Receptors for hyaluronic acid and poliovirus : expression and role in glioma invasion

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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

CD15s/CD62E interaction mediates the adhesion of non-small cell lung cancer cells on brain endothelial cells:implications for cerebral metastasis

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell–brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell–brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s–CD62E interaction in brain metastasis

Receptors for Hyaluronic Acid and Poliovirus: A Combinatorial Role in Glioma Invasion?

Background: CD44 has long been associated with glioma invasion while, more recently, CD155 has been implicated in playing a similar role. Notably, these two receptors have been shown closely positioned on monocytes. Methods and Findings: In this study, an up-regulation of CD44 and CD155 was demonstrated in established and earlypassage cultures of glioblastoma. Total internal reflected fluorescence (TIRF) microscopy revealed close proximity of CD44 and CD155. CD44 antibody blocking and gene silencing (via siRNA) resulted in greater inhibition of invasion than that for CD155. Combined interference resulted in 86 % inhibition of invasion, although in these investigations no obvious evidence of synergy between CD44 and CD155 in curbing invasion was shown. Both siRNA-CD44 and siRNA-CD155 treated cells lacked processes and were rounder, while live cell imaging showed reduced motility rate compared to wild type cells. Adhesion assay demonstrated that wild type cells adhered most efficiently to laminin, whereas siRNA-treated cells (p,0.0001 for both CD44 and CD155 expression) showed decreased adhesion on several ECMs investigated. BrdU assay showed a higher proliferation of siRNA-CD44 and siRNA-CD155 cells, inversely correlated with reduced invasion. Confocal microscopy revealed overlapping of CD155 and integrins (b1, avb1 and avb3) on glioblastoma cell processes whereas siRNAtransfected cells showed consequent reduction in integrin expression with no specific staining patterns. Reduced expression of Rho GTPases, Cdc42, Rac1/2/3, RhoA and RhoB, was seen in siRNA-CD44 and siRNA-CD155 cells. In contrast t

Reduced expression of CD44 and CD155 following siRNA-KD in SNB-19 cells.

<p><b>a</b>, siRNA-KD of CD44 and CD155 was confirmed by Western blotting using both wild type (<i>wt</i>) and non-targeting siRNA treated cells as controls. GAPD-siRNA was used as a positive control to validate the knockdown effect. <b>b</b>, Expression of CD44 (<b>top panels</b>) and CD155 (<b>bottom panels</b>) in non-targeting siRNA treated cells (<b>control/left panels/L</b>) and relevant siRNA-KD (<b>right panels/R</b>) cells; scale bars: 25 µm. In control cells (<b>L</b>), CD44 is uniformly distributed with intense staining at the edges of the cells (arrows) whereas CD155 is well distributed with dense staining zones at the leading edges of the cells (arrows). CD44/CD155 staining was reduced in the siRNA-KD cells with clearly altered morphology (<b>R</b>). <b>c</b>, CD44/CD155-KD was confirmed by flow cytometry. Expression levels of CD44 and CD155 were significantly reduced in siRNA-KD SNB-19 cells as indicated by percentage of positive cells and fluorescence fold. * indicates statistical significance compared to non-targeting siRNA treated cells (control).</p

Expression of F-actin and integrins and their co-localisation with CD44/CD155 on GBM cells.

<p><b>a</b>, Co-staining of F-actin (green/<b>left panels/L</b>) or β₁-integrin (green/<b>right panels/R</b>) with CD44 (red/<b>top panels</b>) and CD155 (red/<b>bottom panels</b>) on wild type UPAB cells. <b>b</b>, Co-staining of α_vβ₁-integrin (green/<b>L</b>) or α_vβ₃-integrin (green/<b>R</b>) with CD44 (red/<b>top panels</b>) and CD155 (red/<b>bottom panels</b>) on wild type UPMC (CD44 staining) and non-targeting siRNA treated SNB-19 cells (CD155 staining). All images in <b>a</b> and <b>b</b> have scale bars of 25 µm. <b>c</b>, Western blotting showed reduced expression of F-actin and integrins (α_v, β1 and β₃) in CD44/CD155-KD SNB-19 cells when compared to non-targeting siRNA treated cells (control).</p

Reduced motility rate in CD44/CD155-KD SNB-19 cells.

<p>Non-targeting siRNA treated SNB-19 cells were used as a control. <b>a</b>, Velocity of cell movement (<b>right panel</b>) with cell tracking (<b>left panel</b>) analysed over a period of 72 h. The speed by which CD44/CD155-KD cells moved was significantly reduced. <b>b</b>, Total distance moved by cells (<b>right panel</b>) with cell tracking (<b>left panel</b>) over a period of 72 h. The distance travelled by CD44/CD155-KD cells was markedly decreased. Start position: white arrow; end position: black arrow. * indicates statistical significance compared to control.</p