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

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.

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.

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.

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.