Figure 1.
Spatial organization of ECs and pericytes within the glioblastoma neovessel wall in relation to the multilaminar VBM.
(A) Colocalization of MMP2 and CD105 on tumoral ECs; (B-D) relative localization of CD31-, CD146-, and MMP2-reactive ECs in tumour vessels characterized by a thick, multilaminar Coll IV VBM. (E-G) The multilayered arrangement of NG2/CSPG4 D2-expressing pericytes and the relative innermost localization of αSMA (E), PDGFRβ (F), and endosialin/CD248 (G) immunolabelled pericytes. (H, I) Perivascular distribution of cell surface-shed fragments of the PG NG2; note in (C) restricted Mel-CAM/CD146 reactivity on the luminal EC plasma membranes, in (E, inset) the alternate αSMA cytoplasmic reactivity and cell and membrane-associated NG2 D2 labelling, and in (F) the activated form of PDGFRβ revealed on the adluminal pericyte plasma membrane. Nuclear counterstaining TO-PRO3. Bars 20 µm.
Figure 2.
Distribution of NG2/CSPG4 isoforms recognized by mAb 2164H5 in glioblastoma and foetal brain.
(A) 2164H5/Coll IV double immunolabelling shows 2164H5-reactive NG2/CSPG4 isoforms close to pericytes fully embedded in the VBM; proteolytic fragments of these isoforms appear dispersed through the stromal ECM (asterisk) and decorate glioblastoma cells. (B) High magnification view of the neovessel marked with a star in (A) in which this specific pericyte subset preferentially localizes in a more abluminal position with respect to the Coll IV-containing VBM, and resides in mural 'pericyte lacunae' (arrows). (C, D) The distribution of NG2/CSPG4 isoforms recognized by mAb 2164H5 in foetal brain closely resembles that seen in glioblastoma neovessels; notably (C), NG2/CSPG4 fragments form a concentration gradient which increases from the ventricular zone (VZ) through the subventricular zone (SVZ) and close to the vessel wall (D, arrowheads). Nuclear counterstaining TO-PRO3. Bars A, C 25 µm; B, D 10 µm.
Figure 3.
Expression of 2161F9-reactive NG2/CSPG4 isoforms by pericyte subsets.
(A-C) Double immunolabellings of glioblastoma lesions with mAb 2161F9 and antibodies to Coll IV, Coll VI, or the NG2 D2 pAb, specifically show that the pericyte subpopulation expressing these isoforms is organized in the innermost pericyte layers of both 'garland' vessels (A) and smaller neovessels characterized by the typical proliferative appearance of EC whose nuclei bulge into the vessel lumen (B, C; arrows). (D-F) Capturing of pericyte precursor-like cells 2161F9-reactive NG2/CSPG4 isoforms within the vessel lumen and adherent to the vessel wall (arrows in E and F, enlargements of D). (G-I) A pericyte subset expressing the NG2/CSPG4 isoform recognized by mAb 2161F9 is also seen in foetal brain, where its specificity is confirmed, allowing a clear-cut view of the pericyte cell body and processes (high magnification of G in G'). Nuclear counterstaining TO-PRO3. Bars A-C 10 µm; D 20 µm; E-G, H, I 10 µm; G' 5 µm.
Figure 4.
Comparative distribution patterns of two different NG2/CSPG4 isoforms revealed in glioblastoma lesions by double labelling with mAbs 2164H5 and 2161F9.
(A-C) mAb 2164H5 (A) primarily discloses cell surface-released fragments of NG2/CSPG4 included in the VBM (arrowheads) and present in the intercellular spaces (asterisk), whereas mAb 2161F9 (B) defines the pericyte layer (arrows); on the merged image (C), the diverse NG2/CSPG4 expression profiles are clearly recognizable. (D) In a tumour area rich in ECM, clone 2161F9 reveals actively dividing pericytes (arrows), shown in detail in (E) (arrow a, prophase and arrow b, telophase), and two tumour cells, enlarged in (F), in advanced anaphase of mitosis (arrowheads); note in (F) NG2/CSPG4 aggregates next to the dividing chromosomes (arrowheads). Nuclear counterstaining TO-PRO3. Bars A-C, 20 µm; D, 25 µm; E, F 10 µm.
Figure 5.
Expression of NG2/CSPG4 isoforms recognized by mAb 2161D7 in glioblastoma and foetal brain.
(A) NG2/CSPG4 isoforms detected by mAb 2161D7 appear ubiquitously distributed through the pericyte layers, decorating the luminal side of glioblastoma neovessels and co-localizing with the outermost VBM Coll IV layers. (B) 2161D7-reactive multilayered pericytes surrounded by Coll VI VBM. (C) A 'naked' tumour vessel with a thin layer of pericytes without a detectable Coll VI VBM. (D-F) Identical double stainings of foetal brain to those performed on glioblastoma sections confirm an analogous spatial arrangement of the pericyte population marked by mAb 2161D7-reactive NG2/CSPG4 isoforms, and additionally highlight the expression of such isoforms on typical foetal OPCs (arrowheads). Nuclear counterstaining TO-PRO3. Bars A, C, F 20 µm; B, D, E 25 µm.
Figure 6.
Expression of NG2/CSPG4 isoforms recognized by mAb 2166G4 in glioblastoma cells.
(A-C) Immunolabelling with mAb 2166G4 shows the specific expression of these NG2/CSPG4 isoforms by the tumour cells, frequently seen forming perivascular cuffs (V in A and B). (D-F) Coincident staining of tumour cell 2166G4-reactive isoforms and Glut-1. (G-I) mAb 2161D7, identifying pericyte-specific NG2/CSPG4 isoforms, does not stain Glut-1-reactive tumoral cells that in this case do not infiltrate the vessel wall (V), providing indirect evidence of NG2/CSPG4 isoform specificity. Nuclear counterstaining TO-PRO3. Bars A 50 µm; B-I 10 µm.
Figure 7.
Immunochemical characteristics of NG2/CSPG4 isoforms and their proteolytic fragments in foetal brain, glioblastoma and neoplastic cells.
Sections from foetal brain and glioblastoma samples were taken adjacently to sections in which abundant neovessels and enrichment of NG2/CSPG4-expressing pericytes were revealed by immunostaining. The sections were then processed for immunoblotting with the anti-NG2/CSPG4 mAbs (see M & M). A375 and M2 melanoma cells, and the recombinant NG2 ectodomain (NG2rec), used for producing the mAbs, were blotted in parallel for reference. Since mAb 2164H5 did not react with the cognate isoform in Western blotting, it was used to immunoprecipitate the isoform, which was then blotted with mAb B5/M28. The co-recognized isoform is likely to be only one of the NG2/CSPG4 variants and their cell surface-shed fragments immunoprecipitated by mAb 2164H5. Immunoblotting of α-actin with a polyclonal antiserum was used for normalization of gel lane loading. The relative position of molecular weight markers is indicated to the left of each panel.