Fig 1: Orthotopic engraftment of patient-derived GBM cells into the zebrafish brain. (A) Left: schematic showing the acquisition of primary GBM cells from patients. Middle: bright-field and fluorescent microscopy of in vitro cultured primary GBM cells (passage 1) from the same patient as 3D organoid, neurosphere and attached single-layer cells. Immunofluorescence staining showing NES and GFAP expression in differently cultured primary GBM cells. Right: fluorescence microscopy of 4 dpi GBM xenografts from primary GBM cells (passage 1). Primary GBM cells were labeled with CFSE before injecting into zebrafish brain. Two patients (GBM#109 Grade IV and GBM#24 Grade IV) were tested. (B,C) Fluorescence microscopy of 4 dpi patient-derived GBM xenografts from GBM patients (GBM#109, B; GBM#24, C), showing the heterogeneous phenotypes (infiltrative or demarcated) of primary GBM xenografts. Top: #GBM109 was defined as highly infiltrative and #GMB24 was defined as less infiltrative by MRI. Pink labeling (pink arrows) in MRI images indicates the edema zone and potential infiltrating areas. Bottom: brain vasculature is highlighted by fake gray color (kdrl:eGFP). Xenografts with more than five individual infiltrating cell clusters (white arrow) were defined as infiltrative, otherwise they were defined as demarcated. The numbers of xenografts with different phenotypes were counted (pie charts). Scale bars: 100 µm.
Fig 2: Extracellular vesicles recapitulate mesenchymal reprogramming effects of endothelial secretome against proneural glioma stem cells.a Schematic of extracellular vesicle (EV) isolation from conditioned media. b Nanoparticle tracking analysis (NTA/Nanosight) of EVs isolated from CME (HUVEC-CM). c Immunoblot for tetraspanins (CD9, CD63, CD81), and EV purity marker, BIP, indicates the absence of cytoplasmic contamination in EV preparations, according to MISEV201837; Ponceau loading control. d Mass spectrometry analysis of the most abundant proteins enriched in HUVEC EVs (EEVs). e EV transfer assay: HUVEC cells were transduced with Cre-mCherry and proneural-GSC157 were transduced with dsRed/LoxP/eGFP lentiviral vectors. EVs from Cre-transduced HUVECs were purified and incubated with dual reporter proneural-GSC-157 for 4 days to observe a red to green fluorescence switch in recipient GSC157 cells. Cre-loxP experiment involving proneural-GSC157 cells treated with OEVs (top panel), Cre-EEVs (middle panel) and culture supernatant (Sup) from Cre-expressing cells (bottom panel). f Morphological differences observed in proneural-GSC157 cells treated with OEVs or HUVEC-EVs (EEVs) after 3 days in culture. g Cell eccentricity ratios measured by the Incucyte software for proneural-GSC157 cells treated with OEV (black line) versus cells treated with EEVs (blue line). h Expression of SOX2, NES, NOTCH1, NICD, and VIM in proneural-GSC157 cells treated with OEVs or EEVs. i Densitometry analysis of NICD expression (activated NOTCH1) in GSC157 cells treated with CMO, HUVEC-conditioned media (CME), CME derived EVs and Sup (n = 3). j Temozolomide (TMZ) dose-response curve for mesenchymal-GSCs and proneural-GSCs. MTS assay quantifying viability of proneural-GSCs after TMZ treatment in cultures pretreated with fresh media (media), OEV or EEV: GSC157 (k), GSC84 (l) and GSC1079 (m). NICD notch intracellular domain, CMO own conditioned media, Sup supernatant, EV extracellular vesicles, OEVs own EVs EEVs endothelial cell derived EVs, TMZ temozolomide, HUVEC human umbilical vein endothelial cells. Source data are provided as a Source Data file.
