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.

Figure 1

Schematic shows the isolation of EMVs from CM of serum-starved human OS 143B and HOS cells.

Figure 2

Osteosarcoma-induced osteolytic and osteoblastic changes in the BME of the BOOM model. A and B are hematoxylin and eosin–stained tumor-bearing tibial sections showing the presence of rapidly expanding tumor within the marrow cavity. The cortical bone is abnormal, thickened, and has uneven or ruffled borders due to active osteoclastic bone resorption. The black arrows indicate resorption pits, and the green arrow indicates a capillary infiltrating into the cortical bone. The tumor-bearing bone also shows several osteocytes (OCY). C shows multinucleate osteoclasts actively remodeling the bone in the osteosarcoma BME. D shows mineralizing osteoblasts as detected by von Kossa staining. E and F show electron micrographs of an active osteoclast in the vicinity of a resorption pit filled with hydrolytic acidic vesicles (E) and extracellular matrix containing numerous EMVs interspersed among collagen fibrils (F).

Figure 3

Immunodetection and localization of MMP-1 and MMP-13 in the tibial sections of the BOOM model. Brown peroxidase staining indicates MMP expression in the tumor and nontumor cells (osteoblasts (OBL); osteoclasts (OCL); and osteocytes (OCY). Primary antibodies (MMP-1, RB-1536; MMP-13, MS-825) were used at 1:100 dilutions.

Figure 4

NTA and TEM of 143B EMVs. A shows NTA measurements of size and concentration of EMVs in three different samples. B shows a screenshot of video from NanoSight LM14 showing light scatter caused by 143 EMVs. C and D show electron micrographs of 143B EMV pellet. C shows the presence of an MVB containing intraluminal vesicles and several EMVs in the size range of 50 to 200 nm. D shows several mineralizing EMVs and the presence of polarized membrane-associated deposition of hydroxyapatitelike crystals.

Figure 5

Determination of ALP activity and detection of mCherry fluorescence in 143B EMVs. A shows bar graphs comparing ALP activity (U/ml) in143B EMVs versus 143B cell lysates (n ≥ 3). B shows detection of mCherry fluorescence in EMVs derived from 143B-luc-mCherry OS cells (× 20). C shows mCherry-positive 143B EMV forward scatter (FSC) and side scatter (SSC) by flow cytometry.

Figure 6

Detection of pro-osteoclastogenic cargo in 143B EMVs by Western blot analysis. Crude lysates of 143B cells (12.5-25 μg) and EMVs (25-40 μg) were analyzed for MMP-1, MMP-13 (A and B), TGF-β, RANKL, and CD-9 (C) expression by Western blot analysis. All samples were analyzed in triplicate.

Figure 7

Calcium mobilization and induction of EMV biogenesis in 143B OS cells in the presence of forskolin and ionomycin. A1 and A2, show morphologic changes within ionomycin-sensitized 143B cells leading to the accumulation of intracellular, vesicles, whereas B1 and B2, show intracellular vesiculation in forskolin-pretreated and ionomycin-sensitized 143B cells as observed by confocal microscopy. C1 and D1, show kinetic changes in the Fura-2 ratio in ionomycin (C1) and in forskolin-pretreated, ionomycin-sensitized (D1) 143B cells. C2 and D2, compare Fura 350/375 between resting versus ionomycin (alone)–treated 143B osteosarcoma cells (C2) and resting versus forskolin-pretreated and ionomycin-sensitized (forskolin + ionomycin combination) 143B osteosarcoma cells (D2).

Figure W1

Comparison of EMV output between 143B, an aggressive osteosarcoma cell line versus HOS, a nonaggressive nonmetastatic cell line. A shows the number of EMVs (× 10⁸/ml) as determined by NTA, and B compares the protein concentration (mg/ml) of EMVs derived from 143B (bEMVs) versus HOS (hEMVs) osteosarcoma cells.

Figure W2

NTA of HOS EMVs. A shows NTA measurements of size and concentration of EMVs in six different samples. B shows a screenshot of video from NanoSight LM14 showing light scatter caused by HOS EMVs.

Figure W3

Estimated values of cytosolic calcium concentrations in 143B osteosarcoma cells in the presence of forskolin and ionomycin, as determined by Grynkiewicz equation. The estimated values of cytosolic calcium concentrations for resting and ionomycin-sensitized 143B cells are 182 and 2030 nM, respectively, and for resting versus forskolin-pretreated and ionomycin-sensitized cells are 298 and 3410 nM, respectively.