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p32105<br />
Proteomics of astrocytic neoplasms - the search for<br />
tumor-vascular/matrix interactions<br />
Dorota Goplen 1 , Uros Raj~evi} 2 , Simone Niclou 2 , Dominique Revets 3 ,<br />
Fred Fack 3 , Rolf Bjerkvig 1 and Jorge Terzis 4<br />
1<br />
NorLux - Neuro Oncology, University of Bergen, Bergen, Norway; 2 NorLux - Neuro<br />
Oncology, CRP-Santé, Luxembourg; 3 Institute of Immunology, LNS, Luxembourg; 4 NorLux<br />
- Neuro Oncology, CHL, CRP-Santé, Luxembourg<br />
The most malicious tumor of the central nervous system (CNS) is glioblastoma<br />
multiforme (GBM). Tumor cell infiltration together with cellular atypia, necrosis and<br />
recruitment of new blood vessels characterize the GBMs. By serial transplantation<br />
of human GBM biopsies into the CNS of immunodefficient nude rats, two different<br />
tumor phenotypes were obtained. The transplanted xenografts initially displayed a<br />
highly invasive phenotype showing no signs of angiogenesis. By serial transplantation<br />
in animals, the phenotype changed to a less invasive, predominantly angiogenic<br />
phenotype.<br />
Extracts from the tumors were sub-fractioned to enrich for hydrophobic<br />
and membrane proteins. Samples were differentially labeled using CyDye<br />
chemistry and co-separated by two dimensional-differential gel electrophoresis<br />
(2D-DIGE). Differentially expressed spots of interest were robotically processed and<br />
the extracted peptide pools were used for protein identification by matrix assisted<br />
laser disorption ionization time-of flight mass spectrometry (MALDI T<strong>OF</strong> MS) and<br />
MALDI T<strong>OF</strong> T<strong>OF</strong> mass spectrometry. Data were analyzed using various bioinformatics<br />
tools. The functional significance of one of the differentially expressed proteins was<br />
assessed using cell migration and invasion assays where the protein was inhibited<br />
by specific inhibitors and a monoclonal antibody.<br />
We focussed on differentially expressed proteins that are potentially involved in<br />
tumor cell invasion and angiogenesis. For two proteins identified in the invasive<br />
phenotype, increased expression was confirmed by immuno-histochemistry and<br />
Western blot. Applied protein inhibitors strongly reduced the invasiveness of cancer<br />
cells, an effect that was reversible after withdrawal of the inhibitor, indicating a<br />
specific non-toxic effect. Additional candidate proteins are validated by antibody<br />
techniques and other proteomic methodologies including quantitative MS as well<br />
as functional approaches. In conclusion, the xenotransplantation model developed<br />
clearly separates the invasive phenotype from the angiogenic phenotype and a<br />
number of potential tumor markers of the two phenotypes can be delineated by the<br />
presented proteomics approach. Proteins were identified to play an important role<br />
in glioma cell invasion, and their action was effectively inhibited by appropriate<br />
inhibitors.