Modeling and Exploring Recurrent Respiratory Papillomatosis with 3D Cell Cultures

Recurrent respiratory papillomatosis (RRP) is a benign but potentially life-threatening tumor of the airway epithelium that is caused by persistent infection with low-risk human papillomavirus (LR-HPV) type 6 or 11. Current management relies on repeated surgical removal of lesions, as no curative antiviral therapy is available, imposing substantial physical and psychological burden on patients. To develop novel therapeutic strategies, it is essential to elucidate the mechanisms by which LR-HPVs persist, replicate, and drive tumor formation. However, the lack of in vitro models that recapitulate the viral life cycle has hindered its progress. In this study, we established primary epithelial cell cultures from surgical RRP specimens using conditional reprogramming (CR) with J2 feeder fibroblasts and a ROCK inhibitor, achieving a high success rate (～90%). These cells retained LR-HPV genomes and were adaptable to three-dimensional (3D) culture, forming spheroid structures that mimicked in vivo tissue architecture and preserved proliferative and undifferentiated phenotypes. Immunostaining and molecular analyses revealed suppressed cGAS-STING pathway activity and reduced type I interferon response in RRP-derived cells, suggesting impaired innate immune surveillance. This 3D CR-based model provides a robust platform for investigating the pathogenesis of LR-HPV and host–virus interactions, potentially accelerating the development of targeted therapies for RRP.