MSACL 2019 EU Abstract

Self-Classified Topic Area(s): Glycomics

Colorectal Cancer Cell Lines Reveal Striking Isomeric Diversity of Human Cancer O-Glycome

Katarina Madunic1, Stephanie Holst-Bernal1, Guinevere S.M. Lageveen-Kammeijer1, Tao Zhang1, Kathrin Stavenhagen1,2, Chunsheng Jin3, Oleg A. Mayboroda1, Niclas Karlsson3, Manfred Wuhrer1
1 Leiden University Medical Center, Center for Proteomics and Metabolomics, Postbus 9600, 2300 RC Leiden, The Netherlands 2 Beth Israel Deaconess Medical Center - Harvard Medical School, Boston 3 DepColorectal cancer cell lines reveal striking isomeric diversity of human cancer O-glycome artment of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg

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Katarina Madunic (Presenter)
LUMC

Presenter Bio: I have received my diploma in 2012. from Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia. During my studies was given an opportunity to do a master thesis project in Genos, Ltd the Europe’s leading glyco-analytical laboratory under supervision of Prof. Gordan Lauc. I worked on high throughput HILIC analysis of fluorescently labelled N-glycans, the project which launched me into the world of glycoscience.
After several years of professional experience in industry, in 2016. I have started my PhD project at Leiden University Medical Centre under supervision of Prof.Dr. Manfred Wuhrer focusing on exploring the glycosylation of colorectal cancer within Marie Curie GlycoCan training network.

Relevant Financial Disclosures (within past 24 months)

Grant/Research Support Marie Curie ITN grant EU

Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above.

Conflict mitigation NOT REQUIRED.

Abstract

INTRODUCTION: Over the past few years many studies have demonstrated the importance of altered glycosylation in tumor progression. These findings may contribute to discovery of biomarkers and treatment targets as well as understanding cancer biology. N-glycosylation of colorectal cancer (CRC) cell lines has been recently characterized revealing the association of antennary fucosylation with cell line differentiation and Caudal Type Homebox 1 (CDX1) expression. However, little is known about O-glycosylation of CRC cell lines due to its complexity, the presence of multiple isomeric structures as well as lack of enzymatic release methods making it overall challenging to perform in-depth analysis. OBJECTIVES: To provide a better understanding of the variation in O-glycosylation phenotypes and their association with other molecular features, an in-depth O-glycosylation analysis of 26 different colorectal cancer cell lines was performed.
METHODS: We have further optimized a high-throughput sample preparation procedure that now allows the release of O-glycans from only 0.5 million cells in a 96-well plate format. Proteins were immobilized on a PVDF membrane that allowed the release of N-glycans with PNGase F digestion prior to the O-glycan release via reductive β elimination. The samples, containing released O-glycans, were analysed on a sensitive nano-porous graphitized carbon liquid chromatography system coupled to an ion trap mass spectrometer via electrospray ionization (nano-PGC-LC-ESI-MS) allowing isomeric separation of the O-glycan species. Isomeric structures could be identified via their cross-ring and glycosidic fragment ions generated in negative ionization mode.
RESULTS: Striking differences were observed between the O-glycome of 26 colorectal cancer cell lines. We have detected many yet undescribed O-GalNAc linked glycans with a total of nearly 180 different structures. The profiles of all cell lines were dominated by sialylated glycan species formed by elongation of mainly core 2 and core 1 structures. Unsupervised principal component analysis based on structural glycan features revealed a separation between well differentiated colon-like and undifferentiated cell lines. Colon-like cell lines were mainly characterized by a prevalence of I-branched and sialyl Lewis X/A epitope carrying glycans, which showed correlation with the expression of relevant glycosyltransferases. In contrast, the majority of the undifferentiated cell lines showed absence of Lewis type epitope expression together with increased blood group antigen H, A and B expression.
CONCLUSION: Major differences were revealed between the O-glycosylation profiles of a panel of 26 colorectal cancer cell lines. The large biological variation observed within this study indicates that when studying glycobiological changes in cancer, cell line variation must be considered.