signs, body weight changes and tumor growth were monitored. Tumor volume was measured twice weekly until study termination. Tumor volume (V) was estimated using the equation V = L × W2/2, where L and W are tumor length and width, respectively.
Surface flow cytometry analysis of MHC-I in vitro.
Pan02 cells were stimulated with recombinant mouse IFN-α (100 ng/mL, Biolegend, San Diego, Catalog number, 752802), IFN-β (100 ng/mL, Biolegend, San Diego, Catalog number, 581302), IFN-γ (100 ng/mL, Biolegend, San Diego, Catalog number, 575302), or vehicle (PBS) for 18 hours. Cells were stained with anti-MHC-I antibody (PE) (Thermo Fisher Science, Waltham, MA, 12-5958-82) or negative isotype control (Biolegend) at 4°C for 20 minutes. After washing with PBS, cells were subjected to flow cytometry on a FACS Canto II (BD Biosciences, San Diego, CA). Data were analyzed using the FlowJo software (BD Biosciences, version 10.6).
Tumor disaggregation and re-stimulation of tumor-infiltrating lymphocytes in vitro.
Pan02 tumors were dissociated using a gentle MACS murine tumor dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Briefly, tumors from treated mice were cut into 3 mm fragments and transferred into a C-tube (Miltenyi Biotec) with Enzyme A, Enzyme D, and Enzyme R in the kit. The samples were placed onto the GentleMACS dissociator according to the manufacturer’s instructions. After disaggregation, the cell suspension was filtered through a cell strainer (70 μm) and washed three times with PBS containing 0.1% BSA. Subsequently, tumor-infiltrating lymphocytes were re-stimulated as described59. Briefly, cells were labeled using a Miltenyi CD8α T cell enrichment kit (Miltenyi Biotec) and isolated using magnetic sorting according to the manufacturer’s protocols. Tissue culture plates were coated with 5 μg/mL anti-CD3 antibody (145-2C11; BioLegend, San Diego, CA) in PBS for 12 hours, and excess antibody was aspirated before T cell addition. Cells were cultured for 48 hours with 1 μg/mL anti-CD28 antibody before the addition of 2 μM monensin for 4 hours for intracellular interferon γ (IFN-γ) staining.
Antibodies and flowcytometry.
Single cell suspensions were obtained from mouse tumors after tumor disaggregation as described in the previous method. The cells were treated with anti-CD16/CD32 antibody to block Fc receptors. Subsequently, the cells were stained with the following antibodies (BioLegend): Brilliant Violet 510-conjugated anti-CD45, FITC-conjugated anti-CD3, APC-Cy7-conjugated anti-CD8a, FITC-conjugated anti-CD8a, PerCP-conjugated anti-PD-1, APC- conjugated anti-CD44, Pacific Blue-conjugated anti-CD69, PE-conjugated anti-KLRG-1, PerCP-conjugated anti-CD25, APC- conjugated anti-CD103, APC-Cy7-conjugated anti-CD4, Pacific Blue-conjugated anti-FOXP3, APC-conjugated anti-FOXP3, PE-conjugated anti-MHC-I, PerCP-conjugated anti-MHC-II, APC-Cy7-conjugated anti-CD11c, Pacific Blue-conjugated anti-CD11b, PE-conjugated anti-XCR-1. The cells were stained for 30 minutes at 4°C in the dark. For intracellular staining, cells were fixed using 4% Paraformaldehyde Phosphate Buffer Solution (Wako, Osaka, Japan) and permeabilized using 0.5% Polyoxyethylene (10) Octylphenyl Ether (Wako, Osaka, Japan) then stained with antibody for 30 minutes at 4°C in the dark. After extensive washing with FACS buffer, the cells were subjected to flow cytometry Canto II flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (BD Biosciences, version 10.6).
Statistical comparisons were performed using the Prism software, version 9.3.1 (GraphPad Software). One-way ANOVA with a post-hoc Tukey’s test was performed to analyze flow cytometry data. Two-way ANOVA with Tukey’s post-test was used for experiments involving analysis of multiple time points. p-values < 0.05 were considered to be statistically significant.
All data generated or analyzed during this study are included in this published article and its supplementary information files.