Sviluppo e caratterizzazione di organoidi di carcinoma mammario derivati da pazienti come piattaforma farmacologica innovativa

The necessity of developing a more representative in vitro model for the study of basic biological processes, as well as for the validation of new therapies, led to advances in 3D in vitro models, like organoid technology. Patient-derived organoids (PDOs) are 3D structures composed of cells derived from human tissue. Briefly, the tissue is digested, and the cells are cultured in a semi-solid matrix that prevents their attachment to the bottom of the well and allows the formation of cell-cell interactions and their organization into 3D structures. These structures are composed of different cell types, similar to the tissue of origin, and therefore can display its cellular heterogeneity. In particular, this model can be derived from cancer tissue, being an in vitro model that is more complex and representative compared to classical 2D cell lines. This advancement is fundamental in supporting in vivo experimentation, aiming to reduce the gap between in vitro and in vivo models. For this purpose, organoids can be applied in pharmacological research as a tool for novel drug screening and therapy validation in oncological patients. Therefore, the aim of this work is to set up and validate a protocol for the generation and propagation of breast cancer organoids from patient-derived tissue specimens. This novel platform should resemble the characteristics of breast cancer tissue and conserve the features of the tissue of origin in terms of histological type and gene expression pattern. Once the complex culture medium, the Advanced DMEM Organoid Medium, containing a variety of components such as growth factors and molecules involved in cell signalling, was set up, a protocol for establishing organoid cultures was developed. Canine-derived breast cancer tissue was used in the preliminary phases of this process as an alternative, ethical and easy to obtain translational model. Two canine-derived tissues were processed with the developed protocol, and both were able to correctly form organoids when in culture. Interestingly, a cell population was found to grow adhered to the bottom of the culture wells in both samples. Flow cytometry analysis was performed to verify the correspondence between the generated organoid cultures and breast tissue. Both CDO 11 IZS and CDO 12 IZS showed the presence of markers characteristic of breast tissue, albeit with different patterns, representing the complexity and heterogeneity found between different cancer tissues. Subsequently, the protocol was applied to human-derived tissues. Due to the different characteristics of the processed tissues in terms of cellular content, only two of them were able to generate a proper organoid culture. The two organoid cultures were also analysed with flow cytometry, proving that they were representative of breast cancer tissue. Only PDO 07 CNR showed the production of a population of cells that adhered to the bottom of the culture wells: it was analysed as well, showing an overall correspondence between these cells and the organoid culture of origin. In PDO 07 CNR, a gene expression analysis was performed: genes characteristic of the histological type of the tumor of origin, ERBB2, and EGFR, were highly expressed. This preliminary result further confirms the similarity between the organoid culture and the tissue from which it derives. Thanks to the developed protocol, we were able to establish breast cancer organoid cultures that are representative of the tissue of origin.