02 BOOK OF ABSTRACTS .indd

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