(0728) Single Cell Signature of Pathological HPV-Driven Proliferation

Introduction: High-risk human papillomavirus (HPV) infection of stratified squamous epithelium causes 5% of all cancers worldwide. Viral pathogenesis includes the deregulation of the cell cycle which drives epithelial dysplasia and hyperplasia through disorganized basal cell hyperproliferation and aberrant replication in the suprabasal epithelium. Therapeutic inhibition of HPV-induced proliferation without toxicity to normal epithelium has been a challenge, as specific features of HPV-induced proliferation have not been sufficiently defined.

Methods: We recently published single cell RNA sequencing (scRNAseq) data obtained from an established 3D in vitro organotypic raft model of persistent HPV16 replication. This model harbors pathological features associated with HPV infection, including excessive basal and aberrant suprabasal proliferation. Cells from matched, isogenic HPV16+ vs HPV16– NIKS organotypic epithelial rafts were jointly analyzed to allow direct comparisons by HPV status.

Results: Pseudotemporal trajectory analysis aligned cells along the epithelial differentiation program, with cycling G2/M phase cells split between two branches. The first branch originated from basal-like cells and the second branch originated from differentiated cells. The corresponding cells were categorized as basal and suprabasal proliferative cells, respectively. scRNAseq analysis defined unique transcriptome signatures for these subpopulations in HPV+ compared to HPV- epithelium. Current transcriptomic analyses aim to define biomarkers for spatial localization in clinical specimens, and candidate drivers which may be therapeutically targeted to eliminate abnormally, but not normally, proliferating epithelial cells in HPV infected epithelium.

Conclusions: scRNAseq enabled isolation of the transcriptome of epithelial subpopulations representing clinically relevant HPV-driven cellular proliferation. These studies enable detailed analyses of HPV-specific transcriptomic signatures for diagnostic markers and therapeutic targets which inhibit pathological proliferative phenotypes.