1. Volume 28, Issue 6, Pages 730-742 (December 2015)
A Tumor Suppressor Function for Notch Signaling in Forebrain Tumor Subtypes 
Claudio Giachino, Jean-Louis Boulay, Robert Ivanek, Alvaro Alvarado, Cristobal Tostado, Sebastian Lugert, Jan Tchorz, Mustafa Coban, Luigi Mariani, Bernhard Bettler, Justin Lathia, Stephan Frank, Stefan Pfister, Marcel Kool, Verdon Taylor 
Cancer Cell 
Volume 28, Issue 6, Pages (December 2015)
DOI: /j.ccell
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2. Cancer Cell 2015 28, 730-742DOI: (10.1016/j.ccell.2015.10.008)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 Hes5 Expression Identifies Subpopulations of Glioma Cells
(A) PDGF-A was infused into the forebrain lateral ventricle (LV) of adult mice for 6 consecutive days and BrdU administered 2 hr prior to sacrifice.
(B) Hes5::GFP construct driving GFP expression from the mouse Hes5 regulatory elements including promoter, exons I–III, introns, the endogenous 3′-untranslated region, and poly-adenylation signal, was used to monitor Notch signaling in the glioma cells in vivo.
(C) PDGF induced glioma-like growths that protrude from the SVZ into the LV of Hes5::GFP mice (arrows).
(D) Hes5::GFP expression (GFP) in PDGF-induced hyperplasias. GFP+BrdU+ are shown by arrows, and GFP-BrdU+ cells are shown by arrowheads.
(E) Nestin and Hes5::GFP expression (GFP) in PDGF-induced hyperplasias.
(F) PDGF-IRES-Cre retrovirus was injected into the brains of perinatal floxed Trp53 mice to simultaneously delete Trp53 and overexpress PDGF-B in proliferating progenitors.
(G) A PDGF+Trp53−/− glioma in the anterior forebrain.
(H) Hematoxylin and eosin (H&E) staining of a PDGF+Trp53−/− glioma.
(I) Cre expression (Cre+) by PDGF+Trp53−/− glioma cells. The vascular endothelial cells within the tumor mass are marked with CD31. Note the glomeruloid microvascular proliferation (asterisks) typical of GBM.
(J) Hes5::GFP expression (GFP) in viral-induced tumor cells (Cre+) (arrows) 2 weeks after glioma induction. Note the GFP+Cre− astrocytes and progenitors attracted to the tumor (arrowheads).
(K) Direct contact (arrowheads) of some Cre+ Hes5::GFP (GFP) expressing cells (arrows) with the tumor vasculature (CD31).
(L) Percentages of all tumor cells (Cre+), proliferating tumor cells (Cre+PCNA+), or quiescent tumor cells (Cre+PCNA−) that express Hes5::GFP.
(M) Representative images of proliferating GFP−PCNA+, proliferating GFP+PCNA+, or quiescent GFP+PCNA− tumor cells.
(N) Hes5::GFP (GFP) and PCNA expression in PDGF+Trp53−/− tumors 2 weeks after glioma induction (% of Cre+ tumor cells).
Error bars represent ±SEM. Scale bars represent 10 μm in (C)–(E), (I)–(K), and (M) and 1 mm in (G) and (H). See also Figure S1.
Cancer Cell  , DOI: ( /j.ccell )
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4. Figure 2
Rbpj Deletion Induces Glioma Formation by Promoting Proliferation
(A) Kaplan-Meier survival curves showing symptom-free survival of viral-induced PDGF+Trp53−/−, PDGF+Trp53−/−Rbpj−/−, and PDGF+Trp53−/−Rbpj+/− glioma mice.
(B) Hes5::GFP (GFP, arrows) and Mash1 (arrowheads) expression in PDGF+Trp53−/− or PDGF+Trp53−/−Rbpj−/− gliomas 2 weeks after tumor induction.
(C) Quantification of Hes5::GFP (GFP) and Mash1 expressing cells in PDGF+Trp53−/− and PDGF+Trp53−/−Rbpj−/− gliomas.
(D) Percentages of all tumor cells (Cre+) and Hes5+ (Hes5::GFP-expressing) or Hes5− (Hes5::GFP-non expressing) tumor cells that express PCNA in PDGF+Trp53−/− and PDGF+Trp53−/−Rbpj−/− gliomas.
(E) Hes5::GFP (GFP) and PCNA expression in PDGF+Trp53−/−Rbpj−/− tumors 2 weeks after glioma induction (% of Cre+ tumor cells).
