Patient-Derived Organotypic Epithelial Rafts Model Phenotypes in Juvenile-Onset Recurrent Respiratory Papillomatosis

Abstract

Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is driven by human papillomavirus (HPV) low-risk strains and is associated with significant morbidity. While previous studies of 2D cultures have shed light on disease pathogenesis and demonstrated the utility of personalized medicine approaches, monolayer cultures lack the 3D tissue architecture and physiology of stratified, sequentially differentiated mucosal epithelium important in RRP disease pathogenesis. Herein we describe the establishment of JoRRP-derived primary cell populations that retain HPV genomes and viral gene expression in culture. These were directly compared to cells from matched adjacent non-diseased tissue, given the known RRP patient-to-patient variability. JoRRP papilloma versus control cells displayed decreased growth at subconfluency, with a switch to increased growth after reaching confluency, suggesting relative resistance to cell-cell contact and/or differentiation. The same papilloma cells grown as 3D organotypic rafts harbored hyperproliferation as compared to controls, with increased numbers of proliferating basal cells and inappropriately replicating suprabasal cells, mimicking phenotypes in the patient biopsies from which they were derived. These complementary model systems provide novel opportunities to elucidate disease mechanisms at distinct stages in JoRRP progression and to identify diagnostic, prognostic and therapeutic factors to personalize patient management and treatment.

Expanded Basal Compartment and Disrupted Barrier in Vocal Fold Epithelium Infected with Mouse Papillomavirus MmuPV1

“…Proliferation index increased to a median of 3.4% in diseased murine laryngeal tissues, and Ki67-expressing cells were found above the basal layer of the epithelium. A similar expansion of proliferating cells has been observed in RRP lesions [ 23 ] and in MmuPV1-induced lesions of the oral cavity and the anogenital tract [ 74 , 75 , 76 , 77 ]. Like the larynx, we found that proliferation and hyperplasia increased in trachea with MmuPV1-induced disease.…”

“…Healthy vocal fold epithelium is characterized by proliferating cells in the basal and parabasal layers and differentiated suprabasal cells [ 20 , 21 , 22 ]. Human RRP lesions are characterized by an expanded basal compartment, proliferating cells throughout epithelial layers, and decreased differentiation [ 23 , 24 , 25 , 26 , 27 ]. High-risk HPVs are known to suppress apoptosis in infected cells [ 28 ], and apoptosis may play a role in vocal fold epithelial diseases [ 29 ].…”

A Novel In Vivo Model of Laryngeal Papillomavirus-Associated Disease Using Mus musculus Papillomavirus

“…Models of low-risk HPV-associated diseases, including RRP, are limited [ 41 ]. Current preclinical models of RRP are limited to in vitro organotypic raft culture and xenografts, which are typically used at early passages to study short-term viral and host gene expression [ 42 , 43 , 44 , 45 ]. In the sole reported experimental infection of an animal larynx with an animal papillomavirus, canine oral papillomavirus (COPV) was injected into the supraglottic laryngeal tissues of canines [ 46 ].…”

HPV Strain Predicts Severity of Juvenile-Onset Recurrent Respiratory Papillomatosis with Implications for Disease Screening

“…Biopsies were obtained and used to establish primary mucosal squamous epithelial cell cultures as previously described [ 36 ]. Importantly, matched biopsy sets of papilloma and non-diseased tissue were obtained per patient.…”

“…2D cell pellets were collected from early passage (passage 2–4) primary cells derived from papilloma biopsies, extracted for RNA, and run by RT-qPCR using HPV-strain specific primers to determine HPV6 versus HPV11 genotype as previously described [ 36 ].…”