Book of abstract 2008

Glioma: combating the invasive cell phenotype
Laura Donovan, Suzanne Birks, Geoffrey Pilkington
Cellular & Molecular Neuro-oncology Group, Institute of Biomedical & Biomolecular Sciences, School
of Pharmacy & Biomedical Sciences, University of Portsmouth, White Swan Road, Portsmouth PO1 2DT
UK
A small subset of cells displaying stem-like properties, maintaining the ability to self-renew
and sustain growth of the tumour has been identified in glioma. The trans-membrane
protein, CD133, has been identified in such cells and shown to promote multi drug
resistance (MDR). It has also been hypothesised that CD133 facilitates brain tumour
cell invasion and that such tumour stem cells may also serve to disseminate the tumour
within the brain. In addition, ganglioside GD3 plays a pivotal role in the regulation of
tumour growth and invasion by facilitating tumour cell adhesion. Although in nonneoplastic
cells build up of GD3 induces mitochondrially-mediated apoptosis this does
not occur in tumour cells due to the acetylation of the terminal sialic acid residue on GD3
to form 9-0-acetyl GD3 (GD3A). We are using glioma biopsy-derived tissue and early
passage cultures to determine a) the migratory properties of CD133+ tumour stem cells
and b) the influence of deacetylation of GD3A on glioma cell apoptosis. Baculoviruses
are a family of insect specific viruses which have recently been shown to effectively drive
the expression of a reporter gene in both dividing and non-dividing mammalian neural
cells whilst remaining safe and non-pathogenic. We will initially determine whether the
baculovirus vector can be modified to target GD3 in human gliomas and give transient and
stable gene delivery. CD133+ and - cells have been segregated, by autoMACS magnetic
cell separation, from low passage biopsy-derived cells and transformed into neurospheres
via the hanging drop method. All cells used were been characterised by flow cytometry
and immunocytochemistry, using appropriate immuno markers. We have extracted
GD3A from bovine buttermilk by solvent extraction and partitioning and ion-exchange
chromatography. The
p4
purity of this is then tested and the GD3/GD3A used in motility
studies. CD133 expression was seen on tumour cells, as well as a 1.43% positive expression
of CD133 via flow cytometry. Cell migration and invasion in CD133+ and CD133-
populations is being studied by Transwell Boyden Chambers and live cell imaging. An
“all human” 3D-invasion model is also being used. The cultured CD133 (+) and CD133
(-) neuropsheres, from brain tumour biopsies, juxtaposed, in vitro, to a spheroid produced
from brain resected from epileptic patients. Invasion is observed via live-cell imaging and
confocal microscopy. GD3 and GD3A has been prepared from buttermilk and used to
determine influence on glioma cell viability and apoptosis. Influence of CD133+ cancer
stem cells within glioma may determine not only therapeutic response but promote
local spread of the neoplasm within the brain. In addition, it has been shown that by
enzymatically cleaving the acetyl group from GD3A restores the pro-apoptotic potential
to that of GD3. We intend to use the Baculovirus transfected with the cDNA of the
acetylesterase enzyme to target GD3A in glioma cells in vitro in attempt to remove the
acetyl group and then assess apoptosis by Western blot analysis and the employment of the
TUNEL and Annexin V assays.
Funded through generous grants from Ali’s Dream and Charlie’s Challenge brain tumour charities.
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