Non-small cell lung cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85 % of all cases. It is characterized by high aggressiveness, difficult treatment, and late detection, which often results in a poor prognosis for patients. Despite the development of numerous new therapeutic options, choosing the optimal treatment remains a challenge, as it is a biologically very heterogeneous disease. Three-dimensional (3D) tumor models, spheroids, are often used for NSCLC studies, as they better mimic the conditions in the tumor microenvironment in patients than adherent cell lines. The aim of the thesis was to optimize the preparation of viable spheroids of repeatable sizes by centrifugation and to characterize cell cultures by analyzing the expression of cell markers. We established spheroids from different numbers of cells from cell cultures LC004 and LC005, obtained from tumor biopsies of a patient with NSCLC. After optimizing the protocol, we established spheroids from 10,000 cells and continued their cultivation in a ClinoStar bioreactor. We monitored the growth of spheroids using light microscopy and analysed diameter and surface area during cultivation. We presented the results graphically and found that the growth of the spheroids was negligible, with a noticeable reduction in spheroid size and significant cell migration into the medium at the end of the cultivation. We characterized the cells of both cell cultures and the MRC-5 cell line by flow cytometry to analyze the percentage of cells positive for the tumor markers cytokeratin 5, cytokeratin 7, p63, napsin A, the epithelial marker EpCAM, and the fibroblast marker αSMA. The results of flow cytometry showed that both LC004 and LC005 cells were not positive for tumor and epithelial markers but were strongly positive for fibroblast marker αSMA. Based on these results, we concluded that the cultures did not contain cancer cells, but rather cancer-associated fibroblasts (CAFs), which was also consistent with the results of spheroid formation. The reduction in spheroid size and cell migration into the medium reflects the less pronounced proliferative nature and weaker intercellular contacts of CAFs. Although we did not detect cancer cells in the culture, the results obtained are important because they highlight the problem of CAF overgrowth in cell cultures and the importance of their exclusion from primary NSCLC cell cultures.
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