Intervention experiments on 4NQO-induced rat oral carcinogenesis model

Male Sprague–Dawley (SD) rats (4 weeks) were fed daily with 0.002% 4NQO (Sigma-Aldrich, Saint Louis, MO, USA) solution in their drinking water. After the 16-week carcinogen treatment, the drinking water was switched to distilled water. The rats intervened by different methods were described as follows.

(1) Saliva intervention (10 rats/group): (A) 4NQO + OSCC patient saliva; (B) 4NQO + OLK patient saliva; (C) 4NQO + HCs saliva; (D) 4NQO + saline. Rats were treated with 200 µL saliva by submucosal injection twice a week at the start of week 8 and ended at week 16. At week 20, the rats were sacrificed, and the tongues were dissected, and a longitudinal mid-lingual incision was made.

(2) S. mutans/S. gordonii intervention (4 rats/group): (A) 4NQO + S. mutans (CFU = 10⁹); (B) 4NQO + S. gordonii (CFU = 10⁹); (C) 4NQO + saline. Rats were treated with 200 µL live S. mutans/S. gordonii by submucosal injection every 5 days at the start of week 8 and ended at week 16. At week 20, the rats were sacrificed and the tongues were dissected for mucosal epithelium and stored at − 80 °C for further metabolic analyses.

(3) KYNA intervention (10 rats/group): (A) 4NQO + KYNA (28 mg/kg); (B) 4NQO + saline; (C) Normal water + KYNA. Rats were treated with KYNA (70 mg/mL) by submucosal injection every 5 days at the start of week 8 and ended at week 16. At week 20, the rats were sacrificed, and the tongues were dissected, and a longitudinal mid-lingual incision was made.

(4) IL-1β/PD-L1monoclonal antibody (mAb) intervention (8 rats/group): (A) 4NQO + IL-1β mAb (200 µg/rat, BE0246, cloneB122, Bioxcell, USA); (B) 4NQO + KYNA + IL-1β mAb; (C) 4NQO + PD-L1 mAb (200 µg/rat, BE0383, clone 368A.4H1, Bioxcell, USA); (D) 4NQO + KYNA + PD-L1 mAb; (E) 4NQO + control IgG (200 µg/rat, BE0091, Bioxcell, USA). Rats were treated with 200 µL KYNA (28 mg/kg) by submucosal injection every 5 days at the start of week 8 and ended at week 16. After that, rats were treated with indicated IL-1β mAb or PD-L1 mAb by submucosal injection twice a week from week 16 to week 20. At the end of week 20, the rats were sacrificed, and the tongues were dissected, and a longitudinal mid-lingual incision was made. Half of the specimens were fixed in 10% buffered formalin, embedded in paraffin, and cut into 5 μm sections for hematoxylin and eosin (H&E) staining to confirm the pathological diagnosis and immunohistochemistry assays. The other half of the specimens were stored at − 80 °C.

All of the animal procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (2017-015A and KQEC-2022-07-01).