New mechanisms involved in the pathogenesis of osteosarcoma

Osteosarcoma is the most common and most often fatal primary bone tumour, especially affecting children and adolescents. It is a highly aggressive tumor that develops mainly in the long bones and metastasizes primarily to the lung. Osteosarcoma cells are known to establish a crosstalk with resident bone cells leading to a deleterious vicious cycle. In this context, we hypothesized that osteosarcoma cells can release in the bone microenvironment transforming extracellular vesicles (EVs) involved in regulating the bone cell proliferation and differentiation, thereby promoting the tumour growth. EVs are small, intact and heterogeneous membrane vesicles that exchange nucleic acids and proteins between cells, inducing phenotypic, biochemical and genetic changes in target cells. Aim of this thesis is to investigate the role of extracellular vesicles as mediators between cancer cells and resident bone cells. We used human osteosarcoma cell lines to set protocols aimed at isolating, visualizing and characterizing EVs. Our results showed that osteosarcoma cell lines produce EVs that are able to induce tumour phenotype in recipient murine fibroblasts NIH3T3, as already described for other cancer cell-derived EVs [1,2]. Indeed, in so treated NIH3T3, we observed an enhanced survival capability under low-serum conditions, high levels of activated survival pathways, an increased migration, the acquired capability to grow in an anchorage-independent manner and a de novo expression of osteoblastic and tumorigenic markers. As regarding bone cells, we used as target cells human osteoblasts, their precursors, mesenchymal stromal cells, and human monocytes as osteoclast precursors. The treatment with osteosarcoma-derived EVs induced an increase of MSC differentiation into mature ALP+ and Alizarin red+ cells. EVs derived from osteosarcoma cells promote the migration and the anchorage-independent growth in normal osteoblasts suggesting a transformation toward a tumour-like phenotype. Finally in monocytes, treated with osteosarcoma EVs, we observed an increased proliferation and the formation of TRAP+ multinucleated osteoclasts, because of presence in EVs of cytokines involved in osteoclast activation and differentiation. These findings highlight the key role of EVs in the crosstalk between the osteosarcoma and bone microenvironment cells, suggesting that further investigation are needed to better define this new field of research. [1] Antonyak MA, et al. Proc Natl Acad Sci U S A. (2011)4852-7 [2] Li B, et al. Oncogene. (2012) 4740-9