Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms. To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode.
Osteosarcoma (OS) is an aggressive bone malignancy with a high relapse rate despite combined treatment with surgery and multiagent chemotherapy. As for other cancers, OS-associated microenvironment may contribute to tumor initiation, growth, and metastasis. We consider mesenchymal stromal cells (MSC) as a relevant cellular component of OS microenvironment, and have previously found that the interaction between MSC and tumor cells is bidirectional: tumor cells can modulate their peripheral environment that in turn becomes more favourable to tumor growth through metabolic reprogramming (1). Stem-like cells were derived from HOS osteosarcoma cell line by using the spherogenic system (2). CSC isolated from HOS (HOS-CSC) were co-coltured with MSC isolated from bone marrow. Cell lysates and supernatants were collected for the analysis of RNA expression and of secreted cytokines, by Q-RT-PCR and specific ELISA assays, respectively. Here, we determined the effects of MSC on OS stemness and migration, two major features associated with recurrence and chemoresistance. Recruitment of MSC to the tumor environment leads to enhanced proliferation of OS stem cells, which increase the expression levels of TGFβ1. The latter, in turn, could be responsible for the activation of NF-kB genes and IL-6 secretion by MSC. Pro-tumorigenic effects of MSC, via IL-6, including induction of HOS-CSC migration and sphere growth, can be counteracted by IL-6 neutralizing antibody. The presence of MSC is also responsible for increased expression of adhesion molecules involved in intra- or extra-vasation. Stromal cells in combination with OS spheres exploit a vicious cycle where the presence of CSC stimulates mesenchymal cytokine secretion, which in turn increases stemness, proliferation, migration, and metastatic potential of CSC. Furthermore, for the first time we identified a novel OS stem cell marker, the Met proto-oncogene, that is frequently overexpressed and is pathogenetically relevant in OS (2 and 3). Altogether, our data corroborates the concept that a comprehensive knowledge of the interplay between tumor and stroma that also includes the stem-like fraction of tumor cells is needed to develop novel and effective anti-cancer therapies.
