Studying the eff​ect of FREM2 protein on glioblastoma cell migration, invasion and cytoskeletal reorganization using nanobodies

Krapež, Gloria (avtor), Jovčevska, Ivana (mentor)

Glioblastoma (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.

• Jezik: Angleški jezik
• Ključne besede: glioblastoma, nanobodies, FREM2, gene silencing, functional assay
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2026
• PID: 20.500.12556/RUL-184054
• Datum objave v RUL: 25.06.2026

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Preučevanje vpliva proteina FREM2 na migracijo, invazijo in preoblikovanje citoskeleta glioblastomskih celic z uporabo nanoteles

Izvleček

Glioblastom (GBM) je najpogostejši in najbolj agresiven primarni maligni možganski tumor pri odraslih, za katerega so značilni hitro napredovanje, znotrajtumorska raznolikost in odpornost na zdravljenje. Kljub zdravljenju, ki vključuje kirurško resekcijo, obsevanje in kemoterapijo s temozolomidom (TMZ), ostaja preživetje bolnikov slabo. Vedno več dokazov kaže, da epitelno mezenhimski prehod (EMT) in reorganizacija citoskeleta prispevata k invaziji, migraciji in odpornosti GBM na terapijo. Kompleks FRAS/FREM, zlasti njegova komponenta FREM2, je vpleten v ohranjanje epitelijske integritete in razvoj, vendar njegova funkcionalna vloga pri GBM ostaja neraziskana. Namen doktorske naloge je pojasniti vlogo FREM2 pri migraciji, invaziji, preoblikovanju citoskeleta GBM celic in drugih procesih, pri čemer sta bila uporabljena 2 pristopa: utišanje gena FREM2 s siRNA in tretiranje celic s FREM2 specifičnim nanotelesom (NB3F18). Uspešno smo optimizirali utišanje FREM2 v več GBM celičnih linijah in človeških astrocitih ter potrdili znižano izražanje tako RNA kot proteina. Funkcionalni testi, vključno z metodami celjenja ran, invazijo skozi membrano (transwell), sferoidni invazivni rob in sledenjem posameznim celicam, niso pokazali zmanjšanja migracije ali invazije po utišanju FREM2 ali tretiranju z NB3F18, kar je ovrglo našo začetno hipotezo. Kljub temu pa je analiza izražanja genov s sekvenciranjem RNA razkrila transkripcijske spremembe in identificirala podskupino 46 diferenčno izraženih genov v vseh GBM celičnih linijah po utišanju FREM2. Analize obogatitve poti so povezale utišanje FREM2 z signalnimi potmi kot so apoptoza, metabolizem, transport veziklov in transport ionov. Apoptotski testi so potrdili povečano apoptozo v celičnih linijah NCH644 in U251MG po sočasnem utišanju FREM2 in kratkotrajni izpostavitvi TMZ. Dolgotrajno tretiranje s TMZ ni privedlo do pričakovane povečanega izražanja FREM2, kar bi potrdilo vpletenost FREM2 v procesu pridobljene odpornosti na TMZ. In vivo eksperimenti na Drosophila melanogaster so pokazali, da manipulacija homologa FREM2, imenovanega Perdido, vpliva na rast gliomskih celic. Nadizražanje Perdida je povečalo, medtem ko sta utišanje Perdida ali tretiranje z NB3F18 zmanjšala število tumorskih celic, kar podpira povezavo med FREM2 in napredovanjem glioma. Rezultati so razširili znanje o vlogi FREM2 v GBM. Čeprav neposrednih učinkov na migracijo in invazijo nismo dokazali, smo ugotovili povezavo med FREM2 in apoptozo ter spremembo v izražanju transkripcijskih genov, ki so pomembni za napredovanje GBM. Uporaba NB3F18 v D. melanogaster je potrdila uporabnost nanotelesa kot vsestransko raziskovalno orodje ter nakazala njihov potencial za akademsko in klinično uporabo.

• Ključne besede: glioblastom, nanotelesa, FREM2, utišanje genov, funkcionalni testi