Developing a tumor microenvironment in rotating human melanoma cell cultures : study of a novel preclinical model

The 3D culture is currently the most promising technique used in preclinical studies for drug testing. One of the main challenges in preclinical in vitro studies is the long-term observations (over 21 days), which can be performed using cell spheroids maintained in bioreactor conditions─tumorspheres. Here, our goal was to develop and characterize two types of human melanoma cell lines: primary (FM55p) and malignant (WM266-4) cultures, in rotating bioreactors as an alternative to in vitro modeling. We proposed potential end points of evaluation: viability tests (PI–propidium iodide imaging of necrotic core, PI/annexin V flow cytometry, trypan blue staining) and modulators of the epithelial-mesenchymal transition pathway (spectral flow cytometry of vimentin, MCAM, and CD44 expression). In addition, with confocal microscopy, we visualized regulators of angiogenesis (VEGF-β) and single-cell spectral flow cytometry (E- and VE-cadherin). The tumorspheres showed the ability to grow for at least 1 month to reach millimeter sizes. Their morphology was improved compared to traditional 3D spheroids, and we observed changes in the tumor microenvironment and the expression of key proteins. Our study demonstrated the utility of tumorspheres in personalized medicine and drug development The advantage of the described models is the creation of prospects for further development of maintaining cell models that are hybrid systems combining the features of spheroids and organoids for preclinical and translational research.