Figure 6. Targeted E6 and E7 Expression Induces Hyperplasia and Dysplasia with Reduced Off-Target Effects.

(A) General strategy for generating conditional and inducible iKH mice by crossing the KRT14-CreER^tam (iK) strain to our knockin HPV16 strain (H).

(B) Representative photographs of iKH animals at 8–10 months.

(C) Comparison of oral volume fold change between LH, KH, and iKH and control littermate mice 42 weeks post-tamoxifen injection reveals that restricting expression to the lingual and oropharyngeal epithelia significantly reduces off-target effects on external oral epithelial volume (n = 3, *p < 0.01).

(D) Quantitative real-time PCR analysis of E6 and E7 transcript copy numbers from dissected whole tongue of iKH and control littermate mice calculated using respective standard curves (n = 3, **p < 0.001, ***p < 0.0001).

(E) Body weight remains unchanged with lingual TAM administration. Comparison of male versus female body weights over the course of the study reveals no significant acute or long-term impact of i.l. TAM delivery either during or following administration, respectively (n = 3–8, mean ± SEM). Yellow shading highlights tamoxifen injection time frame. Blue shading highlights daily animal monitoring performed immediately post-injection followed thereafter by periodic weight and health assessment.

(F) Histologic analysis of epithelial tissues. Representative sagittal sections from adult tongue and cutaneous epithelia were formalin fixed, embedded, and H&E stained. Black arrowheads denote suprabasal mitoses. Magnification, 400×; bar, 50 µm.

(G) Comparison of cutaneous epithelial thickness fold change between LH, KH, and iKH and control littermate mice reveals that restricting expression to the lingual epithelia significantly reduces general cutaneous epithelial manifestations (n = 3, mean ± SEM; **p < 0.001, *p < 0.01).

(H) Increased expression of a E7/E2F1 target within iKH tongues. IF analysis of representative sections from adult iKH tongues stained for the HPV16 biomarker MCM7. White arrowheads denote suprabasal events. Magnification, 400×; bar, 50 µm.

(I) Targeted expression of E6 and E7 together with 4NQO treatment drives oropharyngeal squamous cell carcinoma development. Schematic overview of the animal experiment (top) and representative images of the gross morphology for tumors (yellow arrow) that develop in the iKHP mice provided 4NQO (20 µg/ml) in the drinking water (bottom). Dotted lines delineate the tumor boundary.

(J) Histologic and immunohistochemical analysis of immune cell infiltration. Representative sections from formalin-fixed, embedded, and H&E-stained iKHP tongues (left) and reveal morphologic and histologic abnormalities consistent with oropharyngeal squamous cell carcinoma. Inset highlights a region with an invasive epithelial island (yellow arrowhead). IHC staining of infiltrating Cd8a+ cytotoxic T cells (right). Magnification, 200×; bar, 200 µm.

Data presented in (C), (D), and (G) are shown as mean ± SEM of a representative experiment (n = 3 experiments). Two-way ANOVA with Bonferroni’s post hoc tests were used to determine significance. Related to Figure S7.