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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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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.
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pone-0110316-g002: 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.

Mentions: Sialoglycoconjugates are restricted to the extracellular compartment which includes the extracellular side of the plasma membrane, the extracellular matrix, secreted proteins, and the subcellular organelles of the secretory and endocytic pathways of cells. In order to establish that these proteins could be tagged with a molecular reporter, we conducted flow cytometry analysis on the model human astrocytoma cell line U373 MG to examine the labeling efficiency of the metabolic oligosaccharide engineering strategy (Fig. 2). Over 95% of the viable cell population was stained with streptavidin-Alexa Fluor 488 whatever the dose of azidosugar used, indicating that AC4ManNAz is metabolized to SiNAz homogeneously into all cells and this from the lowest dose of 10 µM. U373 MG cells treated with AC4ManNAz exhibited a dose-dependent increase in fluorescence that indicated the accumulation of biotin moieties on the cell surface, whereas untreated cells showed only a background level of fluorescence after exposure to the biotin-phosphine probe. The background fluorescence was identical to that observed with U373 MG cells that were not exposed to any reagents and thus represents autofluorescence of cells and not nonspecific uptake of the biotin-phosphine probe or streptavidin-Alexa Fluor 488 (data not shown). As quantified by flow cytometry, the AC4ManNAz–treated U373 MG cells displayed about 280–450 fold greater Alexa488-specific fluorescence intensity than the control vehicle-treated cells. Reliable fluorescence labeling was achieved at a 10 µM concentration of azidosugar, however optimal results were obtained at a concentration ranging from 25 to 50 µM. No increase in labeling was observed at concentrations higher than 50 µM. A significant decrease in labeling occurred at concentrations higher than 50 µM owing to the limited solubility of AC4ManNAz. At all concentrations, AC4ManNAz showed no cellular toxicity as assessed by morphology and 7-AAD staining. Altogether, these results demonstrate that AC4ManNAz was efficiently incorporated into cellular glycans with no apparent adverse effect on U373 MG cell survival.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110316-g002: 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.
Mentions: Sialoglycoconjugates are restricted to the extracellular compartment which includes the extracellular side of the plasma membrane, the extracellular matrix, secreted proteins, and the subcellular organelles of the secretory and endocytic pathways of cells. In order to establish that these proteins could be tagged with a molecular reporter, we conducted flow cytometry analysis on the model human astrocytoma cell line U373 MG to examine the labeling efficiency of the metabolic oligosaccharide engineering strategy (Fig. 2). Over 95% of the viable cell population was stained with streptavidin-Alexa Fluor 488 whatever the dose of azidosugar used, indicating that AC4ManNAz is metabolized to SiNAz homogeneously into all cells and this from the lowest dose of 10 µM. U373 MG cells treated with AC4ManNAz exhibited a dose-dependent increase in fluorescence that indicated the accumulation of biotin moieties on the cell surface, whereas untreated cells showed only a background level of fluorescence after exposure to the biotin-phosphine probe. The background fluorescence was identical to that observed with U373 MG cells that were not exposed to any reagents and thus represents autofluorescence of cells and not nonspecific uptake of the biotin-phosphine probe or streptavidin-Alexa Fluor 488 (data not shown). As quantified by flow cytometry, the AC4ManNAz–treated U373 MG cells displayed about 280–450 fold greater Alexa488-specific fluorescence intensity than the control vehicle-treated cells. Reliable fluorescence labeling was achieved at a 10 µM concentration of azidosugar, however optimal results were obtained at a concentration ranging from 25 to 50 µM. No increase in labeling was observed at concentrations higher than 50 µM. A significant decrease in labeling occurred at concentrations higher than 50 µM owing to the limited solubility of AC4ManNAz. At all concentrations, AC4ManNAz showed no cellular toxicity as assessed by morphology and 7-AAD staining. Altogether, these results demonstrate that AC4ManNAz was efficiently incorporated into cellular glycans with no apparent adverse effect on U373 MG cell survival.

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