Isoform-level transcriptome analysis for enriched cancer stem cells from patient-derived ovarian cancer cell lines

Background: In high-grade serous ovarian cancer (HGSOC), the abilities of tumor cells to resist cytotoxic treatment and relapse are largely driven by ovarian cancer stem cells (CSCs). CSCs are a population of cells within a tumor that maintain stem-like qualities, such as self-renewal and asymmetric division, but also have the capacity to produce metastasis and drive drug resistance​. Despite extensive research over the past two decades, no specific CSC target has been identified in ovarian cancer, and no drug has proven effective in neutralizing the CSC population. Therefore, identifying the molecular mechanisms underlying CSC development in HGSOC is urgently needed, as eradicating CSCs represents one of the most formidable challenges in oncology, yet also offers one of the most transformative opportunities to alter the course of cancer therapies. Based on the emerging evidence of linking differential isoform usage to the regulatory mechanisms of developmental process, we hypothesize that CSCs exploit isoform diversity to sustain stemness, drive therapy resistance, and shape prognosis while gene-level programs shape general differentiation status. Methods: We employed CSC-enriched sphere culture system to emphasize the features of CSC population from 3 patient derived high grade serous ovarian carcinomas (PD-HGSOC) with different metastatic abilities and tumor aggressiveness. For these cell lines, we generated RNA-seq data for genome-wide transcriptome profiling to investigate stemness and differentiation status comparing standard culture condition and CSC-enriched spheres. In this study, we analyzed RNA-seq data in gene-level as well as in isoform-level with the most up-to-date transcript annotation to identify patient-specific CSC features and define the mechanism of cancer related stemness and plasticity. Results: We analyzed gene-level transcriptome profiles focusing on stemness signatures in PD-HGSOC and CSC-enriched spheres. We observed that CSCs exhibited markedly higher stemness characteristics and identified differentially expressed gene sets across PD-HGSOCs. We noted that each cell line had a distinct subset of genes regulating stemness which suggested multiple mechanisms of CSC development and maintenance. In result of isoform-level analysis, we found that a large proportion of gene-level expression changes were driven by the differential expression of a single predominant isoform. Importantly, our isoform-level analysis enabled the identification of differential isoform usage that would not be detected at the gene level expression analysis, as changes in isoform composition can occur while total gene expression remain unchanged. These results are instrumental for foundational understanding of CSC plasticity and for next generation drug design, increasing target specificity and reducing adverse effects.