R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2

通过R26-CAG-LSL-Kras*G12C-IRES-Luc（NM-KI-200323）小鼠与R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2（NM-KI-234663）小鼠交配获得。

Fig.1 Body weight changes. Tamoxifen induction of Kras*G12C and HPV-E6/E7 expression in Krt14-CreERT2 mice led to visible tumor development within 4 weeks. Mild body weight loss was observed in the treatment group (n=6, with one death on day 25), likely reflecting tumor-induced morbidity. No significant weight changes were seen in Vehicle controls (n=4). These findings confirm successful tumorigenesis following tamoxifen induction in this model. R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2 mice (8 weeks old) were treated with Tamoxifen (100 mg/kg, i.p., every other day, three times total) or Vehicle. Mice were terminated 4 weeks post-treatment (n=4-6 per group). Data are mean ± SEM.

Fig.2 Tumor visualization by IVIS and gross observation at day 21. By day 21 post-tamoxifen induction, IVIS imaging confirmed robust tumor growth in the treatment group. Notably, visible tumors developed at ear tag sites, suggesting that local epithelial proliferation during wound healing may promote or accelerate Kras/HPV/Krt14-CreERT2-driven tumorigenesis. R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2 mice were treated with Tamoxifen (100 mg/kg, i.p., every other day, three times total) or Vehicle. At day 21 post-treatment, IVIS imaging showed significant tumor bioluminescence at mutiple site in Tamoxifen-treated mice, indicating robust tumor growth. Additionally, visible tumors were observed at ear tag sites in the Tamoxifen group. (n=4-6 per group).

Fig.3 Mice ear and ear tumor histopathology. R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2 mice (8 weeks old) were treated with Tamoxifen (100 mg/kg, i.p., every other day, three times total) (G2) or Vehicle (G1). Mice were terminated 4 weeks post-treatment (n=4-6 per group). 

G2: An epithelial tumor exhibiting mixed exophytic and endophytic growth is observed within the auricular skin and associated soft tissues. The tumor is continuous with the epidermis and squamous epithelium resembling the follicular infundibulum; it displays multifocal to coalescing proliferation, forming irregular papillary, nested, cord-like, and trabecular structures that infiltrate deep into the dermis and subcutaneous tissue. The tumor cells are primarily of squamous epithelial origin (black arrows), appearing polygonal or ovoid with moderate to abundant eosinophilic cytoplasm; intercellular bridges are visible. Prominent keratinization is evident within tumor nests, manifesting as keratin pearl formation, dyskeratosis, central keratin deposits, surface hyperkeratosis, and focal parakeratosis (blue arrows). The tumor cells exhibit disordered arrangement and loss of polarity, with localized proliferation of basaloid cells. Nuclear atypia is moderate, characterized by nuclear enlargement, hyperchromasia, anisokaryosis, visible nucleoli, an increased nuclear-to-cytoplasmic ratio, and increased mitotic activity (which is notably brisk in some areas). A marked stromal reaction is present, with abundant fibroblasts and collagen deposition between tumor nests, indicating a desmoplastic response. Mild to moderate inflammatory cell infiltration—comprising mainly lymphocytes, plasma cells, and scattered neutrophils—is observed in the peritumoral and stromal regions, accompanied by focal hemorrhage in some areas.

Fig.4 Mice uterus histopathology. R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2 mice (8 weeks old) were treated with Tamoxifen (100 mg/kg, i.p., every other day, three times total) （G2） or Vehicle （G1）. Mice were terminated 4 weeks post-treatment (n=4-6 per group). 

G2-194: Focal thickening of the mucosal folds is observed in the uterus and cervix/cervicovaginal junction, accompanied by an irregular luminal contour.There is focal to multifocal thickening of the cervical/vaginal-type squamous or transitional epithelium (black arrows); mild elongation of rete ridges is noted, with focal mild invagination or budding of the epithelium into the lamina propria.The submucosa and cervical stroma are markedly thickened, containing numerous spindle-shaped cells and collagen fibers arranged in fascicles or whorls; cellular atypia is inconspicuous and mitotic activity is low, suggesting reactive fibroblastic/smooth muscle-like stromal hyperplasia.The stroma exhibits mild to moderate chronic inflammatory cell infiltration—predominantly lymphocytes and plasma cells—with scattered neutrophils observed focally (green arrows).

G2-195: Mucosal folds and endometrial glands are visible in the uterine horn; the glands are arranged relatively regularly, and the glandular epithelium shows no obvious neoplastic atypia.The mucosa is thickened in the central-to-left cervical/vaginal region, exhibiting localized irregular folding or papillary hyperplasia.The cervical/vaginal squamous epithelium is markedly thickened (black arrow), characterized by hyperplasia of basal and parabasal cells, increased epithelial stratification, elongation of rete ridges, localized epithelial disarray, and reduced cellular polarity.There is no definitive evidence of invasion into the deep stroma in the form of irregular tumor nests, cords, or keratinizing carcinomatous nests breaching the basement membrane.

Fig.5 Mice ovary histopathology. R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2 mice (8 weeks old) were treated with Tamoxifen (100 mg/kg, i.p., every other day, three times total) （G2） or Vehicle （G1）. Mice were terminated 4 weeks post-treatment (n=4-6 per group). 

Fig.6 Mice lung histopathology. R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2 mice (8 weeks old) were treated with Tamoxifen (100 mg/kg, i.p., every other day, three times total) （G2） or Vehicle （G1）. Mice were terminated 4 weeks post-treatment (n=4-6 per group). 

Fig.7 Mice eyeball histopathology. R26-CAG-LSL-Kras*G12C-IRES-Luc/R26-CAG-LSL-HPV-E6-CAG-LSL-HPV-E7-IRES-Luc/Krt14-CreERT2 mice (8 weeks old) were treated with Tamoxifen (100 mg/kg, i.p., every other day, three times total) （G2） or Vehicle （G1）. Mice were terminated 4 weeks post-treatment (n=4-6 per group).