Fig. 2. Characterization of 4NQO-induced murine oral squamous cell model.

a Left panel, C57Bl/6 mice were implanted with 1 × 10⁶ of either 4MOSC1 or 4MOSC2 cells into the tongue. Tongue lesions when the tumor volume reached ~100 mm³. Middle panel, representative H&E staining of histological tissue sections from mouse tongues with 4MOSC1 or 4MOSC2 tumors. Right, representative pictures of tumors stained to show expression of cytokeratin 5 (CK5, green) and DAPI (blue) (n = 3 mice per group). b Top panel, representative H&E stain of a nonmetastatic cervical lymph node from mice with 4MOSC1 tumors. Bottom panel, representative H&E stain of a metastatic cervical lymph node from mice with 4MOSC2 tumors. Metastatic growth of 4MOSC2 cells into the lymph node is depicted with a dotted line in the bottom area (n = 5 mice per group). c Representative tumor tissue sections stained for LYVE-1 by immunohistochemistry in 4MOSC1 or 4MOSC2 tumors (n = 3 mice per group). d Absolute number of immune cells infiltrating 4MOSC1 or 4MOSC2 tumors. Shown is the average number of live cells infiltrating per mm³ of tumor (n = 3 mice per group). e Immunofluorescent staining of CK5 and CD8 to show squamous cell character of the lesion and CD8 infiltration in mice with 4MOSC1 or 4MOSC2 tumors, respectively (n = 3 mice per group) (CK5, green; CD8, red; DAPI, blue). 4MOSC1 or 4MOSC2 tumors were isolated from mice and mechanically and enzymatically digested. Single-cell suspension was then stained with CD45, Nk1.1, CD3, CD8, CD44, PD-L1, PD-1, CTLA-4, LAG-3, and TIM-3 fluorescent labeled antibodies and analyzed by flow cytometry. Shown are representative flow cytometry plots of f the frequency of tumor cells (CD45 negative) expressing PD-L1 and g the frequency of CD8⁺/CD44⁺ cells expressing inhibitory receptors PD-1, CTLA-4, LAG-3, and TIM-3 in individual tumors (n = 4 mice per group). Contour plots of lymphocytes from tumor (green), and corresponding cervical lymph nodes (blue), and blood (red) are overlaid and the frequencies of tumor CD8⁺/CD44⁺ T cells expressing each inhibitory receptor are shown (n = 4 mice per group).