Patient-derived organoids as a platform to study cervical cancer and viral infection

Cervical cancer is a common gynecological malignancy, often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study cervical epithelium and the cancers derived thereof. Here, we describe a long-term culturing protocol for ecto- and endocervical epithelia, which generates 3D organoids that stably recapitulate the two tissues of origin. In the example of successful HSV-1 infection, we show that the organoid-based cervical models serve as promising platforms for studying sexually transmitted pathogens. Starting from Pap-brush material, a small biobank of patient-derived tumoroids was established that retained the causative HPV genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutics and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches. Purpose: Transcriptome analysis was performed on organoids/tissues derived from human ecto- and endocervix as well as associated malignancies to characterize the new organoid-based platform. Methods: Ectocervical organoids and tumoroid lines were cultured in M7 media, whereas endocervical organoids in M7+4 media prior harvesting for RNA extraction (see paper for details). Additionally, RNA was extracted from healthy ecto-and endocervical tissue biopsies as well as from primary cells collected via Pap brush method from cervical cancer patients. The libraries were prepared by the Utrecht Sequencing Facility (USEQ) based on polyA enrichment. Sequencing was performed on an Illumina NextSeq500 by using 75-bp single-end or paired-end sequencing. Reads were aligned to the human reference genome (GRCh37) using Burrows-Wheeler Aligner (BWA) (v0.5.9). Viral sequences of different HPV subtypes were detected in the RNA-seq data of the different tumoroid lines and tissues by using VirusSeq-CLI, a wrapper around the VirusSeq pipeline. Results: Ectocervical and endocervical organoids display distinct transcriptomic profiles that can be maintained in cultures for long term. Similarily, the the gene expression profile of tumor organoids clusters apart from healthy counterparts and shows evident tumor-like characteristics. Tumor organoids show expression of causative HPV oncogenes. mRNA profiles of primary tissues and organoids were generated by Deep Sequencing (Illumina NextSeq500 by using 75-bp single- or paired-end sequencing). Transcriptome profiles were generated from 3 biologically independent organoid lines derived from healthy ectocervix and endocervix of patients undergoing hysterectomy due to other than cervix related conditions (organoids in both early and late passages were sequenced). In total of 9/12 established tumoroid lines were sequenced (3 of the lines in both early and late passages). Additionally, 3 tissue samples were sequenced from normal ecto- and endocervix as well as from 3 independent cervical squamous cell carcinomas.