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Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

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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).
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pone-0110316-g003: 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).

Mentions: Subsequently, confocal microscopy analysis of fluorescent streptavidin labeled cells was performed to specifically assess the cellular location of the azido-containing glycoconjugates (Fig. 3). As expected, U373 MG cells subjected to metabolic incorporation of AC4ManNAz followed by Staudinger ligation with biotin phosphine were intensely stained with streptavidin-Alexa Fluor 488 on the cell surface, with no apparent intracellular staining (Fig. 3A and 3B). Consistent with flow cytometric findings, staining was completely dependent on the concomitant presence of AC4ManNAz and Staudinger phosphine probe confirming that the labeling via azide-containing glycoconjugates is highly specific and that azide-bearing cell surface glycans were readily imaged by using streptavidin-Alexa Fluor 488 (Fig. 3C and 3D). Furthermore, there was no evidence of cell surface labeling for the control groups, confirming that background fluorescence staining is negligible (Fig. 3E). Taken together, the results suggest that AC4ManNAz was selectively, efficiently, and homogenously incorporated into cell surface sialoglycoconjugates.


Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4216004&req=5

pone-0110316-g003: 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).
Mentions: Subsequently, confocal microscopy analysis of fluorescent streptavidin labeled cells was performed to specifically assess the cellular location of the azido-containing glycoconjugates (Fig. 3). As expected, U373 MG cells subjected to metabolic incorporation of AC4ManNAz followed by Staudinger ligation with biotin phosphine were intensely stained with streptavidin-Alexa Fluor 488 on the cell surface, with no apparent intracellular staining (Fig. 3A and 3B). Consistent with flow cytometric findings, staining was completely dependent on the concomitant presence of AC4ManNAz and Staudinger phosphine probe confirming that the labeling via azide-containing glycoconjugates is highly specific and that azide-bearing cell surface glycans were readily imaged by using streptavidin-Alexa Fluor 488 (Fig. 3C and 3D). Furthermore, there was no evidence of cell surface labeling for the control groups, confirming that background fluorescence staining is negligible (Fig. 3E). Taken together, the results suggest that AC4ManNAz was selectively, efficiently, and homogenously incorporated into cell surface sialoglycoconjugates.

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Show MeSH
Related in: MedlinePlus