Development of a Novel Mouse Model of Spontaneous High-Risk HPVE6/E7-Expressing Carcinoma in the Cervicovaginal Tract

Abstract

Current preclinical models for cervical cancer lack important clinical and pathologic features. To improve upon these models, we aimed to develop a novel, spontaneous HPV16-expressing carcinoma model that captures major aspects of HPV-associated cancer in the female genital tract. This novel preclinical model features (i) expression of HPV oncogenes E6 and E7 in the tumors in female reproductive tract of mice, (ii) spontaneous progression through high-grade squamous intraepithelial lesion (HSIL) to carcinoma, and (iii) flexibility to model cancers from different high-risk HPV genotypes. This was accomplished by injecting plasmids expressing HPV16 E6/E7-luciferase, AKT, c-myc, and Sleeping Beauty transposase into the cervicovaginal tract of C57BL/6 mice followed by electroporation. Cell lines derived from these tumors expressed HPV16 E6/E7 oncogenes, formed tumors in immunocompetent mice, and displayed carcinoma morphology. In all, this novel HPV-associated cervicogenital carcinoma model and HPV16E6/E7-expressing tumor cell line improves upon current HPV16-E6/E7-expressing tumor models. These tumor models may serve as important preclinical models for the development of therapeutic HPV vaccines or novel therapeutic interventions against HPV E6/E7-expressing tumors. SIGNIFICANCE: This study describes the development of a clinically relevant mouse model of cervicovaginal carcinoma that progresses from high-grade lesions and recapitulates key features of human HPV⁺ cervical cancer.

Figure 1.

Generation of spontaneous HPV⁺ cervicovaginal carcinoma model in C57BL/6 mice. Mice received transient CD3 cell depletion at day −3, −2, −1, 7, and 14. On day 0, C57BL/6 mice received 10 μg of each DNA plasmid (total injection volume 20 μg) as a submucosal injection in the vaginal tract, followed by electroporation. A, Schematic of experimental design. B, Schematic of plasmids used to induce HPV⁺ cervicovaginal tumors via oncogene integration. Arrows, direction of plasmid integration. C, Representative images of the development of AMES-16–induced cervicovaginal tumor post electroporation as measured by IVIS Spectrum imaging. Bioluminescence was recorded by IVIS Spectrum after intraperitoneal injection of luciferin solution. D, Tumor growth as monitored by bioluminescence imaging. E, Survival rate shown by percentage. Mice were considered dead due to tumor when tumor diameter >15 mm, and the mice were subsequently sacrificed. **, P = 0.0023; ***, P ≤ 0.0001.

Figure 2.

Representative images of HPV16⁺ spontaneous cervicovaginal tumor. Mice received transient CD3 cell depletion at day −3, −2, −1, 7, and 14. On day 0, C57BL/6 mice received 10 μg of each AMES-16 DNA plasmid (total injection volume 20 μg) as a submucosal injection in the vaginal tract, followed by electroporation. A and B, Representative H&E of tumor displaying well-differentiated (A) or poorly differentiated SCC (B). C and D, RNA ISH of HPV16 E6 for corresponding tumors shown in A and B. E, IHC of tumor proliferation marker Ki-67. F, IHC of c-myc. G, IHC of carcinoma marker CK14. H, IHC of AKT.

Figure 3.

T-cell characterization in HPV16⁺ spontaneous cervicovaginal tumor. A, Time course of T-cell recovery post depletion. Mice received transient T-cell depletion via administration of intraperitoneal injection of 200 μg anti-CD3 antibody daily 3 days prior to AMES-16 electroporation and weekly for 2 weeks after electroporation. B, Tumor-bearing mice were sacrificed 6 weeks post AMES-16 electroporation and tumor immune infiltrate was evaluated by flow cytometry. Bar graph summary of AMES-16—induced cervicovaginal tumor-infiltrating lymphocytes. C, Bar graph summary of exhaustion markers expressed by tumor-infiltrating lymphocytes (TIL) 6 weeks post AMES-16 electroporation.

Figure 4.

Anatomical orientation and lesion progression from HSIL to SCC in spontaneous HPV⁺ cervicovaginal carcinoma model. At each time point following the AMES-16-plasmid electroporation, Three mice were sacrificed and their reproductive tracts were harvested and fixed in formalin for sectioning and histologic analysis. Representative images of the histologic examination were selected. A, Magnification (×2) of mouse reproductive tract 3 weeks post AMES-16 DNA electroporation anatomically labeled. B, Magnification (×40) showing cervical HSIL in black box with orientation as shown by cervix in A. C, Magnification (×40) showing vaginal HSIL in dotted black box as shown in lower left corner of A. D, Magnification (×20) showing vaginal HSIL in dashed black box and carcinoma in black circle with orientation as shown in A. E, Development of SCC from HSIL. Left, emergence of HSIL (see box) corresponding with luminescence values approximately 1 × 10⁶. Middle, appearance of SCC (see box) and corresponding with luminescence values approximately 1 × 10⁷. Right, appearance of HSIL and invasive carcinoma corresponding with luminescence values approximately 1 × 10⁸. F, Left, representative H&E of HSIL lesion 2 weeks post AMES-16 DNA electroporation. Right, HPV16 RNA (black arrows) colocalizes with HSIL lesion at week 2 post AMES-16 DNA electroporation with no evidence of HPV RNA expression in dermal compartment. HPV16 RNA was performed by RNAscope ISH.

Figure 5.

Characterization of Tal3 cell line–induced tumor size and penetrance in immunocompetent C57BL/6NTac mice. A, E6 expression in the Tal3 cell line was measured by RT-PCR and gel electrophoresis. C57BL/6NTac mice were subcutaneously injected with 8 × 10³, 8 × 10⁴, 8 × 10⁵, or 8 × 10⁶ Tal3 cells in the vaginal tract. B, Tumor growth curve of submucosal injection of indicated doses of Tal3 cells/mouse. N = 5. C, Representative image of Tal3 tumor as measured by IVIS Spectrum imaging. Bioluminescence was recorded by IVIS Spectrum after intraperitoneal injection of luciferin solution.

Figure 6.

Characterization of SCC, tumor markers, and tumor infiltrate by Tal3 cell line. C57BL/6NTac mice were submucosally injected with 8 × 10⁵ Tal3 cells in the vaginal tract. A, Representative H&E of Tal3 cell line displaying SCC morphology. B, IHC of c-myc. C, IHC of AKT. D, IHC of tumor proliferation marker Ki-67. E, Mice were sacrificed 3 weeks post submucosal injection of Tal3 and tumor-infiltrating lymphocytes (TIL) were evaluated by flow cytometry. A bar graph result summary of tumor-infiltrating lymphocytes. F, Bar graph summary of exhaustion markers expressed by Tal3 tumor-infiltrating lymphocytes.