In Depth O-Glycosylation Structural Analysis of Colorectal Cancer Cell Lines Katarina Madunic (Presenter) Leiden University Medical Center Presenter Bio: Katarina Madunic received her diploma in 2012. from Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia. She fell in love with glycans while working on master thesis project about high throughput HILIC analysis of fluorescently labelled N-glycans in Genos Ltd. Croatia. In August 2016. she started her PhD project at Leiden University Medical Centre under supervision of Prof.Dr. Manfred Wuhrer focusing on exploring the glycosylation of colorectal cancer within GlycoCan consortium.

Authors: Katarina Madunic1, Stephanie Holst-Bernal1, Kathrin Stavenhagen1,3, Chunsheng Jin2, Niclas Karlsson2, Manfred Wuhrer1
1 Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands 2 Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg 3 Beth Israel Deaconess Medical Center - Harvard Medical School, Boston

Altered glycosylation has been widely described in cancer. However, little is known about O- glycosylation due to the complexity of the analytical approaches. Many studies used single cell lines to investigate glycosylation changes in cancer without taking into account the differences between cell lines. During this study O-glycosylation signatures of 25 colorectal cancer cell lines were examined by nano-PGC-LC-ESI-MS, allowing the differentiation between isomeric structures and their association with different cell phenotypes. Our results have shown profound differences between O-glycomes of the cell lines revealing that single cell line studies may not provide representative results.

Introduction

Colorectal cancer is the second leading cause of cancer death in Europe (1). Altered glycosylation has been widely described in cancer and over the past few years many studies have demonstrated the importance of altered glycosylation in tumour progression (2). Although some studies have been performed on ex vivo tumor tissues (3), others have often used in vitro models such as cell lines to study molecular mechanisms underlying cancer onset and progression (4). While the correlation between primary tumors and cancer cell lines has been demonstrated on the genetic level (7), the potential to use them as a model system for cancer related glycobiological changes still needs to be evaluated. Recently, a study characterized the N-glycan profiles of different cell lines derived from primary tumors and metastatic sites, revealing that different N-glycans could be associated with different cell phenotypes (5). Unfortunately, little is known about O-glycosylation of cell lines due to its complexity, the presence of multiple isomeric structures as well as a complicated sample preparation procedure, making it overall very challenging. During this study, an in-depth O-glycosylation analysis of ten different colorectal cancer cell lines was performed, investigating their potential as glycobiological model systems with aim to provide a better understanding of the diversity between different cell lines, such as the variation in glycosylation phenotypes and their association with other molecular and phenotypic features.

Methods

We have optimized and applied a 96-well plate membrane based method (5) for preparation of released O-glycans from solely 0.5 million cells. Immobilization of proteins on a PVDF membrane allowed the release of N-glycans by PNGase F digestion followed by O-glycan release via reductive beta elimination (6). Samples were analyzed 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). PGC allowed isomeric separation of the O-glycan species and, in combination with negative mode fragmentation spectra, isomeric structures could be identified via their cross ring fragment ions.

Results

Until now we have processed ten different cell lines (HT29, HCT-8, SW620, SW480, HCT-116, C10, LS180, LS174T, LS411N, T84) and revealed a total of 80 different O-glycan structures. The oligosaccharides varied from 2 up to 14 residues and core 2 glycans were the main core structures in all the ten cell lines. Low amounts of core 3 and core 4 structures were detected in only few cell lines.The majority of the structures were sialylated, carrying both α2,6- and α2,3-linked N-acetylneuraminic acids next to very low amounts of sulphated O-glycans. We observed profound differences between the cell lines. The dominant glycan structures in low mucin producing cell lines were core 2 mono- or disialylated structures with in some cases attachment of a fucose residue. Structures carrying more than one fucose residue were dominating the spectra of the high mucin producing cell lines LS174T and LS180, often expressing sialyl-Lewis epitopes. These cell lines also expressed small amounts of sialyl-Tn antigen while the other cell lines did not reveal those at all.

Conclusions & Discussion

In conclusion, major differences were observed between the O-glycosylation profiles of the ten colorectal cancer cell lines suggesting that cell lines should be well characterized and selected before using a single cell line to study glycobiological changes in cancer. Furthermore, combining different cell lines could be beneficiary to tackle and simulate the tumor heterogeneity. This study will be expanded to a total of 25 colorectal cancer cell lines in order to evaluate their potential as glycobiological model systems.

(1) Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D.M., Forman, D. and Bray, F., 2015. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer, 136(5).

(2) Adamczyk, B., Tharmalingam, T. and Rudd, P.M., 2012. Glycans as cancer biomarkers. Biochimica et Biophysica Acta (BBA)-General Subjects, 1820(9), pp.1347-1353.

(3) Hinneburg, H., Korać, P., Schirmeister, F., Gasparov, S., Seeberger, P.H., Zoldoš, V. and Kolarich, D., 2017. Unlocking cancer glycomes from histopathological formalin-fixed and paraffin-embedded (FFPE) tissue microdissections. Molecular & Cellular Proteomics, 16(4), pp.524-536.

(4) Sethi, M.K., Thaysen-Andersen, M., Smith, J.T., Baker, M.S., Packer, N.H., Hancock, W.S. and Fanayan, S., 2013. Comparative N-glycan profiling of colorectal cancer cell lines reveals unique bisecting GlcNAc and α-2, 3-linked sialic acid determinants are associated with membrane proteins of the more metastatic/aggressive cell lines. Journal of proteome research, 13(1), pp.277-288.

(5) Holst, S., Deuss, A.J., van Pelt, G.W., van Vliet, S.J., Garcia-Vallejo, J.J., Koeleman, C.A., Deelder, A.M., Mesker, W.E., Tollenaar, R.A., Rombouts, Y. and Wuhrer, M., 2016. N-glycosylation profiling of colorectal cancer cell lines reveals association of fucosylation with differentiation and caudal type homebox 1 (CDX1)/Villin mRNA expression. Molecular & Cellular Proteomics, 15(1), pp.124-140.

(6) Jensen, P.H., Karlsson, N.G., Kolarich, D. and Packer, N.H., 2012. Structural analysis of N-and O-glycans released from glycoproteins. Nature protocols, 7(7), p.1299.

(7) Domcke, S., Sinha, R., Levine, D.A., Sander, C. and Schultz, N., 2013. Evaluating cell lines as tumour models by comparison of genomic profiles. Nature communications, 4, p.2126.