Clinical samples

The resected tumor tissues (n = 15) from OSCC patients and normal tissues (n = 10) from patients who underwent orthognathic surgery were obtained from Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Nanjing University. These patients who underwent orthognathic surgery have good oral hygiene and no confirmed periodontal diseases were found through periodontal examination. Sterile surgical samples were freshly collected and formalin-fixed and paraffin-embedded for further hematoxylin–eosin staining (H&E) and fluorescent in situ hybridization (FISH) validation. We obtained written informed consent from all patients. The study protocol was approved by the Ethics Committee of Nanjing Stomatological Hospital, Affiliated Hospital of Medical School, Nanjing University (IRB approval number: 2018NL-008 (KS) & NJSH-2023NL-18) and the Animal Ethical and Welfare Committee of Nanjing University (IACUC-D2202108 & IACUC D2303084).

Fluorescent in situ hybridization (FISH)

Spectrum-Yellow FISH labeled with RNAscope™ Probe-B-P. intermedia-16SrRNA-C1 (ACD, 1197771-C1, GCCTA-ATACCCGATG-TTGTCCACAT-ATGGCATCTG-ACGTGGACC) was performed according to the manufacturer’s instructions. The specific probe detecting P. intermedia was designed and developed based on the NCBI database (Additional file 1: Fig S1). Fluorescence microscopic analysis was conducted with a confocal microscope. The Panoramic 250 Flash system (3DHISTECH) was used to scan the slides.

Fig. 1

Flow chart of the experiments. P. intermedia detection in OSCC and normal tissues, in vitro study of SCC7 cells, and in vivo study in a murine transplanted tumor model of OSCC

Bacterial strain

P. intermedia strain ATCC 25611 was purchased from BeNa Culture Collection (Beijing, China) and grown in Columbia blood agar plates under anaerobic conditions at 37 °C for 3–4 days. The monoclonal colonies were then picked up and inoculated in sterilized brain heart infusion broth (BHI) supplemented with yeast extract (1 mg/mL), hemin (5 μg/mL), and vitamin K (1 μg/mL) until the cells reached the logarithmic growth phase.

Cell culture

The mouse OSCC cell line SCC7 (Cellcook Biotech Co., Ltd. Guangzhou, China) was cultured at 37 °C with 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium including 10% fetal bovine serum (FBS) and 1% penicillin (1 × 105 U/L)-streptomycin (100 mg/L) solution (PS).

Transmission electron microscopy (TEM)

A transmission electron microscope (TEM) was used to visualize the morphology of intracellular bacteria. SCC7 cells were seeded with complete cell culture medium (1640 medium with 10% FBS and 1% PS). Before P. intermedia invasion, the cells were washed twice with phosphate-buffered saline (PBS), and antibiotic-free medium was added. P. intermedia was collected during the logarithmic phase, washed twice with PBS, and added to the cells at an MOI of 10, and the cells were incubated in the cell incubator for 3 h. After infection, the cells were washed with PBS 3 times, and 2.5% glutaraldehyde (Sigma‒Aldrich) was added for 5 min at 25 °C protected from light. All cells were observed using a Hitachi electronic microscope at 80 kV.

Scanning electron microscopy (SEM)

SCC7 cells were added to slides and allowed to adhere overnight. The infection steps were the same as described for TEM. After 3 h, the cells were fixed with 2.5% glutaraldehyde (Sigma‒Aldrich) in PBS. The cells were dehydrated with a gradient of increasing alcohol concentrations (40–120%). Then, they were dried using the critical point method (Quorum, Model K 850). The samples were gold-sputtered (Hitachi, MC1000) to generate conductivity and then coated with iridium fast coating (5 nm) on a Leica EMACE600 and imaged on a Hitachi SU8100 field-emission SEM.

Cell proliferation assay

Cell Counting Kit‐8 (CCK‐8) assays (Dojindo Molecular Technologies, XiongBen, Japan) were used to assess the proliferation ability of SCC7 cells. SCC7 cells were seeded into 96‐well plates at a concentration of 3 × 103 cells each well in 100 μl of medium, and after adherence overnight, we treated the cells according to the experimental design. Heat-killed P. intermedia (1, 10, 50, 100 MOIs) was added to SCC7 cells for 24, 48, 72 h, respectively. There were 5 replicate wells in each group. Afterward, 10 µL of CCK‐8 solution was added to each well of the plate. After 2 h of incubation at 37 °C with 5% CO2, the absorbance was measured at 450 nm by a microplate reader. OD values were analyzed as the mean ± standard deviation.

Cell invasion assay

Transwell upper chambers (8-μm pore size; Corning Incorporated, Toledo, NY, USA) were pre-coated with Matrigel (Corning Incorporated). 3 × 104 SCC7 cells were suspended in 200 μL medium and then plated on the upper chambers with Matrigel. Then, 10% FBS was added in 500 μL medium, which was prepared onto the lower chambers. After culture for 24 h, the invaded SCC7 cells on the lower surface of the chamber were fixed by paraformaldehyde solution for 30 min at room temperature and stained using 0.1% crystal violet (Solarbio) for 30 min. Finally, the number of cells on the lower surface of the membrane were observed and counted using a microscope (Olympus) in five random fields.

