Patient-derived method for rapidly constructing cervical organoids with native immune and mutation profiles.

e17531 Background: Persistent infection with high-risk human papillomavirus(HR-HPV), particularly HPV16, is a major risk factor for the development of cervical intraepithelial neoplasia (CIN), which can progress through various stages and eventually lead to cervical cancer. Traditional in vitro models often fail to capture the complex immune interactions that occur within tissue. To address this limitation and better understand HPV-associated cervical lesion progression, we have developed patient-derived organoids that represent five distinct stages, from early CIN to cervical cancer. Methods: These organoids were derived from various stages of HPV infection: HPV-negative & Normal tissue, HPV16-positive & Normal tissue, HPV16-positive & LSIL, HPV16-positive & HSIL, and HPV16-positive & CA. Cervical tissue specimens were soaked in a washing solution for 3 minutes, followed by three washes in a 50 mL tube with 5 mL of solution each time. Cleaned tissue blocks were cut into 1 mm³ microtissue blocks and placed in a 10 cm culture dish with 100–200 μL of culture medium. The blocks were then resuspended in 1 mL of medium and transferred to a new dish with 15 mL of medium. The medium was partially refreshed every 5–7 days to maintain organoid growth. Organoid growth was monitored, and transcriptome sequencing was performed to analyze gene expression profiles. Whole-exome sequencing (WES) and single-cell RNA sequencing were used to assess genetic variations and immune cell compositions. HE staining was employed to evaluate histological characteristics. Results: The primary culture of cervical organoids resulted in the formation of three-dimensional spherical structures with organoid-like characteristics. HE staining of the organoids showed structural similarities to the source tissue. Whole-exome sequencing (WES) of organoids derived from HPV16-negative normal tissue, HPV16-positive&HSIL, and HPV16-positive&CA showed high mutation overlap rates, indicating that the organoids maintain a high degree of similarity to the tissue in terms of mutation profiles. This indicates that the organoid culture system preserves most of the tissue-originated mutations. Single-cell RNA sequencing analysis of organoids from HPV16-positive normal tissue and HPV16-positive cancerous tissue showed that the organoids effectively retain the immune microenvironment of the source tissue. Conclusions: This study demonstrates the successful construction of cervical organoids representing five distinct stages of cervical lesions, each with a preserved native immune microenvironment. These organoids provide a more physiologically relevant model for investigating HPV infection and immune responses at different stages of disease progression.