Studying the eff​ect of FREM2 protein on glioblastoma cell migration, invasion and cytoskeletal reorganization using nanobodiesKrapež, Gloria (Avtor) Jovčevska, Ivana (Mentor) glioblastomananobodiesFREM2gene silencingfunctional assayGlioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by fast progression, high intratumor heterogeneity, and resistance to therapy. Despite treatment, which includs surgical resection, radiotherapy, and chemotherapy with temozolomide, patient survival remains poor. GBM progression is supported by epithelial mesenchymal transition (EMT) and cytoskeletal reorganization leading to GBM invasion, migration, and therapy resistance. The FRAS/FREM extracellular matrix complex, particularly its FREM2 component, has been implicated in epithelial integrity and development, yet its functional role in GBM remains unexplored. Throughtout this doctoral thesis, we examined the role of FREM2 in GBM cell migration, invasion, cytoskeletal reorganization, and more, using both small interfering RNA (siRNA) silencing approach and a FREM2 specific nanobody (NB3F18). We successfully optimized siRNA silencing of FREM2 in multiple GBM and human astrocyte cell lines, confirming downregulation at RNA and protein levels. Functional assays, including wound healing, transwell invasion, spheroid formation, and single cell tracking, showed no consistent reduction in migration or invasion following FREM2 silencing or NB3F18 treatment, disproving our initial hypothesis. We continued the search with gene expression profiling using RNA sequencing (RNA-seq). With gene expression profiling we identified 46 differentially expressed genes in GBM cell lines with silenced FREM2 compared to controls. Pathway enrichment analyses linked FREM2 silencing to apoptosis, metabolism, vesicle trafficking and ion transport pathways. Apoptotic assays confirmed increased apoptosis in cell lines NCH644 and U251MG after combined FREM2 silencing and short-term TMZ exposure. Prolonged TMZ treatment did not show expected FREM2 upregulation, which could imply the role of FREM2 in TMZ resistancy. In vivo experiments in Drosophila melanogaster demonstrated that manipulation of the FREM2 homolog Perdido affected glioma cell growth: overexpression of Perdido enhanced, while silencing of Perdido or NB3F18 treatment reduced tumor cell numbers, showing a link between FREM2 and glioma progression. These results provide an insight into the role of FREM2 in GBM. While we did not show direct effects on migration and invasion, FREM2 appears to influence apoptosis and transcriptional gene expression changes relevant to GBM progression. The use of NB3F18 in D. melanogaster validated nanobodies as versatile research tools and suggested their potential for academic and clinical application.20262026-06-25 07:15:18Doktorsko delo/naloga184054sl