Enzyme-linked immunosorbent assay (ELISA)

Analysis was performed on cell culture supernatants or serum to determine the concentrations of cytokines, including IL-17A, IL-6, IL-10, IL-1β, PD-L1, IFN-γ and TNF-α. All ELISA kits were purchased from Multiscience Biotechnology Co., Ltd. (Hangzhou, Zhejiang, China). SCC7 cells treated with heat-killed P. intermedia for 12 h were cultured using the abovementioned method and grouped according to the MOIs of 1, 10, 50, and 100.

Murine transplanted tumor model of OSCC

C57BL/6 J mice (6 weeks old, female) were housed in a stable environment with a 12:12 h light/dark cycle and had free access to food and water. The humidity was kept between 55 and 65% and the temperature was maintained between 23% and 25 °C. To eliminate interference from other bacteria in the oral cavity, mixed antibiotics containing metronidazole (1 mg/ml; Sigma‒Aldrich), neomycin (1 mg/ml; MCE), ampicillin (1 mg/ml; MCE), and vancomycin (0.5 mg/ml; MCE) were administered in drinking water for 3 days before injection. SCC7 cells resuspended in PBS (3 × 105, 5 μl/per) were submucosally injected into the buccal mucosa to establish a OSCC transplanted tumor model [14]. P. intermedia strain ATCC 25611 was collected during the logarithmic phase, washed twice with PBS, inactivated in an 80 °C water bath for 2 h, and diluted with PBS (3 × 102 CFU, 5 μl/per). The experimental mice were divided randomly into the control group and heat-killed P. intermedia group (6 mice per group). All mice received submucosal injection of SCC7 suspension liquid (3 × 105, 5 μl/per). Each mouse in the heat-killed P. intermedia group was injected with heat-killed P. intermedia (3 × 102 CFU, 5 μl/per) into the palpable tumor 7 days after SCC7 cell injection. Tumor volume and body weight were measured at 2 day intervals until the end of the experiment. Tumor volume = maximum diameter × vertical diameters^2/2. Fourteen days after the first injection, the mice were sacrificed for serum and tumor quantification. All animal experiments were performed in compliance with ethical regulations.

Hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence

Mouse tumor specimens were formalin-fixed, dehydrated, paraffin-embedded and cut into 5 μm sections using routine protocols for staining with H&E. Images were captured by microscopy (Olympus CX23; Olympus Corporation, Tokyo, Japan). The Panoramic 250 Flash system (3DHISTECH) was used to scan the slides.

Immunohistochemistry was processed in the same way as described above and then stained with IL-17A (Abcam, ab91649), IL-23 (NOVUS BIO, NBP1-76697), IL-6 (Abcam, ab290735), KI-67 (Abcam, ab237728), matrix metalloproteinase-9 (MMP-9) (Abcam, ab283575), GABBR2 (Abcam, ab75838), FASL (ABclonal, A0234), P63 (ABclonal, A19652), CD8α (Abcam, ab217344), FOXP3 (CST, 98377S), F4/80 (Abcam, ab6640), CD206 (Abcam, ab64693) and DAPI (Abcam, ab104139). Images were taken using a microscope or a confocal laser scanning microscope. The panoramic 250 Flash system (3DHISTECH) was used to scan the slides. All immunohistochemistry assays included negative controls and positive controls. The IHC-positive areas were stained brown in the cytoplasm, and the average optical density (AOD) [AOD = Integrated Optical Density (IOD) SUM/Area SUM] was measured using ImageJ (IHC toolbox plugin). The relative AOD values were calculated using the normalization method. At least three random fields of each slide were selected.

Tumor RNA extraction, mRNA sequencing, and transcriptome analysis

Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then, the libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

Differential expression analysis was performed using DESeq25. A Q value < 0.05 and fold change > 2 or fold change < 0.5 were set as the thresholds for significantly differentially expressed genes (DEGs). Hierarchical cluster analysis of DEGs was performed using R (v 3.2.0) to demonstrate the expression pattern of genes in different groups and samples. The radar map of the top 30 genes was drawn to show the expression of upregulated or downregulated DEGs using the R packet ggradar.

Based on the hypergeometric distribution, GO, KEGG pathway, Reactome and WikiPathways enrichment analyses of DEGs were performed to screen the significantly enriched terms using R (v 3.2.0). R (v 3.2.0) was used to draw the column diagram, chord diagram and bubble plot of the significantly enriched terms.

Gene Set Enrichment Analysis (GSEA) was performed using GSEA software8-9. The analysis used a predefined gene set, and the genes were ranked according to the degree of differential expression in the two types of samples. Then, we tested whether the predefined gene set was enriched at the top or bottom of the ranking list.

Statistical analysis

SPSS 20.0 (IBM, NY, USA) was used for data analysis. ImageJ and GraphPad Prism 9.0 (Graph Software Inc.) were used for image analysis. Data are expressed as the mean ± SD. The difference in measurement data between two groups was assessed using a two-tailed/one-tailed Student’s t test. Multiple group comparisons were conducted using one- or two-way analysis of variance (ANOVA). The level of statistical significance was set at p < 0.05.