Error bars represent ±SEM. Scale bars represent 10 μm. See also Figure S2.
Cancer Cell  , DOI: ( /j.ccell )
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5. Figure 3
Low HES5 Expression Correlates with Glioma Malignancy and Poor Survival in Grades II–III Astrocytoma and Proneural GBM Patients
(A) Kaplan-Meier survival plots for grades II–III astrocytoma and GBM patients with differential HES5 expression (REMBRANDT data set). The low or high levels are defined from the mean HES5 expression value in each tumor type.
(B) Percentages of grades II–III astrocytomas and GBMs that show down or upregulated HES5 expression compared to non-tumor brain tissue (REMBRANDT data set).
(C) HES5 expression in grades II–III astrocytoma, grades II–III oligodendroglioma, GBM, and non-tumor brain tissue (REMBRANDT data set). Up or downregulated are ≥2-fold changes over the mean HES5 expression in non-tumor brain tissue. The ≥2 up or down cutoff are shown (gray). The whiskers are plotted according to the Tukey method and notches represent ±1.58 IQR/sqrt(n).
(D) Hes5::GFP expression at early and late stages of tumor development in mouse PDGF+Trp53−/− gliomas. Error bars represent ±SEM.
(E) Kaplan-Meier plots for human GBM patients with differential HES5 expression (TCGA database) grouped by GBM subtype. The low or high levels are defined from the mean HES5 expression in each tumor type.
See also Figure S3.
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6. Figure 4
PDGF+Trp53−/−Rbpj−/− GBMs Maintain a Similar Expression Pattern as PDGF+Trp53−/− GBMs but Acquire Some Histological Features of sPNETs
(A) H&E staining of late stage PDGF+Trp53−/− and PDGF+Trp53−/−Rbpj−/− gliomas showing pseudopalisading necrosis and neuroblastic rosettes.
(B and C) Examples of pseudopalisading necrosis and microvascular proliferation (asterisks) in PDGF+Trp53−/− gliomas (B) and rosette formation in PDGF+Trp53−/−Rbpj−/− gliomas (C). The Cre+ cells are viral infected and transformed.
(D) PDGF+Trp53−/− and PDGF+Trp53−/−Rbpj−/− gliomas and contralateral control material were isolated from PDGF-IRES-Cre infected mice and prepared from microarray analysis. The tumor cells were identified by GFP expression from the Rosa-CAG::GFP Cre-reporter locus.
(E) Separation of tumor samples by hierarchical clustering of pair-wise correlation coefficients using 50% of the most variable genes in the data set. The heatmap shows a comparison of PDGF+Trp53−/− (n = 4) tumors, PDGF+Trp53−/−Rbpj−/− (n = 4) tumors, and control brain tissue (n = 4).
(F) GSEA of PDGF+Trp53−/− and PDGF+Trp53−/−Rbpj−/− gliomas. The gene expression profiles were compared with the TCGA GBM subtype signatures using a Correlation Adjusted MEan RAnk gene set test (CAMERA). The plots show -log10 of rank-based p values of the four GBM subtype signatures in the tumor versus control comparison. Note the mixed proneural and mesenchymal features in both PDGF+Trp53−/− and PDGF+Trp53−/−Rbpj−/− mouse tumors are shown.
Scale bars represent 100 μm. See also Figure S4.
Cancer Cell  , DOI: ( /j.ccell )
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7. Figure 5 Noch1 and Notch2 Receptors Inhibit Glioma Formation
(A) Kaplan-Meier survival curves showing symptom-free survival of mice with PDGF-IRES-Cre viral-induced PDGF+Trp53−/−, PDGF+Trp53−/−Notch1−/−Notch2−/−, and PDGF+Trp53−/−Notch2ICD gliomas.
(B) H&E staining of a PDGF+Trp53−/−Notch1−/−Notch2−/− glioma.
(C) H&E staining showing pseudopalisading necrosis and neuroblastic rosettes in a PDGF+Trp53−/−Notch1−/−Notch2−/− tumor.
(D) Kaplan-Meier survival curves of mice with PDGF+Trp53−/−Rbpj−/−Notch2ICD and PDGF+Trp53−/−Rbpj−/− tumors.
Scale bars represent 1 mm in (B) and 50 μm in (C). See also Figure S5.
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8. Figure 6
Rbpj and Trp53 Deletion Synergize to Promote a Premalignant State
(A and B) To delete Rbpj and/or Trp53 in Hes5+ adult NSCs, adult Hes5::CreERT2 mice carrying floxed Trp53 alleles, floxed Rbpj alleles, or both, and a Cre-reporter for lineage tracing were treated with TAM (A) and were analyzed 14 days or 60 days later (B).
