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Gastrointestinal (GI) cancer is the most common group of malignancies and many of its types are among the most deadly. Various glycoconjugates have been used in clinical practice as serum biomarker for several GI tumors, however, with limited diagnose application. Despite the good accessibility by endoscopy of many GI organs, the lack of reliable serum biomarkers often leads to late diagnosis of malignancy and consequently low 5-year survival rates. Recent advances in analytical techniques have provided novel glycoproteomic and glycomic data and generated functional information and putative biomarker targets in oncology. Glycosylation alterations have been demonstrated in a series of glycoconjugates (glycoproteins, proteoglycans, and glycosphingolipids) that are involved in cancer cell adhesion, signaling, invasion, and metastasis formation. In this review, we present an overview on the major glycosylation alterations in GI cancer and the current serological biomarkers used in the clinical oncology setting. We further describe recent glycomic studies in GI cancer, namely gastric, colorectal, and pancreatic cancer. Moreover, we discuss the role of glycosylation as a modulator of the function of several key players in cancer cell biology. Finally, we address several state-of-the-art techniques currently applied in this field, such as glycomic and glycoproteomic analyses, the application of glycoengineered cell line models, microarray and proximity ligation assay, and imaging mass spectrometry, and provide an outlook to future perspectives and clinical applications. ; We acknowledge the support from the European Union, Seventh Framework Programme, Gastric Glyco Explorer initial training network: grant number 316929. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (FCOMP-01-0124- FEDER028188) and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/ LA0003/2013, PTDC/BBB-EBI/0786/2012, and PTDC/BBBEBI/0567/2014. AM acknowledges the grant received from FCT, POPH (Programa Operacional Potencial Humano), and FSE (Fundo Social Europeu) (SFRH/BPD/75871/2011). MB acknowledges the University of Girona for pre-doctoral fellowship.

In the scenario of personalized medicine, targeted therapies are currently the focus of cancer drug development. These drugs can block the growth and spread of tumor cells by interfering with key molecules involved in malignancy, such as receptor tyrosine kinases (RTKs). MET and Recepteur d'Origine Nantais (RON), which are RTKs frequently overactivated in gastric cancer, are glycoprotein receptors whose activation have been shown to be modulated by the cellular glycosylation. In this work, we address the role of sialylation in gastric cancer therapy using an innovative 3D high-throughput cell culture methodology that mimics better the in vivo tumor features. We evaluate the response to targeted treatment of glycoengineered gastric cancer cell models overexpressing the sialyltransferases ST3GAL4 or ST3GAL6 by subjecting 3D spheroids to the tyrosine kinase inhibitor crizotinib. We show here that 3D spheroids of ST3GAL4 or ST3GAL6 overexpressing MKN45 gastric cancer cells are less affected by the inhibitor. In addition, we disclose a potential compensatory pathway via activation of the Insulin Receptor upon crizotinib treatment. Our results suggest that cell sialylation, in addition of being involved in tumor progression, could play a critical role in the response to tyrosine kinase inhibitors in gastric cancer. ; This research was funded by the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was also funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585 and POCI-01-0145-FEDER-007274); and National Funds through the Foundation for Science and Technology (FCT), under the projects: PTDC/BBB-EBI/0567/2014 and PTDC/BBB-ECT/2518/2014. We acknowledge the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 748880 awarded to MB. We thank ...

Cellular glycosylation plays a pivotal role in several molecular mechanisms controlling cell⁻cell recognition, communication, and adhesion. Thus, aberrant glycosylation has a major impact on the acquisition of malignant features in the tumor progression of patients. To mimic these in vivo features, an innovative high-throughput 3D spheroid culture methodology has been developed for gastric cancer cells. The assessment of cancer cell spheroids' physical characteristics, such as size, morphology and solidity, as well as the impact of glycosylation inhibitors on spheroid formation was performed applying automated image analysis. A detailed evaluation of key glycans and glycoproteins displayed by the gastric cancer spheroids and their counterpart cells cultured under conventional 2D conditions was performed. Our results show that, by applying 3D cell culture approaches, the model cell lines represented the differentiation features observed in the original tumors and the cellular glycocalix underwent striking changes, displaying increased expression of cancer-associated glycan antigens and mucin MUC1, ultimately better simulating the glycosylation phenotype of the gastric tumor. ; We acknowledge the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 748880 awarded to M.B. This work was funded by FEDER funds through the Operational Program for Competitiveness Factors—COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274) and National Funds through the Foundation for Science and Technology (FCT), under the projects: PTDC/BBB-EBI/0567/2014 (to CAR) and UID/BIM/04293/2013; and the project NORTE-01-0145-FEDER-000029, supported by the North Portugal Regional Program (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF).

