Comment[ArrayExpressAccession]	E-GEOD-44561
MAGE-TAB Version	1.1								
Public Release Date	2014-06-04
Investigation Title	Effect of Notch1 pathway activation on high-grade glioma cells								
Comment[Submitted Name]	Effect of Notch1 pathway activation on high-grade glioma cells
Experiment Description	In this study, we explored the transcriptomic consequences of strong activation of the Notch pathway in embryonic human neural stem cells and in gliomas. For this we used a forced expression of the Notch intracellular domain (NICD).  Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signalling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration which was accompanied by the sharp reduction of neural stem cell transcription factor expression (ASCL1, OLIG2, SOX2) while HEY1/2, KLF9, SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation.  Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRb and a-smooth muscle actin (aSMA)) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (HB-EGF, IL8, PLGF), metalloprotease (MMP9) and adhesion proteins (VCAM-1, ICAM-1, ITGA9). In xenotransplantation experiments, contrasting with the infiltrative and poorly-vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly-disseminating but highly-vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties. Embryonic human neural progenitors obtained from Lonza, the U87 glioma cell line, and glioma cancer stem cells (Gb4 and Gb7) characterized in Guichet et al. (Glia, 2013, 61(2), 225-39) were infected with lentiviruses expressing YFP or YFP-IRES-NICD (activated form of Notch receptor). After 48h, RNA were extracted for Affymetrix microarray analysis.								
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl							
Person Last Name	hugnot	Hugnot	Guichet	Rothhut					
Person First Name	jean philippe	Jean	Pierre	Bernard					
Person Mid Initials		P	O						
Person Email	hugnot@univ-montp2.fr								
Person Affiliation	inserm inm u1051								
Person Address	inserm inm u1051, hopital saint eloi 80 avenue augustin fliche, montpellier, languedoc, France								
Person Roles	submitter								
Protocol Name	P-GSE44561-1	P-GSE44561-5	P-GSE44561-6	P-GSE44561-2	P-GSE44561-9	P-GSE44561-3	P-GSE44561-8	P-GSE44561-4	P-GSE44561-7
Protocol Description	Raw data were controlled using Affymetrix software such as AGCC and Expression Console (www.affymetrix.com). Primary analysis was performed with an external MAQC A sample to validate robustness of data, regarding Affymetrix recommendations and following Institut Curie's genomic platform (Paris, France). Probeset mean, RLE means, AUC were in agreement with Affymetrix recommendations. ID_REF =  VALUE = Log2 RMA signal intensity	100ng of total RNA was amplified according to the Affymetrix GeneChip® Whole Transcript (WT) Expression assay using the Ambion® WT Expression Kit and Affymetrix labelling kit, which generate sense and labeled DNA.	Targets were prepared with 5.5µg of labelled DNA, and hybrized onto Affymetrix Human Gene ST 1.1 array plates using Affymetrix GeneTitan systems. The GeneTitan performed automatically hybridization, washes, stainings, and scanning of arrays.	Cells were infected with lentiviruses expressing YFP or YFP-IRES-NICD (activated form of Notch receptor). After 48h, RNA were extracted.	Glioma cancer stem cells isolated from glioblastoma were grown as neurospheres in defined medium.	Embryonic human neural stem cells obtained from Lonza company were grown as neurospheres in defined media.	U87 high-grade glioma cell line (from ATCC) was grown in 10% serum.	Total RNA were extracted with Qiagen RNeasy kits. Quality checks (RIN>8.7) were performed on an Agilent Bioanalyzer.	The arrays were scanned according to Affymetrix recommendations and GeneTitan robotic procedures.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	growth protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	LENTIVIRUS	CELL TYPE							
Experimental Factor Type	lentivirus	cell type							
Comment[SecondaryAccession]	GSE44561								
Comment[GEOReleaseDate]	2014-06-04								
Comment[ArrayExpressSubmissionDate]	2013-02-21								
Comment[GEOLastUpdateDate]	2014-06-05								
Comment[AEExperimentType]	transcription profiling by array								
SDRF File	E-GEOD-44561.sdrf.txt