title: Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.
abstract: BACKGROUND
  Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells.
  
  
  RESULTS
  Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines.
  
  
  CONCLUSIONS
  The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.
authors.affiliation: Department of Clinical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA. milan.milovancev@oregonstate.edu
authors.forename: Milan
authors.initials: M
authors.lastname: Milovancev
authors.forename: Ian
authors.initials: I
authors.lastname: Hilgart-Martiszus
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McNamara
authors.forename: Cheri P
authors.initials: CP
authors.lastname: Goodall
authors.forename: Bernard
authors.initials: B
authors.lastname: Seguin
authors.forename: Shay
authors.initials: S
authors.lastname: Bracha
authors.forename: Samanthi I
authors.initials: SI
authors.lastname: Wickramasekara
mesh_terms: D000818:Animals; D019921:Biotinylation / Q000662:veterinary; D015153:Blotting, Western / Q000662:veterinary; D001859:Bone Neoplasms / Q000378:metabolism / Q000662:veterinary*; D045744:Cell Line, Tumor; D004283:Dog Diseases / Q000378:metabolism*; D004285:Dogs; D015972:Gene Expression Regulation, Neoplastic; D013058:Mass Spectrometry / Q000662:veterinary; D008565:Membrane Proteins / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D012516:Osteosarcoma / Q000378:metabolism / Q000662:veterinary*; D020543:Proteome / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary
chemical_list: D008565:Membrane Proteins; D020543:Proteome