Book of abstract 2008

CD44 and CD155 expression on human glioma in vitro: a flow
cytometric, immunocytochemical and TIRF microscopy study of
invasion indicators
M. Navarros Rego, N. Karatas, G. Whitaker, P. B. Nunn, P. Warren, G. J. 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
The poliovirus receptor, CD155 (PVR) is expressed on neoplastic glia and has already
been used in therapeutic targeting of glioma. Recently CD155 has been proposed as
playing a key role in glioma motility and invasion. CD44 is a cell adhesion molecule,
originally described as the lymphocyte homing receptor, which is has two isoforms with
respective molecular weights of 80-90kDa and 150kDa. The lower molecular weight
isoform mediates attachment to hyaluronic acid (HA), which is present in relatively
high concentration within the brain. CD44 is over-expressed in CNS and mediates both
mediates glioma cell adhesion and invasion. Moreover, CD155 resides proximal to CD44
on the cell membrane of monocytes. The interactive role of the two molecules, CD44 and
CD155, therefore merits investigation in the context of brain tumour invasiveness. Our
aims were, therefore, to evaluate the expression levels of, and spacial relationship between,
CD44 and CD155 in cultured glioma at high and low passage.
High and low passage in vitro cultures of glioma were immunocytochemically stained using
CD44 and CD155 antibodies and imaged by epi-fluorescence. Quantitative analysis was
obtained by flow cytometry and the special relationship between the two epitopes on the
cell surface was elucidated by total internal reflected fluorescence (TIRF) microscopy.
Immunocytochemistry showed both CD44 and CD155 to be expressed at high and low
passage numbers of glioblastoma in vitro. Double staining revealed both antigens to be
expressed on the cell membrane at close but distinct sites. Flow cytometry revealed a
higher expression level of CD44 compared with CD155 on all cultures tested.
Having shown that these two molecules are co-expressed and are closely apposed on the
cell membranes of glioma cells and are both involved in migration and invasion will are
now aiming to carry out live cell imaging and Transwell Boyden chamber assays. In these
experiments the influence of monoclonal antibody blocking and siRNA knock-down for
both molecules, singularly and together will be used to establish any synergy or additive
effect of CD44 and CD155 on glioma invasion.
Acknowledgements: M Navarros Rego was supported by a Leonardo da Vinci Scholarship.
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