Geoff Pilkington

NG2 proteoglycan promotes angiogenesis-dependent tumor growth in CNS by sequestering angiostatin

During embryogenesis, the NG2 proteoglycan is expressed on immature capillary vessels, but as the vessels mature they lose this expression. NG2 is up‐regulated in high‐grade gliomas, but it is not clear to what extent it contributes to malignant progression. Using a combination of high spatial and temporal resolution functional magnetic resonance imaging and histopathological analyses, we show here that overexpression of NG2 increases tumor initiation and growth rates, neovascularization, and cellular proliferation, which predisposes to a poorer survival outcome. By confocal microscopy and cDNA gene array expression profiles, we also show that NG2 tumors express lower levels of hypoxia inducible factor‐1α, vascular endothelial growth factor, and endogenous angiostatin in vivo compared with wild‐type tumors. Moreover, we demonstrate that NG2‐positive cells bind, internalize, and coimmunoprecipitate with angiostatin. These results indicate a unique role for NG2 in regulating the transition from small, poorly vascularized tumors to large, highly vascular gliomas in situ by sequestering angiostatin.

Cancer stem cells in the mammalian central nervous system

Abstract. Malignant tumours intrinsic to the central nervous system (CNS) are among the most difficult of neoplasms to treat effectively. The major biological features of these tumours that preclude successful therapy include their cellular heterogeneity, which renders them highly resistant to both chemotherapy and radiotherapy, and the propensity of the component tumour cells to invade, diffusely, the contiguous nervous tissues. The tumours are classified according to perceived cell of origin, gliomas being the most common generic group. In the 1970s transplacental administration of the potent neurocarcinogen, N‐ethyl‐N‐nitrosourea (ENU), enabled investigation of the sequential development of brain and spinal neoplasms by electron microscopy and immunohistochemistry. The significance of the primitive cells of the subependymal plate in cellular origin and evolution of a variety of glial tumours was thereby established. Since then, the development of new cell culture methods, including the in vitro growth of neurospheres and multicellular tumour spheroids, and new antigenic markers of stem cells and glial/neuronal cell precursor cells, including nestin, Mushashi‐1 and CD133, have led to a reappraisal of the histological classification and origins of CNS tumours. Moreover, neural stem cells may also provide new vectors in exciting novel therapeutic strategies for these tumours. In addition to the gliomas, stem cells may have been identified in paediatric tumours including cerebellar medulloblastoma, thought to be of external granule cell neuronal derivation. Interestingly, while the stem cell marker CD133 is expressed in these primitive neuroectodermal tumours (PNETs), the chondroitin sulphate proteoglycan neuronal/glial 2 (NG2), which appears to denote increased proliferative, but reduced migratory activity in adult gliomas, is rarely expressed. This is in contrast to the situation in the histologically similar supratentorial PNETs. A possible functional ‘switch’ between proliferation and migration in developing neural tumour cells may exist between NG2 and ganglioside GD3. The divergent pathways of differentiation of CNS tumours and the possibility of stem cell origin, for some, if not all, such neoplasms remain a matter for debate and continued research, but the presence of self‐renewing neural stem cells in the CNS of both children and adults strongly suggests a role for these cells in tumour initiation and resistance to current therapeutic strategies.

INSIDIA: a FIJI Macro Delivering High-throughput and High-content Spheroid Invasion Analysis†

Time‐series image capture of in vitro 3D spheroidal cancer models embedded within an extracellular matrix affords examination of spheroid growth and cancer cell invasion. However, a customizable, comprehensive and open source solution for the quantitative analysis of such spheroid images is lacking. Here, the authors describe INSIDIA (INvasion SpheroID ImageJ Analysis), an open‐source macro implemented as a customizable software algorithm running on the FIJI platform, that enables high‐throughput high‐content quantitative analysis of spheroid images (both bright‐field gray and fluorescent images) with the output of a range of parameters defining the spheroid “tumor” core and its invasive characteristics.

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

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.

Role of the GAS6-AXL receptor-ligand pairing in migration of human glioma cells

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.