Table 1.
Current in vitro and preclinical developments for investigational targeted toxins in glioblastoma.
Table 2.
Current clinical trials for investigational targeted toxins in glioblastoma (HTTP://www.clinicaltrials.gov).
Figure 1.
Profiling cell surface sialoglycoproteins via the bio-orthogonal chemical reporter strategy combined with quantitative shotgun proteomics.
The strategy consists of six steps: 1) metabolic labeling of cells with Ac4ManNAz; 2) chemoselective conjugation of azide-labeled glycans with a biotin-linked phosphine; 3) cell lysis; 4) affinity enrichment of the biotin-tagged proteins on streptavidin-conjugated beads; 5) on-beads trypsin digestion of affinity-captured sialoglycoproteins; 6) identification using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using a nano-LC-LTQ Orbitrap mass spectrometer platform and sequence database searching, and quantitative differential analysis of the sialoglycoproteins using label-free analysis with DIFFTAL (DIFferential Fourier-Transform AnaLysis) software algorithm.
Figure 2.
Flow cytometric analysis of azide-modified surface sialoglycoconjugates on U373 MG cells.
U373 cells were incubated with increasing concentrations of Ac4ManNAz (10 to 100 µM) or vehicle for two days and then probed with biotin phosphine (50 µM) for 1 h at room temperature. Next, non-permeabilized cells were stained with Alexa Fluor 488 (AF488)-conjugated Streptavidin. After washing, cells were permeabilized, stained with 7-amino-actinomycin D viability dye (7-AAD), and subsequently analyzed by flow cytometry. (A) FACScan dot plots generated by the analysis of 10,000 events show Streptavidin Alexa Fluor 488 versus 7-AAD cell staining. (B) 7-AAD+: percentage of non-viable 7-AAD positive cell population; AF488+/7-AAD-: percentage of AF488 positive viable cell population; AF488 MFI: AF488 mean fluorescence signal associated with viable cell in arbitrary units (AU). (C) Dose-dependent incorporation of Ac4ManNAz into U373 MG cell surface sialoglycoconjugates. Data are representative of at least three independent experiments.
Figure 3.
Confocal microscopy images of azide-modified surface sialoglycoconjugates on live U373 MG cells.
U373 cells were incubated with 25 µM Ac4ManNAz (A – C) or vehicle (D, E) for two days and subjected (A, B, D) or not (C, E) to Staudinger ligation with biotin phosphine (50 µM) for 1 h at room temperature. Next, cells were stained with Streptavidin Alexa Fluor 488 at 4°C for 20 min and, after washing, fixing, and staining for the nucleus with the DAPI dye, imaged. Merged indicate that the images of cells labelled with Alexa Fluor 488 (λem = 520 nm) and DAPI (λem = 460 nm) are merged and shown in green and blue, respectively. Scale bars, 20 µm (A, C – E) and 10 µm (B).
Figure 4.
Affinity purification of biotinylated cell surface sialoglycoproteins.
U373 MG cells, primary GBM cells (SIDs), adult (AL) and fetal (ASG6 and ASG7) astrocytes, and neural progenitor cells (NPC) were metabolically labelled with 25 µM Ac4ManNAz for 2 days and conjugated with 50 µM biotin-phosphine capture reagent. Azide-tagged and biotin-conjugated sialoglycoproteins from total cell lysate were captured by streptavidin beads, separated by SDS-PAGE and visualized using streptavidin-HRP conjugate and enhanced chemiluminescence (upper panels). Shown are the results from 5 µg of total cell lysate (Input, IN), 5 µg of the flow through material (FT) that did not bind to the beads, and 5% of the eluted material (EL) from 1 mg (SIDs and AL), 600 µg (ASG6 and ASG7 lysates combined into a single pooled ASG6&7 lysate) and 50 µg (NPC) of protein that bound to the beads. (A) Western blot analysis of U373 MG input, flow-through and eluate fractions. (B) Western blot analysis of SIDs, AL, ASG6, ASG7 and NPC input fractions. (C) Western blot analysis of SIDs, AL, NPC and ASG6&7 flow through and eluate fractions. Prior immunodetection, proteins blotted onto membranes were stained to reveal the total protein expression profile of each sample (lower panels).
Figure 5.
Classification of identified sialoglycoproteins by GO annotation.
(A) Cellular components were assigned using Ingenuity Pathway Analysis Knowledge Base (http://ingenuity.com/), the Human Protein Reference Database (http://www.hprd.org/), and the Gene Ontology (GO) Consortium (http://geneontology.org/). (B) Molecular functions were assigned using the PANTHER classification system (http://www.pantherdb.org/). Of the 843 unique sialoglycoproteins confidently identified from the ten samples, 485 (58%) and 121 (14%) proteins could be assigned to the “plasma membrane” (GO:0005886) and “extracellular space” (GO:0005615) GO categories, respectively. A total of 819 molecular function hits were allotted to the 606 cell surface proteins. Of these, approximately one-third (34%) correspond to receptor activity including all major classes of cell surface receptor proteins.
Figure 6.
Top 65 cell surface sialoglycoproteins overexpressed in GBM patient tumors (SIDs) compared to human astrocytes.
Biostatistics analysis of average differences in sialoglycoprotein mean intensities (“Effect sizes”), between multiple replicate samples of tumor tissues (“tumor experimental group”) and fetal and adult astrocytes (“astrocyte reference group”) was performed using a one-way analysis of variance (ANOVA). Effect sizes (Log2 scale) of proteins which are up-regulated (Effect size >3 and p-value <0,05) in the tumor experimental group versus the astrocyte reference group are plotted against protein median intensities (linear scale) in the tumor experimental group. The x-axis shows the median intensity values on a logarithmic scale. Among the 65 proteins up-regulated in tumors, 52 were detected in all four tumor samples (blue diamonds), while 8 and 5, were found in at least three (red squares) or two (green triangles), among the four tumor samples, respectively.
Table 3.
Top 52 cell surface sialoglycoproteins overexpressed in GBM tumor cells.
Figure 7.
Gene expression of glioblastoma-associated cell surface sialoglycoproteins.
The expression of 23 genes was analyzed by quantitative real-time PCR in GBM patient tumors and adult astrocytes, as described in the Materials and Methods section. The relative expression levels from at least triplicate experiments were averaged and expressed as means ± standard deviation (SD) (A) Heat map visualization. Bar at top gives color scale of relative expression from the mean expression: Green: Down-regulation of gene expression; Dark: No change; Red: Up-regulation of gene expression. A significant modification of gene expression was defined as a >2 down- (dark green) or >3 up-regulation (dark red). (B) Histograms represent mean expression values for each gene ± SD.