Background: The development of models to study chemotherapy resistance is essential for improving our understanding of disease. This need is particularly acute in mucinous ovarian carcinoma (MOC), a rare subtype of ovarian cancer with limited treatment options and poor prognosis at advanced stages. Our testing of standard-of-care therapies on patient-derived MOC organoids revealed variable responses, particularly to paclitaxel and irinotecan. Exploring these heterogeneous treatment responses is crucial for developing more effective therapeutic strategies. Here, we tested cellular barcoding to trace the competitive dynamics of MOC organoid clones upon chemotherapy-mediated selective pressure.
Methodology: We barcoded three MOC organoids with SPLINTR lentiviral library to ensure each cell received a unique identifier. Clonal composition was assessed by DNA sequencing at baseline. Next, we assessed clonal responses to paclitaxel and irinotecan. MAC-Seq (multi-analysis of cells) analyses were conducted on MOC organoids treated with different doses of either paclitaxel and irinotecan for 48h and Gene Set Enrichment Analysis (GSEA) was utilised to detect the enriched pathways.
Results: We generated MOC organoid models transduced with a barcode library. MAC-Seq was able to detect differentially expressed genes and the barcodes within each organoid line after treatment. We found that clonal diversity in each MOC organoid line tested was unique. After 48h chemotherapy exposure, a reduction in barcode number was observed in treated organoids compared to DMSO. The distribution of individual barcodes was altered, indicating changes in clonal dynamics. Moreover, variable clonal dynamics were observed between the different MOC lines examined. KEGG Pathway analysis revealed that differentially expressed genes were highly enriched for metabolic processes, JAK-STAT signalling pathway, RNA binding and DNA damage/repair. Ongoing work will longitudinally track resistant clones through prolonged sequential treatments using MAC-Seq.
Conclusion: Cellular barcoding is a powerful method to trace the clonal dynamics of MOC organoids in response to chemotherapy. MAC-Seq technology offers a scalable platform for high-throughput sequencing to detect transcriptomic as well as barcode changes during chemotherapy. By identifying gene expression states that predict clone survival, this research lays the groundwork for selecting optimal therapies targeting the most aggressive resistant clones within tumours.