Animal model

We conducted a controlled comparative experimental study using 120 Syrian golden male hamsters (Mesocricetus auratus, 5 weeks old, weighing 80–110 g). They were obtained from VACSERA, Cairo, Egypt. The animal study was approved by the Alexandria University review committee and the procedures followed are in accordance with institutional guidelines (IRB#00010556-IORG0008839). The hamsters were weighed once per week throughout the whole period of the experiment. They were housed in show box cages (Technoplast, Italy) one per box under the same condition on a regular alternating lighting cycle (12:12 light: dark).

To establish an oral cancer model, we chemically induced OSCC by painting the left buccal pouch, only to facilitate eating by the other side, of the hamsters with 7, 12 dimethylbenz [a] anthracene carcinogen (DMBA, Sigma Aldrich, 57,976 Germany). We used hamster buccal pouch as our oral cancer model due to the similarities between its lining mucosa and the epithelium covering hard palate, tongue and gingiva of human oral cavity. Furthermore, multiple correspondence to human OSCC were found regarding morphology, molecular markers expression, and finally DNA mutations [18].

During the carcinogenesis phase, we used DMBA along with a carbamide peroxide as a promoter, 5 days per week (alternate days, 3 days for DMBA and 2 days for the promoter) [19]. We specifically used this promoter to decrease the induction period from 16 weeks to less than 12 weeks and obtaining a well-developed intraoral OSCC exophytic masses. This was done to minimize the handling procedures throughout the experiment with the animals and the carcinogen. Carcinogenesis was evaluated microscopically 4 weeks after induction by sacrificing cohorts of 2 hamsters every 2 weeks and evaluating lesions with H&E staining until well-established OSCCs were detected. Moreover, hamsters with tumor size greater than 2 cm in diameter were dropped from the study and euthanized before the predetermined time point (4 weeks).