# In Vitro Model Characterizing Carcinogenic Progression of HPV-Positive Oropharyngeal Cancer

## TL;DR

This paper introduces a lab model to study how HPV16 causes oropharyngeal cancer, aiming to improve early detection and treatment.

## Contribution

A novel in vitro model using 3D organotypic raft cultures to mimic HPV16-driven oropharyngeal cancer progression.

## Key findings

- The model successfully mimics structural and biochemical changes in HPV16-transfected oral epithelium from precancerous to malignant states.
- The system allows monitoring of viral genome status and oncogenic/metabolic markers linked to HPV16-driven OPSCC.
- Validation with clinical datasets confirms the model's utility for biomarker discovery and early detection strategies.

## Abstract

High-risk human papillomavirus (hrHPV), particularly HPV16, is a major driver of oropharyngeal squamous cell carcinoma, now the most common HPV-related cancer in the U.S., surpassing cervical cancer. Despite available vaccines, low uptake and shifting sexual behaviors have contributed to rising incidence, with projections exceeding 30,000 cases annually by 2029. Early detection remains difficult due to the absence of precursor lesions and long latency between infection and symptom onset. To address these limitations, we developed an in vitro HPV16 oral cancer model using the 3D organotypic raft culture system. This model mimics the progression of HPV16-transfected oral epithelium from precancerous to malignant states and allows for detailed monitoring of structural and biochemical changes. Validated using established markers of high-grade lesions, the model allows us to study the early progression of HPV16-driven disease and supports the search for reliable biomarkers to improve diagnosis and guide treatment.

Background/Objective: Human papillomavirus (HPV) represents the most widespread sexually transmitted infection globally, with high-risk strains such as HPV16 driving a rising incidence of oropharyngeal squamous cell carcinoma (OPSCC), particularly in developed countries like the United States and United Kingdom. In the U.S., HPV16-associated OPSCC has surpassed cervical cancer as the most common HPV-related malignancy. Despite the availability of preventive vaccines, uptake remains suboptimal among adolescents and shifting sexual behaviors have contributed to increased disease burden. Early detection remains a major clinical challenge due to the absence of defined precursor lesions and the extended latency between viral exposure and disease onset. Most patients present with advanced-stage disease and no prior clinical history of pre-malignancy, limiting access to early-stage samples and hindering biomarker discovery. Methods: To address these limitations, we developed an in vitro HPV16 oral cancer model, using the three-dimensional organotypic raft culture system that simulates the progression of HPV16-transfected oral epithelium from precancerous states to malignant phenotypes. Results: Using HPV16-transfected human tonsil keratinocytes, we generated stratified and differentiated epithelia that mimic the biochemical and structural changes observed in vivo. This system enables detailed monitoring of epithelial differentiation, biochemical shifts, viral genome status, and key oncogenic and metabolic markers associated with HPV16-driven OPSCC. By aligning expression profiles with clinical datasets, we validated the model through the measurement of virologic markers linked to infection and progression, as well as tissue markers indicative of carcinogenic transformation. Conclusions: This model offers a promising tool for refining early detection strategies and evaluating potential clinical biomarkers, ultimately aiming to improve diagnostic precision and therapeutic outcomes in HPV-associated OPSCC.