Integration of Oncogenes via Sleeping Beauty as a Mouse Model of HPV16+ Oral Tumors and Immunologic Control

Abstract

Human papillomavirus type 16 (HPV16) is the etiologic factor for cervical cancer and a subset of oropharyngeal cancers. Although several prophylactic HPV vaccines are available, no effective therapeutic strategies to control active HPV diseases exist. Tumor implantation models are traditionally used to study HPV-associated buccal tumors. However, they fail to address precancerous phases of disease progression and display tumor microenvironments distinct from those observed in patients. Previously, K14-E6/E7 transgenic mouse models have been used to generate spontaneous tumors. However, the rate of tumor formation is inconsistent, and the host often develops immune tolerance to the viral oncoproteins. We developed a preclinical, spontaneous, HPV16⁺ buccal tumor model using submucosal injection of oncogenic plasmids expressing HPV16-E6/E7, NRas ^G12V , luciferase, and sleeping beauty (SB) transposase, followed by electroporation in the buccal mucosa. We evaluated responses to immunization with a pNGVL4a-CRT/E7(detox) therapeutic HPV DNA vaccine and tumor cell migration to distant locations. Mice transfected with plasmids encoding HPV16-E6/E7, NRas ^G12V , luciferase, and SB transposase developed tumors within 3 weeks. We also found transient anti-CD3 administration is required to generate tumors in immunocompetent mice. Bioluminescence signals from luciferase correlated strongly with tumor growth, and tumors expressed HPV16-associated markers. We showed that pNGVL4a-CRT/E7(detox) administration resulted in antitumor immunity in tumor-bearing mice. Lastly, we demonstrated that the generated tumor could migrate to tumor-draining lymph nodes. Our model provides an efficient method to induce spontaneous HPV⁺ tumor formation, which can be used to identify effective therapeutic interventions, analyze tumor migration, and conduct tumor biology research. Cancer Immunol Res; 6(3); 305-19. ©2018 AACR.

Figure 1. Generation of spontaneous HPV buccal tumors in immunocompromised mice

(A) Schematic of plasmids used to induce oral tumors by oncogene transfection. (B) Immunocompromised, athymic nude mice (NCr strain, n=5/group) received plasmids once through submucosal injection followed by electroporation in the buccal area. Different combinations of plasmids are indicated in the legend. The bioluminescence image was recorded by IVIS Spectrum after IP injection of luciferin solution. (C) Percent tumor-free survival. Three independent experiments were performed. P values were calculated by log-rank test (C). P values < 0.05 were considered significant. Data are presented as mean ± SD.

Figure 2. Generation of spontaneous HPV buccal tumors in immunocompetent mice

(A) C57BL/6NCr, Balb/c, or FVB/NCr mice (n=5/group) received submucosal plasmid injections, followed by electroporation. The plasmids contained HPV16-E6/E7, luciferase, NRas^G12V, and SB100 transposase. Bioluminescence intensity was recorded by an IVIS Spectrum. (B) Schematic diagram of treatment protocol. Control C57BL/6NCr mice (n=5): plasmid transfection only; CD3-depleted C57BL/6NCr mice (n=5): 100 μg anti-CD3 IP for a total of three doses at one-day intervals before plasmid transfection; CD4-depleted C57BL/6NCr mice (n=5): began with 100 μg anti-CD4 IP for a total of three doses at one-day intervals before receiving one dose weekly after plasmid transfection; CD8-depleted C57BL/6NCr mice (n=5): began with 200 μg anti-CD8 IP for a total of three doses at one-day intervals before receiving two doses weekly after plasmid transfection. (C) Bioluminescence kinetics of buccal tumors in different groups. Three independent experiments were performed. Data are presented as mean ± SD.

Figure 3. Bioluminesence signal as index for spontaneous HPV buccal tumor growth

C57BL/6NCr mice (n=5) were CD3-depleted using 100 μg anti-CD3IP daily for three consecutive days, followed by submucosal plasmid injection and electroporation in the buccal area one day after the final antibody injection. Plasmids contained HPV16-E6/E7, luciferase, NRas^G12V, and SB100 (10 μg/plasmid). (A) Relationship between bioluminescence kinetics of buccal tumor and percentage of CD3⁺ T cells over time. Black star: visualized overt tumor. Peripheral blood from tail arteries was taken for flow cytometry to check CD3⁺ T cells percentage over time. (B) The bioluminescence intensity plotted against tumor volume. Three independent experiments were performed. Data are presented as mean ± SD.

