High‐Throughput Lectin Microarray‐Based Analysis of Live Cell Surface Glycosylation

Liu, Yu; Tao, Sheng Ce; Zhu, Heng; Schneck, Jonathan P.

doi:10.1002/0471140864.ps1209s63

Cited by 8 publications

(7 citation statements)

References 19 publications

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“…The surface of vertebrate cells are modified with a wide variety of glycans with considerable structural diversity [ 39 ], forming a dense glycocalyx at the cellular surface. Altered glycosylation can be indicative of malignancy, and STn is known to be present in more than 80% of human carcinomas [ 21 ].…”

Section: Discussionmentioning

confidence: 99%

Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau

et al. 2018

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Recurrent ovarian cancer (OvCa) is thought to result in part from the inability to eliminate rare quiescent cancer stem cells (CSCs) that survive cytotoxic chemotherapy and drive tumor resurgence. The Sialyl-Thomsen-nouveau antigen (STn) is a carbohydrate moiety present on protein markers of CSCs in pancreatic, colon, and gastric malignancies. We have demonstrated that human OvCa cell lines contain varying levels of cells that independently express either STn or the ovarian CSC marker CD133. Here we determine co-expression of STn and CD133 in a subset of human OvCa cell lines. Analyses of colony and sphere forming capacity and of response to standard-of-care cytotoxic therapy suggest a subset of OvCa STn+ cells display some CSC features. The effect of the anti-STn antibody-drug conjugates (ADCs) S3F-CL-MMAE and 2G12-2B2-CL-MMAE on OvCa cell viability in vitro and in vivo was also assessed. Treatment with S3F-CL-MMAE reduced the viability of two of three OvCa cell lines in vitro and exposure to either S3F-CL-MMAE or 2G12-2B2-CL-MMAE reduced OVCAR3-derived xenograft volume in vivo, depleting STn+ tumor cells. In summary, STn+ cells demonstrate some stem-like properties and specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate both STn+ CSC and STn+ non-CSC populations.

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Section: Discussionmentioning

confidence: 99%

Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau

et al. 2018

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“…Caution must be exerted however, as the expression of a putative cell surface galactosyltransferase (Penno et al, ) and the presence of glycosidases (Porwoll et al, ) in the culture medium released by dead cells may influence terminal galactosylation of cell surface proteins, affecting the extent of correlation between cell surface lectin binding and MAb β1,4‐galactose levels. When taken in to account, by exploiting the advantages of cell surface glycan screening in a live‐cell lectin microarray format (Li et al, ; Tao et al, ; Tateno et al, ), there is potential to couple the technology to microscale cell culture systems and high throughput product titre measurement (Amanullah et al, ; Legmann et al, ; Silk et al, ) to rapidly identify culture conditions that result in cell lines with favourable productivity and desirable glycan processing. Rapid screening of cell lines in this way permits a level of feedback control, identifying promising production cell line candidates with ideal glycan processing early in development which can then undergo further manipulation during process optimisation.…”

Section: Discussionmentioning

confidence: 99%

CHO cell line specific prediction and control of recombinant monoclonal antibody N‐glycosylation

Grainger

James

2013

Biotech & Bioengineering

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Here we demonstrate that it is possible to predict and control N-glycan processing of a secreted recombinant monoclonal antibody during manufacturing process development using a combination of statistical modelling and comparative measurement of cell surface glycans using fluorescent lectins. Using design of experiments--response surface modelling (DoE-RSM) methodology to adjust the relative media concentrations of known metabolic effectors of galactosylation (manganese, galactose, and uridine) we have shown that β1,4-galactosylation of the same recombinant IgG4 monoclonal antibody produced by different CHO cell lines can be precisely controlled in a cell line specific manner. For two cell lines, monoclonal antibody galactosylation could be increased by over 100% compared to control, non-supplemented cultures without a reduction in product titre and with minimal effect on cell growth. Analysis of galactosylation effector interactions by DoE-RSM indicated that Mn²⁺ alone was necessary but not sufficient to improve galactosylation, and that synergistic combinations of Gal and Urd were necessary to maximize galactosylation, whilst minimizing the deleterious effect of Urd on cell growth. To facilitate rapid cell culture process development we also tested the hypothesis that substrate-level control of cellular galactosylation would similarly affect both cell surface and secreted monoclonal antibody glycans, enabling facile indirect prediction of product glycan processing. To support this hypothesis, comparative quantitation of CHO cell surface β1,4-galactosylation by flow cytometry using fluorescent derivatives of RCA and ConA lectins revealed that substrate-controlled variation in monoclonal antibody galactosylation and cell surface galactosylation were significantly correlated. Taken together, these data show that precision control of a complex, dynamic cellular process essential for the definition of protein product molecular heterogeneity and bioactivity is possible. Moreover, real-time, or near real-time control can be enabled by facile, rapid measurement of cell surface biomarkers of cellular biosynthetic capability.

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“…Generally, a lectin microarray based on glycan binding to the arrayed lectins is used to conduct high-throughput glycosylation pattern analyses of proteins . This method enables glycosylation pattern analysis of glycoproteins, such as cell surface proteins, without liberating glycans from proteins. , Therefore, this technique is widely used for various research fields as a high-throughput screening tool. However, the information on glycan expression obtained with this technique depends on the kind of lectin.…”

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confidence: 99%

Rapid Quantitative Profiling of N-Glycan by the Glycan-Labeling Method Using 3-Aminoquinoline/α-Cyano-4-hydroxycinnamic Acid

et al. 2012

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Protein glycosylation is a crucial phenomenon for understanding protein functions, since its patterns and degree are associated with many biological processes, such as intercellular signaling and immune response. We previously reported a novel glycan-labeling method using a 3-ainoquinoline/α-cyano-4-hydroxycinnamic acid (3-AQ/CHCA) liquid matrix for highly sensitive detection by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS). In the present study, we examined the practicality of this method for qualitative and quantitative glycan profile analysis. We first investigated the reproducibility of the data for 16 N-glycans prepared from human epidermal growth factor receptor type 2 (HER2). All of the data obtained in intra-assays and interassays were highly correlated with statistical significance (R(2) > 0.9, p < 0.05). In addition, the HER2 glycosylation pattern differed significantly between different breast cancer cell lines SK-BR-3 and BT474 in a comparative analysis of profile data. Finally, the quantitative capability of this method was examined by using PA-labeled monosialylated N-glycan as an internal standard (IS). Using IS for AQ-labeled neutral and sialylated standard glycans, the ion peak intensity was highly linear (R(2) > 0.9) from 0.5 to 5000 fmol. Furthermore, using IS for HER2 N-glycans, all of the N-glycans were highly linear with their dilution factors (R(2) > 0.9). These results suggest that our developed AQ labeling method enabled rapid qualitative and quantitative analyses of glycans. This glycan analysis method should contribute to the field of biomarker discovery and biomedicine in applications such as quality control of biotechnology-based drugs.

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High‐Throughput Lectin Microarray‐Based Analysis of Live Cell Surface Glycosylation

Cited by 8 publications

References 19 publications

Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau

Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau

CHO cell line specific prediction and control of recombinant monoclonal antibody N‐glycosylation

Rapid Quantitative Profiling of N-Glycan by the Glycan-Labeling Method Using 3-Aminoquinoline/α-Cyano-4-hydroxycinnamic Acid

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