Fig 3: Mesenchymal reprogramming of proneural glioma cell stemness by endothelial conditioned media.Time-dependent changes in clonogenicity (ELDA) of GSCs in the presence of endothelial conditioned media (CME) or control media (CMO), compilation of limiting dilution assays across three proneural GSC lines: GSC1079 (a), GSC157 (b), GSC528 (c) and three mesenchymal GSCs lines: GSC1123 (d), GSC83 (e), GSC1005 (f); black lines represent cells treated with fresh media (FM), gray lines are indicative of cells treated with CMO, and blue lines represent cells treated with CME; n = 3 each group and each time point. g TCGA data mining of proneural versus mesenchymal genetic signatures across 51 patients diagnosed with GBM. Proneural hallmarks include, but are not limited to, NES, NOTCH1 and SOX2. Mesenchymal hallmarks include, but are not limited to, VIM, TGM2 and CD44. Green and purple represent mesenchymal (MES) and proneural (PN) genetic signatures, respectively. h Volcano plot showing enriched mesenchymal markers detected in the mass spectrometry (MS) of CME treated GSC157 cells relative to CMO treated GSC157 cells. i Expression of proneural hallmarks (NES and SOX2) and mesenchymal hallmark (TGM2) in proneural cells (GSC157, GSC1079) and mesenchymal cells (GSC83, GSC1005) after 7 day treatment with CMO, or CME. Flow cytometry of mesenchymal-GSC83 (j) and proneural-GSC157 (k) for CD44-APC. Dashed line, IgG control; blue curve, cells treated with CMO; red curve, cells treated with CME. l Kaplan–Meier symptom-free survival curve of tumor bearing mice injected with proneural-GSC157 cells pre-treated with either fresh media, CMO or CME; n = 5 mice per group; mice intracranially injected with cells pretreated with either fresh media (media) - dashed line, with CMO—black line, or with CME—blue line. m Wound healing/cell migration assay of proneural-GSC157 cells grown as monolayer and treated with CMO or CME throughout 48 h. n Quantification of the wound healing assay for CMO or CME treated GSC157 cells. GSC glioma stem cells, CMO own conditioned media, CME conditioned media derived from endothelial cells. Source data are provided as a Source Data file.
Fig 4: Dynamic proximity between glioma stem cells and endothelium.a–f Human GBM tissue co-stained for the endothelial cell (EC) marker, CD31, and for GSC marker, NES. White arrowheads point to NES+ GSCs juxta-positioned along CD31+ blood vessels in the GBM tissue. g Schematic of steps taken to obtain live mouse tissues for high resolution confocal imaging of GSCs and ECs in mouse tumor xenografts. Phase tile image (h) and Green fluorescence tile image (i) of a vibratome cut 200 µm thick coronal section of mouse brain injected with GSC157 proneural-GSCs. Lycopersicon lectin-stained blood vessels in the tumor hemisphere (TH; j) and contralateral hemisphere (CH; m) of the mouse tumor xenografts. GFP labeled GSC157 cells in the tumor hemisphere (k) and contralateral hemisphere (n) of the mouse brain. Merged Lycopersicon lectin /GFP with insets showing GSC proximity with ECs and cellular morphologies of GFP+ cells in the tumor hemisphere (l) and contralateral hemisphere (o). p Quantification of the number of GFP+ proneural glioma stem cells found in close proximity to-, or not near the vicinity of endothelial cells. q Cartoon representation of the use of mouse aortae to generate sprouting endothelial-tumor cell ‘dynamic’ co-cultures. Green cells represent GFP+ GSC83 that were found to migrate towards sprouting ECs. q–t Three days after GFP+ GSCs were placed with aortic ring, GFP+ GSCs migrated towards ECs and adopted more elongated cellular morphologies. Green arrowheads indicate the presence of GFP+ GSCs and red arrowheads point towards ECs. GSC glioma stem cells, ECs endothelial cells, TH tumor hemisphere, CH contralateral hemisphere, EC endothelial cells.
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