(P 65) PROFILING EXTRACELLULAR VESICLES AND NANOPARTICLES IN OSTEOSARCOMA

Objective: Osteosarcoma is a malignant bone tumor primarily arising in osteoblasts, the cells responsible for bone formation. This type of cancer predominantly affects adolescents, although it also occurs in older adults. Standard treatment for osteosarcoma typically involves a combination of surgery and chemotherapy. Despite the intense chemotherapy regimens, recurrent and metastatic patients' outcomes have remained poor. Extracellular vesicles (EVs) have become highly significant in cancer research due to their essential involvement in intercellular communication and their potential as biomarkers for diagnosis and therapy. Recent discoveries of unique nanoparticles called exomeres and supermeres have broadened the repertoire of particles produced by mammalian cells and involved in long-range cell-to-cell communication. Initial work in carcinoma models has shown that these particles can significantly increase chemoresistance of tumors by the way of their oncoproteins’ content.

Methods: High-grade KHOS and U2OS osteosarcoma cell lines and human fetal osteoblasts (hFOB1.19) were cultured in serum-free conditioned media for 48h. Subsequently, EVs and nanoparticles are isolated from osteosarcoma and osteoblast cell lines using a multi-step ultracentrifugation method based on size and density. Nanoparticle tracking analysis (NTA) is employed to analyze the particles’ sizes; however, its ability to detect nanoparticles smaller than 30nm, such as exomeres and supermeres is limited. Transmission electron microscopy (TEM) is used to validate the particles' sizes, providing visualization and characterization of EVs and nanoparticles based on their diameters performed on gradient-purified samples, including small EVs, exomeres, and supermeres. To obtain the proteomic profile of each type of particles, we used an untargeted mass spectrometry approach.

Results: Osteosarcoma cell lines and normal osteoblasts all produce EVs of the expected sizes. Transmission electron microscopy reveals the presence of large and small EVs in their respective fractions. Importantly, TEM images of exomeres and supermeres fractions reveal for the first time the presence of particles produced by osteoblasts and osteosarcoma cells. This is the first time that non-transformed mammalian cells are shown to produce exomeres and supermeres. Previous work had focused on carcinoma-derived cell lines notorious for high-secretory activity.

Conclusion: This study presents the isolation and characterization of unique extracellular nanoparticles termed exomeres and supermeres. These nanoparticles display evident differences in terms of their size, morphology, and composition. Osteoblasts and osteosarcoma cells' secretory activity contribute majorly to the disease pathogenesis, influencing the microenvironment to promote bone destruction. Our work shows that alongside EVs and soluble cytokines, exomeres and supermeres hold the potential to function as agents of intercellular communication and can significantly impact the tumor microenvironment.