Spremljanje preoblikovanja citoskeleta glioblastomskih celic v prisotnosti nanoteles usmerjenih proti transmembranskemu proteinu FREM2

Kladnik, Špela (avtor), Jovčevska, Ivana (mentor)

Glioblastomi so gliomi IV. stopnje in so najpogostejša oblika primarnih malignih tumorjev centralnega živčnega sistema. Danes je to še vedno bolezen s slabo prognozo, saj živijo pacienti v povprečju le 15 mesecev po diagnozi. Standardno zdravljenje obsega kirurško odstranitev tumorja, ki ji sledi kemoterapija s temozolomidom (TMZ). V večini primerov pride do ponovnega pojava tumorja, zaradi česar pacienti običajno podležejo bolezni v manj kot enem letu. Zato je ta oblika raka še vedno ena izmed najbolj smrtonosnih. Zaradi agresivne in invazivne narave glioblastoma ter visoke smrtnosti je bolezen čedalje pogostejša izbira za iskanje novih tarč, ki bi omogočile zdravljenje in izboljšale možnost preživetja. V magistrskem delu smo raziskovali povezavo med proteinom FREM2 in preoblikovanjem celičnega citoskeleta ter vplivom na celično migracijo in invazijo. Transmembranski protein FREM2 (angl. FRAS1-related extracellular matrix protein 2) je prisoten v glioblastomskih celicah in ima pomembno vlogo pri gibljivosti celic. Za delo smo uporabljali komercialno dostopne glioblastomske celične linije U87MG in U251MG. Vpliv proteina FREM2 na preoblikovanje celičnega citoskeleta in na celično migracijo ter invazijo smo preučevali z dvema metodama: 1) utišanje gena FREM2 s siRNA, in 2) tretiranje z nanotelesi, usmerjenimi proti proteinu FREM2. Opazovali smo celično migracijo pri utišanih oziroma tretiranih in kontrolnih celičnih linijah, natančneje, če utišanje proteina FREM2 vpliva na hitrost celične migracije in invazije. Izvedli smo tudi tretiranje z nanotelesi NB3F18, specifičnimi za protein FREM2. Ob vezavi nanoteles se spremeni konformacija proteina in s tem tudi njegova funkcija. Z imunofluorescenčno mikroskopijo smo opazovali 3 različne komponente citoskeleta: vimentin, β-aktin in F-aktin. Želeli smo testirati, če tretiranje celic z nanotelesi enako vpliva na organizacijo citoskeleta kot utišanje gena FREM2 s siRNA. Pri vimentinu opazne razlike ni bilo, smo pa predvidevali, da je vpliv na aktinski citoskelet večji in bi razlike opazili tam. Ker smo imeli pri eksperimentih z β-aktinom in F-aktinom kar nekaj težav, preoblikovanja aktinskega citoskeleta nismo dokazali in bi bile potrebne še nadaljnje optimizacije in raziskave.

• Jezik: Slovenski jezik
• Ključne besede: glioblastom, FREM2, migracija, invazija, preoblikovanje citoskeleta, vimentin, aktin
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2024
• PID: 20.500.12556/RUL-163134
• Datum objave v RUL: 02.10.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Monitoring reorganization of glioblastoma cell cytoskeleton in the presence of nanobodies targeting the transmembrane protein FREM2

Izvleček

Glioblastoma is stage IV. glioma and is the most common form of a primary brain tumor of the central nervous system. Up to this day, it still classifies as one of the worst types of cancers due to its poor prognosis, as most patients live for only about 15 months after diagnosis. The standard and most effective treatment is surgical removal of the tumor, followed by chemotherapy with temozolomide (TMZ). In most cases, there is a recurrence of the tumor, which is the reason most patients succumb to the disease in less than a year. That is the main reason why this form of cancer is one of the deadliest. Due to the aggressive and invasive nature of glioblastoma and its high mortality, the disease is an increasingly common choice for finding new targets that would enable treatment and improve the chance of survival. In this thesis we were focusing on one of those targets, the transmembrane protein FREM2 (FRAS1-related extracellular matrix protein 2), which has an important role when it comes to glioblastoma cell mobility. For our experiments, the glioblastoma cell lines U87MG and U251MG were used. We were researching the effect that FREM2 gene silencing has on cell migration and invasion. We successfully silenced the FREM2 gene with the use of siRNA. The effect of FREM2 silencing on cell migration and invasion was also monitored. For experiments with immunofluorescence, we also treated cells with nanobodies NB3F18. When nanobodies bind to the FREM2 protein, they change its conformation and its function. We wanted to see if nanobody-treatment effects cytoskeleton remodeling. We were monitoring cytoskeletal proteins vimentin and actin, to observe changes in cytoskeleton reorganization in treated cells. Furthermore, we were comparing cytoskeleton of both cells treated with nanobodies and cells treated with siRNA to those of normal glioblastoma cells. No difference with vimentin was examined, but we assumed there would be some difference with actin cytoskeleton. Unfortunately, we had some problems when conducting experiments with β-actin and F-actin and were unable to confirm our theory. The experiments would need further optimisation and research.

• Ključne besede: glioblastoma, FREM2, migration, invasion, cytoskeleton reorganization, vimentin, actin