Tissue cell fixative

Tumor tissues fixed in 4% tissue cell fixative (Solarbio, Dalian, China) were paraffinized and sliced into sections with 4-μm thickness. These were then dehydrated across an ethanol (100%, 95%, 85%, and 75%) gradient. This was followed by treatment with 25 μL of goat serum (BOSTER, Wuhan, China) to inhibit endogenous peroxidase and then heated for antigen retrieval in the presence of 0.1% sodium citrate buffer (pH 6.0). Next, we incubated the sections with primary antibodies (Table S1) overnight, followed by secondary antibodies (ProteinTech, Wuhan, China) with horseradish peroxidase for 1 h. The final analysis was performed after treating the sections with DAB Horseradish Peroxidase Color Kit. For HE staining, sections were stained in line with guidelines of hematoxylin and eosin kit (Solarbio, Dalian, China). The IHC and HE samples were semi-quantitatively or quantitatively evaluated by two independent researchers who were blinded to group allocation.

A three-channel confocal laser scanning microscope (FLUOVEWFV1000, Olympus, Japan) was used to observe the morphological characteristics and formation speed of the biofilm. COMSTAT 2.0 microbial envelope analysis software was used to quantitatively analyse the biofilm tissue structure.
A sterilized slide (φ = 14 mm) was placed in a flat-bottom 24-well plate (Corming, USA), 100 μL QS bacterial solution (OD_600nm = 0.3) and 2 mL liquid 2216E liquid medium were added to each well, and the cells were cultured at 30 °C for 3 days. The slide was washed with 1 × PBS solution to remove free living bacteria and fixed with 4% tissue cell fixative (Solarbio, Beijing, China) for 2 h. After washing with 1×PBS solution, the slide was stained with 20 μmol/L PI (Solarbio, Beijing, China) in the dark for 25 min and washed with 1 × PBS solution. Two-dimensional observation and three-dimensional scanning of the biofilm on the slide were carried out using a three-channel confocal laser scanning microscope with an objective lens (UPlansSApo 20 × 0.75). The scanning images were processed by Olympus FV10-ASW (version 4.0) and reconstructed into 3D images in which the excitation and emission light wavelengths were 559 nm and 570–670 nm, respectively.

Gelatin (biochemical reagent) and 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2,959, 98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Phosphate buffered saline (PBS, pH = 7.4, without calcium and magnesium) was purchased from Biological Industries. Methacrylic anhydride (MA, 94%) was purchased from Sigma-Aldrich. DME/F-12 1:1 (1X), fetal bovine serum (FBS), penicillin- streptomycin solution and trypsin 0.25% (1X) solution were purchased from Hyclone. 4% tissue cell fixative was purchased from Solarbio.