Elucidating the Function of Exosomes released by Osteosarcoma: Insights into Osteoclast Communication

Introduction: Currently, several studies are being conducted to understand the role of exosomes released by tumor cells, elucidating their involvement in cellular signaling that coordinates important cellular processes during tumor progression. It is believed that osteosarcoma cells control their progression by regulating the activity of osteoblasts and osteoclasts through the release of exosomes. Therefore, exosomes hold significant potential as an alternative strategy for tumor diagnosis and treatment. Aim: The aim of this study is to delineate the role of exosomes released by osteosarcoma cells in tumor progression, by understanding their communication with osteoclast cells. Methods: Exosomes released by osteosarcoma and normal osteoblast cells were isolated by ultracentrifugation at 30.000 RPM for 1 hour. The size, particle number and morphology of the exosomes were assessed by nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM). Subsequent to exosomes characterization, their role in osteoclast activity were investigated. The evaluation of exosome entrance into cells was conducted by culturing osteoclast cells with exosomes labeled with a lipophilic membrane dye (PKH67, SIGMA) for 24 hours and visualizing under microscopy. The exosomes' impact on osteoclast differentiation was determined using the tartrate-resistant acid phosphatase (TRAP) assay whereby exosomes were introduced into macrophage cells medium, during three days of osteoclast cells differentiation. Results and conclusion: The exosomes  isolated exhibited the expected size and morphology as described in the literature, being around 100 nm. Following the confirmation of their ability to enter the cells, a variability in osteoclast cells were observed in the presence of exosomes, with bigger osteoclast cells compared to control (no exosomes introduction). The size of osteoclast cells may impact their resorption activity. Collectively, these experiments contribute to a better understanding of the effects of osteosarcoma-released exosomes on osteoclastogenesis.