(C and D) Proliferation (C, PCNA, BrdU) and expression of Mash1 and Dcx (D) in the SVZ of mutant mice 14 days after TAM treatment. Note the small neoplastic-like lesions emanating from the SVZ into the striatum that are mainly composed of Mash1+ progenitors and Dcx+ neuroblasts (arrows). The expression of GFP from the Rosa-CAG::GFP Cre-reporter (C) indicates derivation from Hes5+ cells.
(E) Hyperplastic proliferative (PCNA+) nodules (arrows) in the striatum proximal to the SVZ in mutant mice 60 days after TAM treatment.
(F) Representative schemes of a 30 μm thick section showing the distribution of PCNA+ and Mash1+ nodules in the striatum of Trp53−/−Rbpj−/− and Rbpj−/− mutant mice after 60 days. The nodules are mainly located in the striatum proximal to the SVZ.
(G) Quantification of striatal nodules in Trp53−/−Rbpj−/− and Rbpj−/− mutant mice.
(H–K) Expression of the stem and early progenitor marker Sox2 (H), intermediate progenitor markers Olig2 (H and I) and Mash1 (I and J), the neuroblast marker Dcx (K), and proliferation markers (PCNA and Ki67 and I and K) in the striatal nodules of Trp53−/−Rbpj−/− mutant mice. The expression of GFP from the Rosa-CAG::GFP Cre-reporter (H, J, and K) indicates derivation from Hes5+ cells (blood vessel, BV, H).
Error bars represent ± SEM. Scale bars represent 10 μm. See also Figure S6.
Cancer Cell  , DOI: ( /j.ccell )
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9. Figure 7
Combined Rbpj and Trp53 Deletion in Hes5+ Cells Leads to Formation of sPNET-like Tumors
(A) Gross appearance of the brains of Hes5::CreERT2 Trp53−/−, Rbpj−/−, and Trp53−/−Rbpj−/− mice 10 months after TAM induction. Note the signs of tumor formation in the olfactory bulb and anterior forebrain of Trp53−/−Rbpj−/− mice (arrows).
(B) Kaplan-Meier survival curves showing survival of control, Trp53−/−, Rbpj−/−, and Trp53−/−Rbpj−/− mutant mice.
(C) Proliferation (PCNA, BrdU) in tumors of Trp53−/−Rbpj−/− mutant mice. The expression of GFP from the Rosa-CAG::GFP Cre-reporter indicates derivation from Hes5+ cells.
(D) Rosette formation in tumors of Trp53−/−Rbpj−/− mutants. The GFP expression from the Rosa-CAG::GFP Cre-reporter indicates derivation from Hes5+ cells and CD31 marks blood vessels.
(E) Two examples of nuclear molding in Olig2+ cells in the tumors of Trp53−/−Rbpj−/− mutants.
(F) Principal component analysis of mouse tumors (Trp53−/−Rbpj−/−, PDGF+Trp53−/−, and PDGF+Trp53−/−Rbpj−/−) and control brain tissue samples using 50% of most variable genes in the microarray data set. The axes represent the two linear combinations of genes that account for most of the variance in the original data set. The Trp53−/−Rbpj−/− tumors were isolated as shown in Figure 4D.
(G) Log2 expression levels of canonical Notch target genes and RBPJ in human sPNETs, grade II–III astrocytomas, and GBMs. The whiskers are plotted according to the Tukey method, notches represent ±1.58 IQR/sqrt(n), and + represents the mean value.
Scale bars represent 1 mm in (A) and 10 μm in (C)–(E). See also Figure S7 and Tables S1, S2, and S3.
Cancer Cell  , DOI: ( /j.ccell )
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10. Figure 8
Proposed Model of the Tumor Suppressive Actions of Notch Signaling during Brain Tumor Initiation and Progression
(A) Within the normal neurogenic lineage, Notch signaling promotes quiescence and maintenance of NSCs and inhibits their transition to fast dividing intermediate progenitors.
(B) Within specific brain tumor subtypes, including grades II–III astrocytoma, proneural GBM, and sPNET, Notch signaling may maintain mitotically quiescent Hes5+ cell populations. The Notch inhibition may favor tumor progression by promoting a shift toward a more proliferative Notch-independent cell state.
(C) A tumor suppressive action of Notch signaling may also reflect its ability to regulate cell fate decisions and lineage choices, in particular the oligodendrocyte lineage.
Cancer Cell  , DOI: ( /j.ccell )
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