Figure 4. Characteristics of spontaneous HPV buccal tumors

(A) Representative pictures of gross buccal tumors arising from oncogene cotransfection in C57BL/6NCr mice. Plasmids contained HPV16-E6/E7, luciferase, NRas^G12V, and SB100. (B) Representative histology sections with H&E stain and special IHC staining with Ki-67 and NRas. (C) Representative images for RNAscope targeting HPV16 RNA and IHC staining with HPV-associated biomarker p16. Three independent experiments were performed.

Figure 5. DNA vaccination generated E7-specific CD8⁺ T-cell responses against spontaneous HPV oral tumors

(A) Schematic diagram of treatment regimens, and the overall time course. C57BL/6NCr mice (n=5) were CD3-depleted, as described, for three consecutive days, followed by submucosal injection and electroporation in the buccal area with plasmids containing HPV16-E6/E7, luciferase, NRas^G12V, and SB100. Ten days after plasmid injection, mice were vaccinated with pNGVL4a-CRT/E7 DNA vaccine or empty pNGVL4a plasmid vector control for a total of four times at four-day intervals. (B) Kaplan-Meier survival analysis of mice. (C) Peripheral blood from tail arteries were examined by flow cytometry for E7-specific CD8⁺ T cells. Percentages were checked over time from two weeks after plasmid injection at one-week intervals. (D) Representative flow cytometry images of the percentage of E7-specific CD8⁺ T cells in PBMCs of tumor-bearing mice treated with pNGVL4a control (top) and pNGVL4a-CRT/E7(detox) (bottom) on day 21 after plasmid injection. A total number of 120,000 cells were acquired. (E) Real-time bioluminescence image of tumor-bearing mice. (F) Line graph depicting the change in tumor volume of tumor-bearing mice after plasmid transfection. Three independent experiments were performed. P values were calculated by log-rank test (B) or two-tailed Student t-test (C). P values < 0.05 were considered significant. Data are presented as mean ± SD.

Figure 6. Analysis of the TME

C57BL/6NCr mice (n=3) were CD3-depleted using 100 μg anti-CD3 IP injected daily for three consecutive days, followed by submucosal plasmid injection and electroporation in the buccal area one day after the final antibody injection. The plasmids contained HPV16-E6/E7, luciferase, NRas^G12V, and SB100 (10 μg/plasmid). Ten days after plasmid transfection, mice were treated with pNGVL4a-CRT/E7(detox) or empty pNGVL4a vector only (20 μg/dose/mouse) via IM injection, followed by electroporation for a total of four times at four-day intervals. Seven days after last vaccination, the mice were euthanized, and the tumor tissues were harvested and analyzed for the presence of various immune cell populations. (A) Representative flow cytometry images (left) and bar graph summary (right) of E7-specific CD8⁺ T cells in the TME. (B) Representative flow cytometry images (left) and bar graph summary (right) of regulatory T cells in TME. (C) Representative flow cytometry images (left) and bar graph summary (right) of myeloid-derived suppressor cells in TME. Three independent experiments were performed. P values were calculated by two-tailed Student t-test. P values < 0.05 were considered significant. Data are presented as mean ± SD.

Figure 7. Metastatic capability in the spontaneous tumor model

Representative mice were presented. (A) Ex vivo bioluminescence assay for the tissues of tumor (left), tumor draining lymph node (TDLN; middle), and spleen (right) of athymic nude mice receiving buccal plasmid injections containing HPV16-E6/E7, luciferase, NRas^G12V, and SB100 followed by electroporation. (B-C) C57BL/6NCr mice received anti-CD3 to deplete T cells for three consecutive days, followed by buccal plasmid injection with HPV16-E6/E7, NRas^G12V, and SB100. (B) Representative image showing the TDLNs and non-TDLNs (NTDLN) of plasmid-transfected mice at the time of euthanization (C) Representative image depicting the luciferase activity of TDLNs and NTDLNs. (D) Luciferase activity after re-implantation of 5 × 10⁵ harvested, proliferating TDLN cells, as well as tumor formation. (E) Representative histology of a tumor formed by re-implantation of TDLN cells. Three independent experiments were performed. P values were calculated by two-tailed Student t-test. P values < 0.05 were considered significant. Data are presented as mean ± SD.