Panoramic slice scanner

Cancer and adjacent normal tissues were formalin-fixed, paraffin-embedded, and prepared as tissue microarrays (TMAs) after hematoxylin and eosin staining and histopathology-guided location. Five-micron-thick TMA sections were deparaffinized and rehydrated in 0.1 M citrate buffer (pH 6.0), followed by high-temperature antigen retrieval in a microwave for 15 min. The sections were incubated overnight at 4 °C with primary antibodies against IRF3 and IRF7 (Abcam, Cambridge, UK), CD4 (Servicebio Technology, Wuhan, China), CD8 (Servicebio Technology), CD19 (Servicebio Technology), CD68 (Servicebio Technology), MPO (Servicebio Technology) and CD21 (Servicebio Technology). The sections were incubated for 30 min with a secondary antibody at room temperature and immunostained based on avidin biotin complex formation, using 3,3′-diaminobenzidine. Hematoxylin was used as a counterstain.
Antigen–antibody complexes in whole samples were detected using a panoramic slice scanner (3DHISTECH, Hungary) and viewed with CaseViewer 2.2 (3DHISTECH). H-scores were calculated to evaluate gene expression levels using Quant Center 2.1 (3DHISTECH): H-score = Σ (PI × I) = (% of weakly stained cells × 1) + (% of moderately stained cells × 2) + (% of strongly stained cells × 3), where PI is the proportion of the positive area, and I is the staining intensity.

Tumour tissues from CDX and PDX were collected and fixed with 4% paraformaldehyde overnight and sectioned into slides at a thickness of 5 μm. For histological analysis, slides were stained with haematoxylin and eosin (H&E). For immunohistochemistry, slides were deparaffinized, rehydrated, and treated with 3% H₂O₂ for 25 min in the dark. After extensive washing, the slides were blocked with 3% BSA in PBS for 30 min and incubated overnight at 4 °C with the indicated primary antibodies, followed by incubation with the appropriate fluorophore-conjugated secondary antibodies. Antibody information is shown in Supplemental Table S4.
TUNEL staining was conducted to evaluate apoptosis in resected tissue according to the manufacturer’s instructions (Servicebio, Wuhan, China). Briefly, after deparaffinization and antigen retrieval, sections were incubated with 1% Triton X-100 in PBS for 20 min at room temperature. Then, the sections were incubated with reaction solution containing 1 µL of TdT, 5 µL of dUTP and 50 µL of equilibration buffer at 37 °C for 2 h in a humidified box. After washing with PBS, the sections were stained with DAPI for 10 min at room temperature. The slides were scanned using a panoramic slice scanner (3DHISTECH, Hungary). ImageJ software was used for quantitation of TUNEL-positive cells.

ScaRNA2 and ATR expression in the TMAs was detected with FISH and immunohistochemistry. The procedures for the scaRNA2 probe hybridization were consistent with the above FISH method. After FITC-TSA incubation, the slides were incubated at 4 °C overnight with ATR primary antibody (Abcam, ab289363, 1:50) in a humidified chamber, followed by the corresponding goat anti-rabbit Cy3 secondary antibody for 1 h at 37 °C in the dark. Image information on the tissue slice was scanned using a panoramic slice scanner (3DHISTECH, Hungary). CaseViewer 2.4 software was employed at 1–400 × arbitrary magnification for observation.