Extracellular Membrane Vesicles Derived from 143B Osteosarcoma Cells Contain Pro-Osteoclastogenic Cargo: A Novel Communication Mechanism in Osteosarcoma Bone Microenvironment

Abstract

The bone microenvironment (BME) is the main hub of all skeletal related pathological events in osteosarcoma leading to tumor induced bone destruction, and decreasing overall bone quality and bone strength. The role of extra-cellular membrane vesicles (EMVs) as mediators of intercellular communication in modulating osteosarcoma-BME is unknown, and needs to be investigated. It is our hypothesis that osteosarcoma-EMVs contain pro-osteoclastogenic cargo which increases osteoclastic activity, and dysregulated bone remodeling in the osteosarcoma-BME. In this study, EMVs were isolated from the conditioned media of 143B and HOS human osteosarcoma cell cultures using differential ultracentrifugation. Nano-particle tracking analysis determined EMVs in the size range of 50-200 nm in diameter. The EMV yield from 143B cells was relatively higher compared to HOS cells. Transmission electron microscopy confirmed the ultrastructure of 143B-EMVs and detected multivesicular bodies. Biochemical characterization of 143B-EMVs detected the expression of bioactive pro-osteoclastic cargo including matrix metalloproteinases-1 and -13 (MMP-1, -13), transforming growth factor-β (TGF-β), CD-9, and receptor activator of nuclear factor kappa-β ligand (RANKL). Detection of a protein signature that is uniquely pro-osteoclastic in 143B-EMVs is a novel finding, and is significant as EMVs represent an interesting mechanism for potentially mediating bone destruction in the osteosarcoma-BME. This study further demonstrates that 143B cells actively mobilize calcium in the presence of ionomycin, and forskolin, and induce cytoskeleton rearrangements leading to vesicular biogenesis. In conclusion, this study demonstrates that 143B osteosarcoma cells generate EMVs mainly by mechanisms involving increased intracellular calcium or cAMP levels, and contain pro-osteoclastic cargo.

Epigenetic alterations in mesenchymal stem cells by osteosarcoma-derived extracellular vesicles

“…For example, in an in vivo mouse study, OS-EVs carrying functional TGFβ (transforming growth factor β) were internalized by MSCs and altered the MSCs phenotype towards a pro-tumorigenic and pro-metastatic phenotype by activating oncogenic IL&-STAT3 signalling pathways, thereby promoting tumour growth and metastasis [21]. In recent years, the composition of OS-EVs cargo has been investigated, indicating the presence of proteins related to tumour progression and metastasis [43,44], and the presence of repetitive DNA, including satellites and transposable elements [45]. However, the impact of OS-EVs on the epigenome of MSCs is still unclear and the consequences of these interactions and their impact on tumour behaviour need to be addressed.…”

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

“…We and others have shown that NTTPPH overexpression results in osteomalacia in long bones, and is localized in EMVs [66,67]. We have previously reported the role of calcium influx and cAMP signaling in EMV biogenesis in OS cells [68]. Whether FGF1 stimulates EMV biogenesis in OS via calcium or cAMP/PKA signaling in OS cells is unknown.…”

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones

“…One study has reported the isolation and characterization of EVs from Ewing's sarcoma cells [18], and another study with 143B osteosarcoma cells has revealed that they secrete EVs capable of stimulating osteoclastogenesis in the surrounding bone microenvironment [19]. However, it is unknown whether OS-derived EVs can participate in intra-tumoral communication, specifically, in inter-clonal cooperation which could modify the metastatic behaviour of OS.…”