Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Nuclear β-catenin in vessels of human astrocytoma does not correlate with WHO grade. (A) TCGA database analyses for Wnt1 , Wnt3a , Wnt5a , Wnt7a , Wnt7b , Dkk1 , and Dkk2 mRNA expression as log2-fold expression. Differences in mRNA expression in GBM compared with normal central nervous system tissue (dashed red line) are shown. (B and C) Paraffin sections of three normal human brains (B) and five different human astrocytoma WHO grades I–IV (C) stained for β-catenin and analyzed for its endothelial, nuclear localization. Insets show individual nuclei in higher magnification. Bars: (B) 14 µm; (C) 20 µm.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Wnt1 expression decreased subcutaneous GL261 tumor growth and increased animal survival. (A) Western blots showing Wnt1 and Dkk1 expression +/−DOX in GL261 cells. (B) sTOP-FLASH assay on human embryonic kidney (HEK293)/GL261 co-cultures without DOX. Monoculture of transfected HEK293 cells served as baseline. Wnt1 and Dkk1 cells were cultured without (−) or with (+) Wnt3aCM (one experiment in triplicate). (C) In vitro proliferation of the control-, Wnt1-, and Dkk1-GL261 line cultured +/−DOX. (D, left) Representative pictures of NUDE mice with subcutaneous tumors −DOX. (right) Tumor volumes ( n = 7/group) of the transplanted glioma cell lines −DOX (*, P < 0.05; **, P < 0.01). (E) H&E-stained paraffin sections revealed reduced necrotic areas for Dkk1-expressing tumors (N). (F, left) Pimonidazole (brown) immunohistochemistry staining revealed tumor hypoxia, hematoxylin counterstaining (blue). (right) Hypoxia in Dkk1 −D compared with the control −D (*, P < 0.05) and Wnt1 −D tumors (**, P < 0.01; n = 4 tumors/group, slices from the center of similar sized tumors). (G, left) Representative vessels, stained for nuclear β-catenin (brown) and hematoxylin (blue) of control −D , Wnt1 −D , and Dkk1 −D tumors. Arrowheads point to β-catenin + nuclei. (right) Quantification of β-catenin + nuclei ( n = 4 tumors/group, 20 vessels/tumor; ***, P < 0.001). Bars: (D) 1 cm; (E and F) 400 µm; (G) 35 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Expressing, Western Blot, Transfection, Cell Culture, In Vitro, Control, Staining, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor-derived Wnt1 reduced and normalized subcutaneous tumor vascularization, whereas Dkk1 caused the opposite effect. (A) Tumor growth in Wnt1 −D compared with Wnt1 +D condition (left; n = 12/group; **, P < 0.01; ***, P < 0.001) and mouse survival (right; n = 12/group). (B) Tumor volume for Dkk1 −D compared with Dkk1 +D condition (left; n = 12/group; *, P < 0.05; **, P < 0.01; ***, P < 0.001) and mouse survival of Dkk1 +/−D tumors (right; n = 12/group). (C, top) IF staining on subcutaneous Wnt1 tumors for CD31/PECAM-1, α-SMA, and TOPRO-3. (bottom left) Vessel density of subcutaneous Wnt1 tumors +/−DOX ( n = 5 tumors/group, 10 pictures/tumor; **, P < 0.01). (bottom right) Association of α-SMA + cells to ECs in Wnt1 +/−D tumors ( n = 5 tumors/group, 10 vessels/tumor; ***, P < 0.001). (D) Same staining and experimental settings as in C for subcutaneous Dkk1 +/−D tumors (*, P < 0.05). (C and D) Insets show vessels in higher magnification. (E) Perfusion with isolectin revealed that vessels from control −D , Wnt1 −D , and Dkk1 −D tumors exhibited blood flow. Bars: (C and D) 400 µm; (E) 200 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Derivative Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Glioma-derived Wnt1 up-regulated endothelial Dll4 , leading to a stalk cell–like gene signature. (A) ISH against Dll4 (black) and hematoxylin (blue) of subcutaneous control −D , Wnt1 −D , and Dkk1 −D GL261 tumors, showing representative small (0–300 µm 2 ; left) and large (>300 µm 2 ; right) vessels. (B, left) Quantification of vessel diameter ( n = 3 tumors/group, 15 pictures/tumor) grouped in categories of 0–30 µm 2 , 31–300 µm 2 , and >300 µm 2 (*, P < 0.05; **, P < 0.01). (right) Quantification of Dll4 + ECs ( n = 6 tumors/group, 5 small and 5 large vessels/tumor; ***, P < 0.001). (C, top) HUVECs co-cultivated with control −D , Wnt1 −D , and Dkk1 −D GL261 cells (asterisks indicate HUVECs). (bottom) qRT-PCR ( n = 6) for human genes regulated by Wnt1 −D and Dkk1 −D compared with control −D co-cultures (red line). (D) qRT-PCR for mouse genes regulated in MBEs stimulated by Wnt3aCM for 18 and 48 h compared with controlCM (red line; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars: (A) 50 µm; (C) 80 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Derivative Assay, Control, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor cell–free Matrigel plug assay confirmed the antiangiogenic effect observed in Wnt1-GL261 tumors. (A) Immunohistochemistry staining for CD31/PECAM-1 (red) and hematoxylin counterstaining (blue) of Matrigel plug sections, supplemented with diluent (control), 400 µg/ml human Wnt1, or 200 µg/ml mouse Dkk1 recombinant proteins. Bar, 200 µm. (B) Quantification of CD31 + area in percentage in Matrigel plugs ( n = 6 plugs/group, 8 representative pictures/plug; **, P < 0.001). Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Matrigel Assay, Immunohistochemistry, Staining, Control, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Tumor-derived Wnt1 normalized vascularization and retained vascular barrier properties in GL261 glioma. (A, left) Representative H&E staining of serial thick sections of tumor-bearing mouse brains. (right) Corresponding IF staining for Podxl and TOPRO-3. (B, top) Quantification of glioma volume based on H&E staining (control −D n = 3, Wnt1 −D n = 6, and Dkk1 −D n = 4; *, P < 0.05). (bottom) Podxl + vessel areas within tumors (control n = 4, Wnt1 n = 6, Dkk1 n = 5; **, P < 0.01). (C) IF staining for CD31/PECAM-1 and desmin ( n = 3 tumors/group, 8 pictures/tumor; *, P < 0.05). (D) Large image reconstructions of IF staining for mouse endogenous IgG (mIgG), Podxl, and TOPRO-3 on brain sections bearing control −D , Wnt1 −D , and Dkk1 −D glioma. Bars: (A) 200 µm; (C) 50 µm; (D, top) 1 mm; (D, bottom) 27 µm. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Derivative Assay, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

