A Mouse Model of Oropharyngeal Papillomavirus-Induced Neoplasia Using Novel Tools for Infection and Nasal Anesthesia

Abstract

Human head and neck cancers that develop from the squamous cells of the oropharynx (Oropharyngeal Squamous Cell Carcinomas or OPSCC) are commonly associated with the papillomavirus infection. A papillomavirus infection-based mouse model of oropharyngeal tumorigenesis would be valuable for studying the development and treatment of these tumors. We have developed an efficient system using the mouse papillomavirus (MmuPV1) to generate dysplastic oropharyngeal lesions, including tumors, in the soft palate and the base of the tongue of two immune-deficient strains of mice. To maximize efficiency and safety during infection and endoscopy, we have designed a nose cone for isoflurane-induced anesthesia that takes advantage of a mouse's need to breathe nasally and has a large window for oral manipulations. To reach and infect the oropharynx efficiently, we have repurposed the Greer Pick allergy testing device as a virus delivery tool. We show that the Pick can be used to infect the epithelium of the soft palate and the base of the tongue of mice directly, without prior scarification. The ability to induce and track oropharyngeal papillomavirus-induced tumors in the mouse, easily and robustly, will facilitate the study of oropharyngeal tumorigenesis and potential treatments.

Keywords: MmuPV1; OPSCC; anesthesia; endoscopy; head-and-neck; mouse; oropharynx; papillomavirus; throat; tumor.

Figure 1

“Mickey’s Space Helmet” maintains isoflurane anesthesia during oral procedures. (A) The oral-access isoflurane nose cone was generated by three-dimensional (3D) printing using the bleach-resistant “Durable Resin.” The elbow adapter (white arrow) bears a small “tooth” that slides into a groove in the port at the top of the cone. This groove is at 90° to, and continuous with, a second circular groove that allows 360° rotation of the adapter relative to the cone. (B) The cone can be used with elastic bands (shown: silicone o-rings) to keep the mouth open via the incisors during infection with the Greer Pick. The tongue can also be restrained using the mandibular band, as shown. (C) The cone is also compatible with endoscopy. (D) The Greer Pick.

Figure 2

Infection of the soft palate with mouse papillomavirus (MmuPV1) elicits tumors. (A) A sagittal cross-section of the maxilla of a nude mouse collected 14 weeks after infection with 10⁸ genome equivalents of MmuPV1. Soft and hard palate tumors are indicated with arrows. (B) The maxilla of an NOD scid gamma (NSG) mouse collected 21 weeks after infection with 8 × 10⁹ genome equivalents of MmuPV1. The soft palate tumor is indicated with an arrow.

Figure 3

Soft palate tumors express viral capsid protein. (A) The soft palate tumor in Figure 2A, stained with hematoxylin and eosin (H&E). (B,C) The tumor in (A) after (B) immunohistochemical (IHC) detection of L1 capsid protein (green: “L1”) and Keratin 14 (magenta; “K14”) and (C) in situ hybridization with probes for MmuPV1 E6 and E7. (D) The tumor in Figure 2B stained with H&E. (E,F) The tumor in (D) after (E) L1 and K14 IHC and (F) in situ hybridization with probes for MmuPV1 E6 and E7.

Figure 4

Infection of the base of the tongue with MmuPV1 elicits dysplastic lesions that express viral protein. Lesions developed at the targeted infection site approximately 2/3 of the distance from the rostral tip to the caudal end of the tongue, approximately 1–2 mm caudal of the median eminence. (A) Panel 1: The tongue of a nude mouse collected 21 weeks after infection with 3 × 10⁹ genome equivalents of MmuPV1. Lines demark the dysplastic epithelium shown in Panel 2, stained with H&E; Panel 3, after L1 and K14 IHC; and Panel 4, in situ hybridization with probes for MmuPV1 E6 and E7. (B) Panel 1: The tongue of an NSG mouse collected 21 weeks after infection with 8 × 10⁹ genome equivalents of MmuPV1. Lines demark the dysplastic epithelium shown in Panel 2, stained with H&E; Panel 3, after L1 and K14 IHC; and Panel 4, after in situ hybridization with probes for MmuPV1 E6 and E7.

Figure 5

Longitudinal endoscopy reveals tumor growth. (A) The soft palate of an uninfected nude mouse injured with a Greer Pick and observed by endoscopy, in conjunction with Mickey’s Space Helmet, 10 weeks after injury. (B,C) The soft palate of a nude mouse observed by endoscopy 14 weeks (B) and 19 weeks (C) after infection with 3 × 10⁹ genome equivalents of MmuPV1.