2.7. Orthotopic xenograft model

All animal experiments were approved by the Animal Research Ethics Committee of Sun Yat‐Sen University (2 020 000 303). All BALB/c nude mice (weight 18‐22 g, male) were purchased from the animal facility of Sun Yat‐Sen University and housed in a specific pathogen‐free environment and treated in accordance with the guidelines of the Institutional Animal Use and Care Committee. First, CAL33 cells were transfected with the luciferase gene (GeneCopoeia Inc, USA). Approximately 0.6 million CAL33 cells suspended in 50 μL PBS in the presence or absence of arecoline were injected into the side of the tongue to investigate the effect of arecoline on the tumorigenesis and metastasis of OSCC in vivo. Tumor size and cervical LN metastasis of OSCC were observed at 1 wk, 2 wk, and 3 wk. After 3 wk, mice were sacrificed. The cervical LNs were isolated for H&E staining. For in vivo scans, mice were anesthetized using a mixture of oxygen/isoflurane inhalation and positioned with legs fully extended. The fluorescence images of mice were acquired using an In Vitro Imaging System (IVIS) Spectrum (PerkinElmer, Waltham, MA, USA). Furthermore, immunohistochemical staining was used to detect the expression of SAA1, cytokeratin, and HLA class I in the xenograft tissues or cervical LN as previously described. ¹⁶ The primary antibodies were SAA1 (Catalog #ab207445, Abcam, 1:1000), pan‐cytokeratin (Catalog #bs1712R, Bioss, 1:200) and HLA class I (Catalog #15240‐I‐AP, proteintech, 1:200).