Journal: eLife

Article Title: EphrinB2 drives perivascular invasion and proliferation of glioblastoma stem-like cells

doi: 10.7554/eLife.14845

Figure Lengend Snippet: ( a ) Representative H&E staining of GSC1 tumours 60 days after intracranial injection into nude mice. Neovascularisation ( i ), focal necrosis ( ii ) and increased cellular density ( iii ) can be observed. ( b ) Representative fluorescent images of GFP-labeled GSC1 tumours stained for the vascular marker CD31 (red) and GFP to identify tumour cells (green, left), the stem cell markers nestin (green, middle) and Sox2 (red, right), as indicated. The arrows indicate examples of GFP-labeled GSC1 that have migrated away from the tumour mass and are invading along the vasculature as single cells or in small groups. Scale bars = 50 μm. ( c ) Heatmap showing Verhaak subtype classification of 4 GSC1 and 3 GSC2-derived tumours in vivo. Colour scale with corresponding normalised enrichment scores is shown on the right. All tumours classified as mesenchymal with a nes >2.6. ( d ) Mean GSEA enrichment plot for the Verhaak mesenchymal gene signature in the GSC lines. Both nom p-val and FDR q-val = 0. ( e ) Intravital 2-photon micrographs of GFP-labeled imNSC1 and GSC1 cells injected into the cortex of wildtype ( Efnb2 WT EC) and endothelial specific Efnb2 knockout mice ( Efnb2 -/- EC) and imaged 7 days later over 6 hr through an intracranial window at a depth of 200 μm. The vasculature was labeled by tail-vein injection of Tx-red conjugated Dextrans (3000 MW). Arrowheads indicate representative perivascular migration patterns for each genotype. Scale bar = 50 μm. ( f ) Quantification of the migrated distance of the cells depicted in ( c ). Each dot represents one cell. n indicates number of animals imaged. One way ANOVA with Tukey post hoc test. ( g ) Left: schematic representation of the experimental set up for in vitro migration assays with endothelial cells. Middle: merged fluorescent and phase contrast still images taken from time-lapse microscopy experiments of GFP-labeled imNSC1 and GSC1 (green) migrating towards brain microvascular endothelial cells (bmvEC, unlabeled cells) at the indicated time points. Right: quantification of boundary length at 60 hr. Students t-test. Scale bar = 500 μm. ( h ) Still fluorescence and phase contrast merged images of GFP-labeled imNSC2 and GSC2 migrating towards bmvEC (unlabeled, left) for 60 hr and quantification (right) as in ( e ). Error bars denote s.e.m., Students t-test. Scale bar = 500 μm. ( i ) Schematic representation of the experimental set up for in vitro migration assays toward recombinant ephrin-B2-Fc (left), phase contrast images (middle) and quantification (right) of imNSC1 and GSC1 migration against coated ephrinB2-Fc pre-clustered with fluorescently-labelled anti-Fc antibodies at 60 hr. Error bars denote s.e.m., Students t-test. Scale bar = 500 μm. Green dots denote boundary of ephrin-B2 coating identified by fluorescence. ( j ) Still images (left) and quantification (right) of GFP-labeled imNSC (GFP) migrating towards bmvEC (unlabeled) treated with control siRNA (Scr) or siRNA against Efnb2 (si Efnb2 ) for 60 hr. Scale bar = 500 μm. Error bars denote s.e.m., Students t-test. For this and later figures dots indicate individual data points and ***p<0.001; **p<0.01 and *<0.05. See also and . DOI: http://dx.doi.org/10.7554/eLife.14845.003 10.7554/eLife.14845.004 Figure 1—source data 1. Raw data for all quantification of NSC/GSC migrated distance and boundary length shown in . DOI: http://dx.doi.org/10.7554/eLife.14845.004

Article Snippet: Primary mouse microvascular endothelial cells were obtained from Caltag Medsystems and subcultured according to the suppliers recommendations (cells were primary cells isolated directly from normal mouse brain, no further authentication performed by the authors, mycoplasma negative as tested by Mycoalert kit, Lonza) Human GSC lines were described previously ( ; ).

Techniques: Staining, Injection, Labeling, Marker, Derivative Assay, In Vivo, Knock-Out, Migration, In Vitro, Time-lapse Microscopy, Fluorescence, Recombinant