Zaynah Maherally

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 early-passage 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 (β1, αvβ1 and αvβ3) on glioblastoma cell processes whereas siRNA-transfected 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 to CD44-knockdown and ‘double’-knockdown cells, no obvious decrease in RhoC expression was observed in CD155-knockdown cells. Conclusions This investigation has enhanced our understanding of cell invasion and confirmed CD44 to play a more significant role in this biological process than CD155. Joint CD44/CD155 approaches may, however, merit further study in therapeutic targeting of infiltrating glioma cells.

TNF-α enhancement of CD62E mediates adhesion of non–small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis

Background CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non–small cell lung cancer (NSCLC). Methods CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. Results CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. Conclusion This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E.

Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood–brain barrier model exemplifies tight-junction integrity

The blood‐brain barrier (BBB) consists of endothelial cells, astrocytes, and pericytes embedded in basal lamina (BL). Most in vitro models use nonhuman, monolayer cultures for therapeutic‐delivery studies, relying on transendothelial electrical resistance (TEER) measurements without other tight‐junction (TJ) formation parameters. We aimed to develop reliable, reproducible, in vitro 3‐dimensional (3D) models incorporating relevant human, in vivo cell types and BL proteins. The 3D BBB models were constructed with human brain endothelial cells, human astrocytes, and human brain pericytes in mono‐, co‐, and tricultures. TEER was measured in 3D models using a volt/ohmmeter and cellZscope. Influence of BL proteins—laminin, fibronectin, collagen type IV, agrin, and perlecan—on adhesion and TEER was assessed using an electric cell‐substrate impedance‐sensing system. TJ protein expression was assessed by Western blotting (WB) and immunocytochemistry (ICC). Perlecan (10 μg/ml) evoked unreportedly high, in vitro TEER values (1200 Ω) and the strongest adhesion. Coculturing endothelial cells with astrocytes yielded the greatest resistance over time. ICC and WB results correlated with resistance levels, with evidence of prominent occludin expression in cocultures. BL proteins exerted differential effects on TEER, whereas astrocytes in contact yielded higher TEER values and TJ expression.—Maherally, Z., Fillmore, H. L., Tan, S. L., Tan, S. F., Jassam, S. A., Quack, F. I., Hatherell, K. E., Pilkington, G. J. Real‐time acquisition of transendothelial electrical resistance in an all‐human, in vitro, 3‐dimensional, blood‐brain barrier model exemplifies tight‐junction integrity. FASEB J. 32, 168‐182 (2018). www.fasebj.org

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.

Drug Repurposing to Circumvent Chemotherapy Resistance in Brain Tumours

The incidence of primary brain tumours in the UK is steadily rising; prognosis for this devastating disease is dismal and not significantly improving. The relative failure for effective therapy may be attributed to heterogeneity with over 120 histological entities. Additionally, the biology is incredibly complex with numerous pathways and cascades working simultaneously or in harmonisation. Moreover, we find that as the tumour evolves, it commandeers alternative pathways for its survival. At its most aggressive form, glioblastoma produces guerrilla cells that trek into the normal brain parenchyma where they are protected by the intact blood-brain barrier (B-BB). The majority of drugs cannot cross the intact B-BB; the dosage required to cross the damaged area around the tumour would be toxic systemically. Effective therapy, therefore, has a number of hurdles against chemotherapy resistance. In this chapter we look at the tumour biology and conventional therapeutic resistance; we then look at the potential of using drugs originally used to treat other diseases. Indeed, repurposed drugs are drawing increasingly more interest, especially as after approximately ten years of development; the new drug failure rate of 25,000:1 costs the industry an astonishing fortune of around

Hyaluronic acid and poliovirus receptors: is dual expression indicative of a combined role in modulating glioma invasion?

2.6 billion per year. Here, we refer to UK regulations and doctor trepidation for prescribing outside of the drug’s licence—‘off-label’—and give examples of some of the repurposed agents which have gained attention within the laboratory and within our current legislation require additional clinical trials for marketing authorisation and prescription without fear of potential litigation.

Silencing of CD44 in Glioma Leads to Changes in Cytoskeletal Protein Expression and Cellular Biomechanical Deformation Properties as Measured by AFM Nanoindentation

INTRODUCTION: We previously reported expression of CD44(Hyaluronic acid/lymphocyte homing receptor) and CD155 (Poliovirus receptor) on established cell lines and early passage cultures of biopsyderived glioma using immunocytochemistry, TIRF microscopy and flow cytometry. Both receptors have been reported to facilitate glioma invasion. METHODS: Antibody blocking and siRNA silencing of CD44 and CD155 were assessed using Transwell assay and live cell imaging for motility and invasion. A ECM cell adhesion array was used to determine adhesive potential of wild type cells versus silenced cells. BrdU cell proliferation assay was carried out to assess proliferative rate of cells following siRNA knockdown of both CD44 and CD155. Interaction and localisation of CD44 and CD155 with F-actin and integrins (b, avb, and avb3) were shown by confocal and TIRF microscopy. RESULTS: CD44 antibody blocking and gene silencing resulted in a higher level of inhibition of invasion than for CD155. Interference with combined CD44/CD155 resulted in 100% inhibition of invasion. Live cell imaging showed reduced speed of motility of knockdown cells over their controls. Wild type cells adhered most efficiently to laminin whereas siRNA-treated cells showed decreased adhesive potential on most of the ECMs used. A higher proliferative rate of siRNA CD44 and siRNA CD155 treated cells was inversely correlated with the reduced invasion of these cells. Confocal microscopy showed distinct overlapping of CD155 and the integrins on filopodia while TIRF microscopy allowed high signal/low noise imaging of double labelled cells. CONCLUSIONS: Joint CD44/CD155 approaches may merit further study in targeting infiltrating glioma cells in therapeutic protocols.Times Cited: 0 Meeting of the British-Neuro-Oncology-Society JUN 23-25, 2010 Glasgow, SCOTLANDINTRODUCTION: The Axl receptor tyrosine kinase (RTK) and its ligand Gas6, have previously been shown to regulate neoplastic cell motility, migration, invasion and proliferation. The aim of this study was to determine the role of Gas6/Axl on migration of human glioma cells. METHODS: Three human glioblastoma derived cell lines of differing origin and degree of heterogeneity,were screened for Axl protein expression by western blot. For investigation of cell migration, serum-starved cells were stimulated with recombinant human Gas6, and scratch wound assays coupled with live cell imaging were performed to monitor the migration rates of these cells. The cells were also analysed for intracellular signalling effects on Axl and phospho-Axl by western blot and protein tyrosine kinase assays. RESULTS: We detected expression of Axl protein in SNB-19 (homogeneous) and UPAB (heterogeneous) cell lines, although not in IN699 (paediatric). It was furthermore demonstrated that Gas6 stimulated the migration of SNB-19 cells over 36 h by a two-fold increase as compared to control-treated cells, resulting in a migration speed of 11.650+0.564 mm/h compared to 6.861+1.264 mm/h. Moreover, a Gas6 concentration-dependent response was observed, revealing a specific promotion of migration (p , 0.05). CONCLUSIONS: Stimulation of the Axl RTK with its ligand Gas6 stimulates migration of human glioma cells, a mechanism that remains to be fully understood. These data can help to understand the mechanisms of local invasion in brain tumours. We wish, therefore, to further investigate Axl activation as well as related signalling pathway activities in glioma cells to help understand the molecular basis of glioma migration and invasion.