Identification of target cells of human papillomavirus 18 using squamocolumnar junction organoids

Abstract

Human papillomavirus 18 (HPV18) is a highly malignant HPV genotype among high‐risk HPVs, characterized by the difficulty of detecting it in precancerous lesions and its high prevalence in adenocarcinomas. The cellular targets and molecular mechanisms underlying its infection remain unclear. In this study, we aimed to identify the cells targeted by HPV18 and elucidate the molecular mechanisms underlying HPV18 replication. Initially, we established a lentiviral vector (HPV18LCR‐GFP vector) containing the HPV18 long control region promoter located upstream of EGFP. Subsequently, HPV18LCR‐GFP vectors were transduced into patient‐derived squamocolumnar junction organoids, and the presence of GFP‐positive cells was evaluated. Single‐cell RNA sequencing of GFP‐positive and GFP‐negative cells was conducted. Differentially expressed gene analysis revealed that 169 and 484 genes were significantly upregulated in GFP‐positive and GFP‐negative cells, respectively. Pathway analysis showed that pathways associated with cell cycle and viral carcinogenesis were upregulated in GFP‐positive cells, whereas keratinization and mitophagy/autophagy‐related pathways were upregulated in GFP‐negative cells. siRNA‐mediated luciferase reporter assay and HPV18 genome replication assay validated that, among the upregulated genes, ADNP, FHL2, and NPM3 were significantly associated with the activation of the HPV18 early promoter and maintenance of the HPV18 genome. Among them, NPM3 showed substantially higher expression in HPV‐related cervical adenocarcinomas than in squamous cell carcinomas, and NPM3 knockdown of HPV18‐infected cells downregulated stem cell‐related genes. Our new experimental model allows us to identify novel genes involved in HPV18 early promoter activities. These molecules might serve as therapeutic targets in HPV18‐infected cervical lesions.

Research progress on the application of organoids in gynecological tumors

“…At present, the main method for modeling CC organoids is to isolate tumor cells from CC tissue. In addition, some studies have also constructed organoids using the matrix gel method by using normal squamous columnar junction tissue from patients undergoing total hysterectomy ( Toyohara et al, 2023 ). In addition, studies have shown that culture media play an important role in constructing organoids of different tissue types ( Huang et al, 2023 ).…”

“…Toyohara et al constructed CC organoids using the matrix gel method, transfected the lentiviral vector (HPV18LCR-GFP vector) into squamous columnar junction (SCJ) organoids from patients, and evaluated the presence of green fluorescence protein (GFP)-positive cells ( Toyohara et al, 2023 ). The results showed that pathways related to cell cycle and viral carcinogenesis were upregulated in GFP-positive cells, while keratinization and mitochondrial autophagy/autophagy-related pathways were upregulated in GFP-negative cells.…”

“…Among the upregulated genes, ADNP , FHL2 , and NPM3 were significantly associated with the activation of the early promoter of HPV18 and the maintenance of the HPV18 gene group. Thus, the initial replication mechanism of HPV18 and the breakthrough in the origin of HPV18-related CC cells were determined ( Toyohara et al, 2023 ).…”

“…Toyohara et al constructed organoids by obtaining normal tissue from the squamous columnar junction area of patients undergoing a total hysterectomy. The specific experimental conditions reported in each study of organoids vary, as detailed in the above sections ( Toyohara et al, 2023 ).…”

Impact of human papillomavirus types on uterine cervical neoplasia

“…Several recent studies have focused on HPV18 target cells, carcinogenesis, and histological differentiation, suggesting that HPV18 may be characterized by latent infection and carcinogenesis in stem cell-like cells. 50,51 HPV generally changes the pattern of HPV gene expression in the differentiated cervical stratified squamous epithelium. E6/E7 is usually expressed in the basal layer, E1^E4, which is involved in interaction with the keratin network, is expressed in the differentiated layer, and L1, which is involved in capsid formation, is expressed in the surface epithelium.…”