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.
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