Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Background: Human papillomavirus (HPV) is an ancient group of small DNA viruses implicated in various human cancers, including oropharyngeal squamous cell carcinoma (OPSCC). Patients with HPV-associated OPSCC generally experience better outcomes than those with non-HPV-associated OPSCC, though the mechanisms driving this divergence remain unclear. DNA viruses like HPV activate the innate immune response through pattern recognition receptors and antiviral sensing pathways, including the cGAS–STING pathway. How these pathways are regulated within the virus-associated OPSCC tumor microenvironment (TME) and their impact on tumorigenesis and clinical presentation are poorly understood. HPV-associated OPSCC provides a unique model to study these antiviral pathways ex vivo. Here, we examine the gene expression of regulatory proteins in the cGAS–STING pathway in HPV-associated OPSCC tonsil tumors compared to contralateral, uninvolved tonsil tissue within the same patient. Additionally, we aim to identify key cellular drivers of the cGAS–STING pathway in the tonsil TME using a single-cell RNA sequencing (sc-RNA seq) pipeline.
Methods: Fresh-frozen tonsil tissue from HPV-associated OPSCC (ipsilateral) and uninvolved tonsils (contralateral) was obtained from a cohort of 17 patients at a single institution. For each patient undergoing transoral robotic surgery (TORS) for HPV-associated OPSCC, both the tumor tonsil and contralateral uninvolved tonsil were removed and studied. RNA was extracted from bulk tissue, and quantitative PCR (qPCR) was performed to assess the relative mRNA expression of select viral and host genes. Fresh tonsil specimens from a smaller cohort of 7 patients were dissociated into single-cell suspensions for subsequent sc-RNA seq and analyzed for gene expression. qPCR data were evaluated for relative fold changes, and sc-RNA seq datasets were processed using Seurat in R to identify unique cell clusters based on established marker genes and quantify the expression of cGAS–STING pathway genes in these cell populations.
Results: Bulk RNA gene expression analysis in our cohort of 17 patients revealed a significantly higher expression (500-3000 fold) of HPV viral genes (E5/E6/E7) in tumor tonsils compared to contralateral controls. This increased viral expression correlated with the upregulation of the viral sensor cGAS and the downstream type I interferon response in tumor specimens. sc-RNA seq analysis in the 7-patient cohort suggests that cGAS upregulation is primarily driven by transformed tumor cells, while the subsequent antiviral type I interferon response is mediated by both tumor cells and myeloid-origin cells, such as macrophages and dendritic cells, in the TME.
Conclusions: HPV-associated OPSCC demonstrates significant upregulation of the cGAS–STING antiviral pathway compared to normal tonsil tissue. This upregulation, along with the subsequent interferon response, appears to be driven by transformed tumor cells and myeloid-origin host cells. This antiviral response and associated inflammatory phenotype may contribute to the improved outcomes observed in HPV-positive OPSCC patients. Further study of these immune regulatory mechanisms could reveal therapeutic targets for both HPV-associated and non-HPV-associated OPSCC patients.