doi: 10.1084/jem.20111580

Figure Lengend Snippet: Pdgfb is a Notch-independent target of β-catenin signaling in ECs. (A) HUVECs co-cultivated with control −D , Wnt1 −D , and Dkk1 −D GL261 cells. qRT-PCR ( n = 4) for human PDGFB regulated by Wnt1 −D or Dkk1 −D (ns) compared with control −D (gray line; *, P < 0.05). (B) qRT-PCR for Pdgfb gene regulated in MBEs stimulated by Wnt3aCM for 18 and 48 h compared with controlCM (gray line; n = 4; *, P < 0.05; ***, P < 0.001). (C, left) qRT-PCR for Axin2 and Pdgfb regulated in β-catenin–deficient ECs transduced with LefΔN-βCTA compared with the vector control (gray line; n = 4; *, P < 0.05; **, P < 0.01). (right) Representative Western blot ( n = 1) for PDGF-B from LefΔN-βCTA ECs compared with the vector control; top band shows the nonreduced (PDGF-BB) and bottom band reduced protein (PDGF-B). Densitometric analysis shows the summarized values for both forms of PDGF-B of samples loaded twice. (D) qRT-PCR of β-catenin–deficient ECs (control) or transduced with LefΔN-βCTA grown for 24 h either on gelatin- or Dll4-coated dishes ( n = 3). Notch pathway induction was monitored by Hes1 expression (*, P < 0.05; **, P < 0.01). Notch signaling was blocked by Dll4-Fc. Error bars indicate SEM.

Article Snippet: Recombinant proteins were as follows: human Wnt1 (PeproTech), mouse Dkk1 as well as human Dll4 (R&D Systems), and Dll4 (mouse):Fc (human; rec.

Techniques: Control, Quantitative RT-PCR, Transduction, Plasmid Preparation, Western Blot, Expressing