"Introduction. Worldwide cancer remains the second-most common cause of death; among bone cancers, Osteosarcoma (OS) is the most common type diagnosed especially in children and young adults1. The lack of specificity for the Cancer Stem Cells (CSCs) subpopulation has been recently identified as the main limitation in conventional therapies2. Moreover, the traditional two-dimensional (2D) in vitro models, employed in the drug testing as well as in biological studies, are affected by a poor in vitro-in vivo translation ability3. This work provides “tumour engineering” osteosarcoma models as new tools to address the disease and improve therapy outcomes.
Methodology. Two different hydroxyapatite-based scaffolds4,5 were used to recapitulate in vivo bone extracellular matrix (ECM); a ceramic scaffold from a direct foaming process and a hybrid biomineralized scaffold were used.
MG63 and SAOS-2 osteosarcoma cell lines were used as parental cells and spheroids. As well-established CSC-enrichment model, the sphere-forming culture6 was used to obtain spheroids under serum free and ultralow-attachment conditions.
The 2D in vitro preliminary evaluation of spheroids and parental cells to confirm CSCs enrichment was performed by gene expression of stemness markers. The phenotype of spheroids under 3D scaffold-based models were investigated by histological, fluorescent and electron microscopy analysis compared to those with parental cells. Stemness (OCT-4, NANOG and SOX-2) and CSC niche-related genes (NOTCH-1, HIF-1α and IL-6) and proteins expression was analysed on spheroids under 3D scaffold-based conditions compared to 2D ones.
Actually, the variability of tumoral properties in serial spheroids passaging7,8 is still being investigated; spheroids serial generations of osteosarcoma cells have been characterized by proliferation, sphere-forming efficiency, migration and invasion ability, and gene and protein expression profile under 2D conditions.
Results. The enrichment of a cell population with stemness properties was confirmed by 2D analysis of gene expression. The morphological evaluation of in vitro 3D scaffold-based models highlights the maintenance of spheroidal phenotype by spheroids. The gene and protein expression confirmed the presence of stemness and CSC-niche markers by 3D scaffold-based spheroids compared to 2D conditions, underling the importance of three-dimensional microenvironments for in vitro studies.
The variability of serial passaging spheroids of osteosarcoma cells was partly reported, giving a starting point to select the right spheroids generation to furtherly improve our tumour models. Moreover, tumour matrix complexity, co-cultures and specific secretome will be included as part of tumour microenvironment.
Conclusions. These 3D in vitro tumour models could improve the predictivity of preclinical studies and enhance the clinical translation, with the ultimate goal to be applied in personalized medicine.
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