Unraveling ovarian cancer stem cell dependencies on miRNA and epigenetic regulation utilizing enhanced 3D culture models

High-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy, largely due to therapy-resistant cancer stem cells (CSCs) that drive recurrence and poor clinical outcomes. This thesis aimed to establish reliable CSC models, uncover novel regulators of stemness, and investigate their mechanistic roles to inform therapeutic strategies. Two 3D culture methods—ultra-low attachment (ULA) spheroids and gelatin methacrylate photomask (GelMA-Pm) encapsulation—were optimized with OVCAR-3 and OVSAHO cell lines and validated in patient-derived samples. OVCAR-3 cells cultured in ULA formed compartmentalized spheroids with enriched CSC and epithelial-to-mesenchymal transition (EMT) traits and multidrug resistance, making this the most robust system. Small RNA sequencing of spheroids identified hsa-miR-548ah-3p as a consistently downregulated and underexplored miRNA. Functional rescue with miRNA mimics reversed CSC characteristics, reduced drug resistance, and impaired migration and invasion. RNA-seq profiling under prolonged mimic treatment highlighted "rescue genes," including VIM, CXCR4, and KIT, integrating with miRNA target predictions. Parallel analyses revealed global 3′UTR lengthening in CSC spheroids, a dormancy-like program that remained responsive to miR-548ah-3p restoration. Complementing these findings, a CRISPR-Cas9 dropout screen with the EPI-KOL epigenetic library identified essential regulators of spheroid survival. Top dependencies converged on DNA damage response (ATM, BRCA1), polycomb and chromatin remodeling (SUZ12, EPC2, EP400, PBRM1), and RNA metabolism (SNRPF, SYNCRIP, PARG). In summary, this thesis integrates 3D culture modeling, miRNA functional studies, APA profiling, and CRISPR screening to reveal that HGSOC CSCs rely on a DNA damage–chromatin–APA axis. Restoration of miR-548ah-3p effectively counteracts CSC traits and drug resistance, nominating it—together with epigenetic regulators—as promising therapeutic entry points. These findings establish optimized 3D culture systems for CSC modeling and uncover mechanistic vulnerabilities that may guide future strategies to overcome resistance in HGSOC.