Tumour invasion is the key element in the high rate of mortality and morbidity in glioma patients. The increased levels of expression of the cell surface adhesion molecules CD44/CD155 on neoplastic cells have been highlighted in several studies; both playing a role in glioma invasion. CD44; originally described as the lymphocyte homing receptor, is a cell adhesion molecule with two isoforms with respective molecular weights of 80-90 kDa and 150kDa. CD44 mediates glioma cell adhesion and invasion through its interaction with hyaluronic acid (HA). CD155, also known as the poliovirus receptor, is a transmembrane glycoprotein, the ectodomain of which mediates cell attachment to the extracellular matrix component, vitronectin. Within the scope of this thesis, an upregulation of CD44 and CD155 has been demonstrated on established cell line (SNB-19) and early passage cultures of biopsy-derived glioma (UPAB and UPMC) using immunocytochemistry and flow cytometry. TIRF microscopy has revealed that CD44 and CD155 are located in close proximity in all the GBM cells studied. CD44 antibody blocking and gene silencing resulted in a higher level of inhibition of invasion than that for CD155 when assessed using the Transwell assay. Interference with combined CD44/CD155 resulted in 86% inhibition of invasion in post-transfected cells. Live cell imaging showed reduced speed of motility and distance travelled in knocked-down cells over their controls. Both siRNA CD44 and siRNA CD155 cells were devoid of filopodia and were rounder in morphology compared to wild type cells. The ECM cell adhesion array demonstrated wild type cells adhered most efficiently to laminin whereas siRNAtreated cells showed decreased adhesive potential on most of the ECMs used. The BrdU cell proliferation assay showed a higher proliferative rate of siRNA CD44 and siRNA CD155 treated cells was achieved and this was inversely correlated with the reduced invasion of these cells. Confocal microscopy showed distinct overlapping of CD155 and the integrins (β1, αvβ1 and αvβ3) on extending processes of the GBM cells whereas siRNAtransfected cells showed consequent reduction in expression level of the integrins with no specific staining patterns. RHO GTPases assay showed reduced expression levels of Cdc42, Rac1/2/3 and RhoA in siRNA transfected CD44 and CD155 cells. No change in expression level of RhoC was observed in siRNA CD155 cells compared to the control cells and the expression levels of RhoB were unchanged in both transfected and control cells. Joint CD44/CD155 approaches may merit further study in targeting infiltrating glioma cells in therapeutic protocols.
|Date of Award||Jan 2011|
|Supervisor||Geoff Pilkington (Supervisor) & Arthur Butt (Supervisor)|