Insights into tumor microenvironment and HPV integrations in cervical cancer pathogenesis revealed by single-cell transcriptome data

Abstract Background HPV infection is prevalent among women, and can lead to severe diseases. This study leverages single-cell RNA-sequencing (scRNA-seq), a cutting-edge technology that allows for cellular-level exploration, to investigate the relationship between cellular heterogeneity and HPV integrations in cervical histopathology.Methods We applied scRNA-seq to examine heterogeneity across normal and three disease stages patients with normal, high-grade squamous intraepithelial lesions (HSIL), microinvasive carcinoma (MIC), and cervical squamous epithelium carcinoma cancer (CSCC) tissues. A pipeline was developed to detect the HPV integration events from scRNA-seq data in this study.Results Our findings revealed an increase in the proportion of squamous epithelial cells and a decrease in the proportion of columnar epithelial cells in the disease progress from normal to CSCC. We identified HPV genes expressed differentially across normal and three disease stages. Notably, we observed HPV integration events occurring more frequently in squamous epithelial cells at single-cell level. An increasing trend in the ratio of HPV-integrated cells was observed in the disease progress from normal tissue to CSCC, which eventually plateaued. We identified several genes, including EGR1, S100A11, S100A8, KRT5, RPL34, ATP1B1, RPS4X and EEF2 that were frequently integrated by HPV across patients. In contrast, genes such as PAN3, BABAM2, SPEN, TCIM-SIRLNT, TEX41-PABPC1P2 and KCNV1-LINC01608 showed frequent integration events across cells. Some of these genes exhibited significant expression differences between tumor and normal squamous epithelial cells. KRT5, ATP1B1, RPS4X, PAN3 and SPEN were novel recurrent HPV-integrated genes we observed at the patient or cell level in this study. Furthermore, we found that HPV genes from various HPV types demonstrated integration preferences in different samples and disease stages.Conclusions These findings uncovered the intricate heterogeneity across normal and three disease stages caused by varying degrees of HPV infection, offering a valuable single-cell perspective on the mechanism of HPV-induced cervical cancer and its clinical relevance.