Dedifferentiation of neurons and astrocytes by oncogenes can induce gliomas in mice

Abstract

Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in humans. Here we show that gliomas can originate from differentiated cells in the central nervous system (CNS), including cortical neurons. Transduction by oncogenic lentiviral vectors of neural stem cells (NSCs), astrocytes, or even mature neurons in the brains of mice can give rise to malignant gliomas. All the tumors, irrespective of the site of lentiviral vector injection (the initiating population), shared common features of high expression of stem or progenitor markers and low expression of differentiation markers. Microarray analysis revealed that tumors of astrocytic and neuronal origin match the mesenchymal GBM subtype. We propose that most differentiated cells in the CNS upon defined genetic alterations undergo dedifferentiation to generate a NSC or progenitor state to initiate and maintain the tumor progression, as well as to give rise to the heterogeneous populations observed in malignant gliomas.

Fig. 1

Glioblastomas induced by a single lentiviral vector. (A) Schematic representation of the lentivector. In the shNF1-shp53 vector, the hairpin targeting NF1 was cloned under the H1 promoter at the 3′ UTR and the hairpin targeting p53 was cloned 5′ to the CMV promoter and under the U6 promoter. (B) Western Blot (WB) showing silencing of both NF1 and p53. NF1 loss resulted in increased phosphorylation of Akt (P-Akt); total Akt expression was used as a control. MEF/Ikk2^−/− cells were infected with the indicated lentivectors. The cell lysate was collected and analyzed by WB. Tubulin detection was used as a loading control. (C) Hematoxylin and eosin (H&E) histology and immunofluorescence of sections from either shNF1-shp53 or H-RasV12-shp53 induced glioblastomas in GFAP-Cre mice (hippocampus injected). Panels i and i′ show images of the tumors (x20) where increased cellularity, vascularity (V), hemorrhage (H) and necrotic areas (N) can be observed. The rest of the panels show all the classical GBM features: necrotic areas (N; panels ii and ii′; x40), perivascular infiltration (indicated by arrows in panels iii and iii′; x40 plus 2 electrical zoom) and multinucleated giant cells (se arrows in panels iv and iv′; x40 plus 2 electrical zoom). Panels v and v′ show by immunofluorescence staining the infiltrative characteristic of the tumor, crossing the midline and migrating to the other hemisphere (blue = DAPI, green = GFP; x5).

Fig. 2

Induction of gliomas by shNF1-shp53 lentiviral transduction of neurons. (A) Photographs (panels i–ii) showing the massive lesion in the brain and H&E staining of shNF1-shp53 induced tumors in the cortex of SynI-Cre mice (iii, magnification, x40; iv, magnification x40 plus two electrical zoom, showing perivascular infiltration (white arrows) and multinucleated giant cell (black arrows). (B) Confocal images showing the presence of both GFP+/RFP− (open arrowhead) and GFP+/RFP+ cells (filled arrowhead) in the tumor. (C) Confocal images showing oncogene/tumor suppressor expression specifically in NeuN+ and Tuj1+ cells. Five days after injection of the virus in the cortex of SynI-Cre transgenic mice, brains were collected and fixed. Sections of the brain were stained with the indicated antibodies. The upper panels show representative confocal images of GFP/RFP before antibodies staining (Scale bar = 75 μm). The lower panels show a representative (see arrows) co-labeling using the antibodies/markers indicated in each panel. When possible DAPI staining was also assessed (in blue). Scale bars = 18.75 μm. N = necrosis.

Fig. 3

Tumorigenesis in the cortex. (A) Confocal microscopy analysis of brain sections 5 days after injection of H-RasV12-shp53 virus in the cortex of GFAP-Cre mouse. Panels show representative (see arrows) co-labeling with the markers indicated in each of the images. Scale bars = 18.75 μm. (B) Maximum intensity projections of large-scale mosaic volumes. Immunolabeling for GFAP and Nestin in tumors at 2, 6, and 8 weeks following injection of lentivirus in the cortex. GFAP staining intensity is seen to decrease with tumor progression, while Nestin labeling increases. Scale bars = 500 microns.

Fig. 4

Mature astrocytes transduced with either shNF1-shp53 or H-RasV12-shp53 virus dedifferentiate to a neural progenitor/stem cell like state. (A) Morphology and staining of cortical astrocytes obtained from GFAP-Cre mice. Confocal microscopy analysis shows double staining for S-100β and GFAP (panels ii–iv), lack of expression of stem/progenitor makers Nestin and Sox2 (panel v) and expression of Cre in all GFAP+ astrocytes (panel vi: Red = GFAP, Green = Cre, Blue = DAPI). (B) Tumors derived from either shNF1-shp53 or H-RasV12-shp53 transduced astrocytes orthotopically transplanted into the hippocampus of NOD-SCID mice; H&E histology and immunohistochemistry. Arrows point to representative Tuj1 +ve cells. (C) i) astrocytes before transduction maintained in media plus serum express GFAP (inset red = GFAP, scale bar = 75 μm). ii) transduced astrocytes in media plus serum (inset red = GFAP), iii and iv) transduced astrocytes transferred to serum free media supplemented with FGF-2. Light microscopy magnification x20. (D) Confocal microscopy analysis of the neurospheres described in (C) panel iv (green = GFP (vector), yellow = Nestin, red = Sox2; scale bar = 75 mu;m).