title: 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.
authors.affiliation: Division of Hematology and Oncology, The University of Kansas Medical Center, Kansas City, KS, USA ; Department of Internal Medicine, The University of Kansas Medical Center, Kansas City, KS, USA ; Department of Orthopedic Surgery, The University of Kansas Medical Center, Kansas City, KS, USA ; Department of Dietetics and Nutrition, The University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: Rama
authors.initials: R
authors.lastname: Garimella
authors.affiliation: Division of Hematology and Oncology, The University of Kansas Medical Center, Kansas City, KS, USA ; Department of Internal Medicine, The University of Kansas Medical Center, Kansas City, KS, USA ; The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: Laurie
authors.initials: L
authors.lastname: Washington
authors.affiliation: Muscle Biology Research Group, School of Nursing and Health Studies, University of Missouri-Kansas City, Kansas City, KS, USA.
authors.forename: Janalee
authors.initials: J
authors.lastname: Isaacson
authors.affiliation: Muscle Biology Research Group, School of Nursing and Health Studies, University of Missouri-Kansas City, Kansas City, KS, USA.
authors.forename: Julian
authors.initials: J
authors.lastname: Vallejo
authors.affiliation: Muscle Biology Research Group, School of Nursing and Health Studies, University of Missouri-Kansas City, Kansas City, KS, USA.
authors.forename: Madoka
authors.initials: M
authors.lastname: Spence
authors.affiliation: Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: Ossama
authors.initials: O
authors.lastname: Tawfik
authors.affiliation: Department of Internal Medicine, The University of Kansas Medical Center, Kansas City, KS, USA ; Kidney Institute, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Rowe
authors.affiliation: Muscle Biology Research Group, School of Nursing and Health Studies, University of Missouri-Kansas City, Kansas City, KS, USA ; School of Medicine, University of Missouri-Kansas City, Kansas City, KS, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Brotto
authors.affiliation: Department of Internal Medicine, The University of Kansas Medical Center, Kansas City, KS, USA ; The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: Raymond
authors.initials: R
authors.lastname: Perez