Optimizacija priprave sferoidov tumorskih celic raka dojke in fibroblastov

Štefin, Blažka (avtor), Mitrović, Ana (mentor)

Rak, kljub napredku v diagnostiki in zdravljenju, ostaja eden največjih zdravstvenih problemov, zato so številne raziskave usmerjene v razumevanje molekulskih mehanizmov raka in iskanje novih terapevtskih možnosti za njegovo zdravljenje. Z napredovanjem razumevanja biologije tumorjev se je pogled na tumorje spremenil, saj jih ne obravnavamo več kot izolirane mase, temveč kot del kompleksnega tumorskega mikrookolja (TMO), ki je postalo ključno pri napredovanju tumorja in pomembno vpliva na nastanek, invazivnost in odpornost na terapijo. Tradicionalni 2D in živalski modeli ne posnemajo ustrezno kompleksnosti tumorjev, zato strmimo k razvoju naprednih 3D celičnih modelov, ki bolje posnemajo TMO in heterogenost človeških tumorjev. V magistrski nalogi smo zato pripravili 3D sferoide so-kultur tumorskih celic raka dojke MCF7 in MDA-MB-231 ter fibroblastov MRC-5. Za pripravo enotnih in ponovljivih sferoidov smo testirali dve različni metodi priprave sferoidov: metodo tekočinskega prekrivanja in metodo viseče kapljice. Ovrednotili smo primernost izbranih celičnih linij za tvorbo 3D sferoidov, potrebo po uporabi ogrodja za njihovo tvorbo, pogoje tvorbe večceličnih sferoidov za posamezen tip celic in njihovo ustreznost za vrednotenje učinka zaviralcev katepsinov. Pokazali smo, da se uporabljene celične linije razlikujejo v svoji sposobnosti tvorbe sferoidov in da lahko s pripravo sferoidov so-kultur izboljšamo tvorbo sferoidov. Na ta način smo pripravili sferoide tudi s celicami MDA-MB-231, ki same ne tvorijo sferoidov. Pri tem smo pokazali, da se pripravljeni sferoidi razlikujejo glede na svoje morfološke lastnosti in da začetno število celic vpliva na velikost pripravljenih sferoidov ter na viabilnost celic v sferoidu. Nadalje smo ugotovili, da lahko tvorbo sferoidov izboljšamo z dodatkom metilceluloze in z uporabo nizko adherentnih plošč pri metodi tekočinskega prekrivanja. V sferoidih so-kultur smo različne populacije celic ločili z uporabo fluorescentnih sond in potrdili, da so se celice MRC-5 združevale v jedru sferoida, tumorske celice pa so se razporedile okrog njih. Za fluorescentne sonde smo predhodno s pretočno citometrijo pokazali, da omogočajo ločevanje populacij celic v so-kulturah tudi po 72 urah. Na koncu smo tudi potrdili, da so pripravljeni sferoidi primeren model za vrednotenje zaviralcev katepsinov, saj je zaviralec katepsina B nitroksolin upočasnil rast sferoidov. V okviru magistrske naloge smo tako pripravili 3D celične modele, ki bolje posnemajo TMO in so primeren model, z uporabo katerega bi lahko izboljšali vrednotenje novih protitumornih terapij.

• Jezik: Slovenski jezik
• Ključne besede: Rak, 3D modeli, sferoidi, tumorsko mikrookolje, protitumorna terapija, cisteinski katepsini
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-168251
• Datum objave v RUL: 05.04.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Optimization of the preparation of spheroids from breast cancer cells and fibroblasts

Izvleček

Despite advances in diagnostics and treatment, cancer remains one of the biggest health problems. Therefore, numerous studies are focusing on understanding the molecular mechanisms of cancer and exploring new therapeutic options for its treatment. With a better understanding of tumour biology, the view of tumours has changed, as they are no longer considered as isolated masses, but as part of a complex tumour microenvironment (TME), which has been recognised as a crucial factor for tumour progression, significantly influencing tumour development, invasiveness, and resistance to therapy. Since conventional 2D and animal models cannot adequately replicate the complexity of tumours, research is shifting towards the development of advanced 3D cell models that can better replicate the TME and the heterogeneity of tumours in humans. For this master’s thesis, we prepared 3D spheroids from co-cultures of the breast cancer cell lines MCF7 and MDA-MB-231 and fibroblasts MRC-5. To prepare uniform and reproducible spheroids, we tested two different methods of spheroid formation: the liquid overlay method and the hanging drop method. We investigated the suitability of the selected cell lines for the formation of 3D spheroids, the need for scaffolds for their formation, the conditions required for the generation of multicellular spheroids for each cell type, and their suitability for evaluating the effect of cathepsin inhibitors. We have shown that the cell lines we used vary in their ability to form spheroids and that spheroid formation can be enhanced by forming spheroid co-cultures. In this way, we were able to prepare spheroids with MDA-MB-231 cells that do not form spheroids themselves. We were able to show that the prepared spheroids differ in their morphological properties and that the initial cell number influences the size of the spheroids as well as the viability of the cells within the spheroid. Furthermore, we found that spheroid formation can be improved by the addition of methylcellulose and the use of low-adherent plates by the liquid overlay method. In the co-culture spheroids, we separated the different cell populations using fluorescent probes and showed that MRC-5 cells accumulated in the spheroid core while tumour cells clustered around them. Using flow cytometry, we confirmed that the fluorescent probes enable the separation of cell populations in the co-cultures even after 72 hours. Finally, we confirmed that the prepared spheroids are a suitable model for the evaluation of cathepsin inhibitors, as the cathepsin B inhibitor nitroxoline decreases spheroid growth. In this master thesis, we have developed 3D cell models that better mimic the TME and serve as a suitable model for better evaluation of new anti-tumour therapies.

• Ključne besede: Cancer, 3D models, spheroid, tumour microenvironment, antitumor therapy, cysteine cathepsins