4) The in vivo studies using the BOOM model were done with the a

4). The in vivo studies using the BOOM model were done with the assistance of the Proof of Concept Laboratory at The University of Kansas Cancer Center, with the approval of the University of Kansas Institutional Animal Care and Use Committee. For histopathologic evaluation, tibias were decalcified in 10% EDTA (pH 7.5) for 2 weeks before sectioning and paraffin embedding. The sections were processed for hematoxylin and eosin staining and immunohistochemistry (IHC). E7080 molecular weight To detect osteoblastic-mediated mineralization

in the tumor tissue, von Kossa staining was done using non-decalcified tumor tissue sections. To detect the immunoexpression of MMPs in the tumor tissue of the BOOM model, MMP-1 and MMP-13 IHC was done using primary antibodies (MMP-1, RB-1536; MMP-13, MS-825) purchased from Lab Vision Thermo Scientific (Kalamazoo, MI), followed by detection. The detection

reagents were purchased from Biocare Medical (Concord, CA) and Dako (Carpinteria, CA). For negative control, primary antibody was excluded, and human placenta tissue sections were used as positive control in MMP IHC. Human osteosarcoma cell lines 143B (highly aggressive and metastatic; k-ras activated) and HOS (nonaggressive and nonmetastatic; k-ras wild type) were purchased from American Type Culture Collection (Manassas, VA). The 143B cells were genetically engineered to express luciferase gene (FUW-Luc-mCherry-puro), and cultured in Dulbecco’s modified Eagle’s medium according

AZD2281 in vivo to the previously described method [2]. The 143B-luc-mCherry cell line was authenticated for its ability to grow in the presence of puromycin in vitro and to proliferate in the tibia of Nu/Nu mice and metastasize to the lungs, as described in the BOOM model [2]. At subconfluence, conditioned media (CM) were prepared by culturing 143B or HOS cells in serum-free media for 24 hours and subjected to differential ultracentrifugation for isolation of EMVs. We used differential ultracentrifugation (low speed followed by ultracentrifugation at 110,000g for 2 hours) to isolate EMVs from the CM prepared from osteosarcoma Unoprostone cells according to the scheme shown in Figure 1. To determine the EMV concentration and size distribution profile of EMVs isolated from CM of osteosarcoma cell cultures, vesicles were analyzed using the NanoSight (Amesbury, UK) NTA 2.3: Nanoparticle Tracking and Analysis instrument and software (release version build 11 RC1, 2012, hardware: LM14). The samples were injected in the sample chamber according to the manufacturer’s recommendations. EMVs were analyzed in phosphate-buffered saline solution under Brownian motion at 22°C to 24°C with laser wavelength at 638 nm. Multiple video frames were captured for 60 seconds per reading. Screen gain remained at 1.0, and detection threshold ranged from 13 to 14. The number of readings for EMVs, at dilutions 1:5000, 1:2000, 1:1000, and 1:100, ranged from 5 to 20 measurements.

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