The surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell... Show moreThe surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell-extracellular matrix interactions. Cancer-induced changes in glycosylation have an impact on the function of major glycoproteins in the human colon, therefore studies focused on colorectal cancer (CRC)-specific glycosylation signatures can provide novel insights into onset and progression of this disease. The major focus of this thesis was to investigate mucin type O-glycosylation signatures of CRC. For this purpose, a protocol for in-depth analysis of N- and O-glycans obtained from cell lines was developed (Chapter 2) using nanoscale porous graphitized carbon liquid chromatography coupled to mass spectrometry (PGC-nano-LC-MS). In Chapter 3 additional conditions were optimized in the MS methodology by using polar protic dopant (methanol and isopropanol) enriched nitrogen gas to increase sensitivity on the MS and tandem MS level. In Chapter 4 we applied the methodology developed in Chapter 2 to the analysis of O-glycosylation signatures of 26 different CRC cell lines. This analysis resulted in the characterization of more than 150 O-glycan structures and increased our understanding of glycan expression in the analyzed cell lines. To gain further understanding in the mechanisms underlying glycomic changes with colon cell differentiation, we explored changes in the cell line glycome and proteome upon spontaneous and butyrate-stimulated differentiation in in vitro cell culture (Chapter 5). By performing an integrative approach, we generated hypotheses about glycosylation signatures of specific cell adhesion proteins, which may play an important role in cancer progression. The localization of glycans on the cell surface and their role in biological processes are important in cancer pathogenesis, making them potential candidates for glycan targeting immunotherapy. Therefore, we further optimized the methodology to enable comprehensive analysis of N- and O-glycans from specific regions of formalin-fixed, paraffin-embedded tissues using laser capture microdissections and applied it for the analysis of selected regions of CRC tissues and their patient-matched colon mucosa controls (Chapter 6). We identified specific tumor-associated carbohydrate antigens (TACAs) that show expression only in the tumor samples, with no or limited expression in the normal colon mucosa. Since TACAs are present in high abundance on the surface of cancer cells which are linked to many different proteins, these are very promising targets for the development of tumor-specific immunotherapy. Show less
Glycan modifications of proteins and lipids form an integral part of the cell’s outermost layer and an array of ligands, adding a high degree of complexity to the cellular phenotype. While... Show more Glycan modifications of proteins and lipids form an integral part of the cell’s outermost layer and an array of ligands, adding a high degree of complexity to the cellular phenotype. While this complexity is an analytical challenge, it also offers a wide range of opportunities for biomarkers and treatment targets. This thesis deals with the analysis of colorectal cancer (CRC)-associated glycomic changes. Current knowledge on CRC-associated glycan changes and their biological role have been reviewed in Chapter 1. In Chapters 2, 3, and 6, we developed novel, high-end methodologies for the glycomic analysis of tissues and cell lines to be able to expand our knowledge on cancer glycomics and to overcome some limitations of current techniques. By applying these new methods, this thesis also covers the characterization of changes in glycosylation in CRC tissues as well as cell lines, thereby contributing to the understanding of CRC biology while identifying cancer-specific signatures underlying CRC development. These signatures can be further explored as potential markers to improve patient care. Additionally, in Chapter 5, we extended our research to pancreatic duct adenocarcinoma and characterized the N-glycome of PDAC cells with different metastatic potential and of a normal pancreatic duct cell line. Show less