Aberrant expression and hyperactivation of the human epidermal growth factor receptor 2 (ErbB2) constitute crucial molecular events underpinning gastric neoplastic transformation. Despite ErbB2 extracellular domain being a well-known target for glycosylation, its glycosylation profile and the molecular mechanisms through which it actively tunes tumorigenesis in gastric cancer (GC) cells remain elusive. We aimed at disclosing relevant ErbB2 glycan signatures and their functional impact on receptor's biology in GC cells. The transcriptomic profile of cancer-relevant glycosylation enzymes, and the expression and activation of the ErbB receptors were characterized in four GC cell lines. Cellular- and receptor-specific glycan profiling of ErbB2-overexpressing NCI-N87 cells unveiled a heterogeneous glycosylation pattern harboring the tumor-associated sialyl Lewis a (SLea) antigen. The expression of SLea and key enzymes integrating its biosynthetic pathway were strongly upregulated in this GC cell line. An association between the expression of ERBB2 and FUT3, a central gene in SLea biosynthesis, was disclosed in GC patients, further highlighting the crosstalk between ErbB2 and SLea expression. Moreover, cellular deglycosylation and CA 19.9 antibody-mediated blocking of SLea drastically altered ErbB2 expression and activation in NCI-N87 cells. Altogether, NCI-N87 cell line constitutes an appealing in vitro model to address glycan-mediated regulation of ErbB2 in GC. ; This work was funded by the project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through COMPETE 2020—Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia)/Ministério da Ciência, Tecnologia e Inovação in the framework of the project "Institute for Research and Inovation in Health Sciences" (POCI-01-0145-FEDER-007274), the project POCI-01-0145-FEDER-016585 (PTDC/BBB EBI/0567/2014), and EU 7th framework programme ITN 316929. Mass spectrometry was performed at the Proteomics i3S Scientific Platform. Microscopy images were obtained at the Advance Light Microscopy i3S Scientific Platform. Henrique O. Duarte was supported by FCT through the FCT PhD Programmes and by Programa Operacional Potencial Humano (POPH), specifically by the BiotechHealth Programme (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), with the reference PD/0016/2012 funded by FCT. Henrique O. Duarte received an individual grant (PD/BI/113948/2015) and a PhD studentship (PD/BD/128407/2017). Meritxell Balmaña has received a funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 748880.

Gastric carcinoma MKN45 cells stably transfected with the full-length ST3GAL4 gene were characterised by glycomic and sialoproteomic analysis. Complementary strategies were applied to assess the glycomic alterations induced by ST3GAL4 overexpression. The N- and O-glycome data were generated in two parallel structural analyzes, based on PGC-ESI-MS/MS. Data on glycan structure identification and relative abundance in ST3GAL4 overexpressing cells and respective mock control are presented. The sialoproteomic analysis based on titanium-dioxide enrichment of sialopeptides with subsequent LC-MS/MS identification was performed. This analysis identified 47 proteins with significantly increased sialylation. The data in this article is associated with the research article published in Biochim Biophys Acta "Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer" [1]. ; We acknowledge the support from the European Union, Seventh Framework Programme, Gastric Glyco Explorer Initial Training Network: Grant number 316929. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (FCOMP-01-0124-FEDER028188) and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/LA0003/2013, PTDC/BBB-EBI/0786/2012, PTDC/BBB-EBI/0567/2014 (CR). This work was also supported by "Glycoproteomics" project Grant number PCIG09-GA-2011-293847 (to DK) and the Danish Natural Science Research Council and a generous Grant from the VILLUM Foundation to the VILLUM Center for Bioanalytical Sciences at the University of Southern Denmark (to MRL). AM acknowledges FCT, POPH (Programa Operacional Potencial Humano) and FSE (Fundo Social Europeu) (SFRH/BPD/75871/2011). The UPLC instrument was obtained with a grant from the Ingabritt and Arne Lundbergs Research Foundation. C.J. was supported by the Knut and Alice Wallenberg Foundation. The mass spectrometer (LTQ) was obtained by a grant from the Swedish Research Council (342-2004-4434).

Background: Changes in glycosylation are known to play critical roles during gastric carcinogenesis. Expression of truncated O-glycans, such as the Sialyl-Tn (STn) antigen, is a common feature shared by many cancers and is associated with cancer aggressiveness and poor-prognosis. Methods: Glycoengineered cell lines were used to evaluate the impact of truncated O-glycans in cancer cell biology using in vitro functional assays, transcriptomic analysis and in vivo models. Tumor patients 'samples and datasets were used for clinical translational significance evaluation. Findings: In the present study, we demonstrated that gastric cancer cells expressing truncated O-glycans display major phenotypic alterations associated with higher cell motility and cell invasion. Noteworthy, the glycoengineered cancer cells overexpressing STn resulted in tumor xenografts with less cohesive features which had a critical impact on mice survival. Furthermore, truncation of O-glycans induced activation of EGFR and ErbB2 receptors and a transcriptomic signature switch of gastric cancer cells. The disclosed top activated genes were further validated in gastric tumors, revealing that SRPX2 and RUNX1 are concomitantly overexpressed in gastric carcinomas and its expression is associated with patients' poor-survival, highlighting their prognosis potential in clinical practice. Interpretation: This study discloses novel molecular links between O-glycans truncation frequently observed in cancer and key cellular regulators with major impact in tumor progression and patients' clinical outcome. ; This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE ( POCI-01-0145-FEDER-016585 ; POCI-01-0145-FEDER-007274 ; POCI-01-0145-FEDER-028489 ) and National Funds through the Foundation for Science and Technology (FCT) , under the projects: PTDC/BBB-EBI/0567/2014 (to CAR), PTDC/MED-ONC/28489/2017 (to AM) and UID/BIM/04293/2013 ; and the project NORTE-01-0145-FEDER-000029 , supported by Norte Portugal Regional Programme ( NORTE 2020 ), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The authors acknowledge the support by Gastric Glyco Explorer Initial Training Network (European Union Seventh Framework Programme GastricGlycoExplorer project, grant number 316929 ). DF acknowledges the FCT PhD Programmes and Programa Operacional Potencial Humano (POPH), specifically the BiotechHealth Programe (Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences), with the reference PD/0016/2012 funded by FCT . Grants were received from FCT, POPH and FSE (Fundo Social Europeu): SFRH/BD/110636/2015 to DF and SFRH/BPD/115730/2016 to FP. We thank Catharina Steentoft and Henrik Clausen for the cell line models and helpful scientific discussions. We are grateful for Nuno Mendes's technical support. The authors acknowledge the support of José Luis Costa and Mafalda Rocha from the i3S Genomics Platform (GenCore), and Maria G Lazaro from the Bioimaging I3S Scientific Platform , member of the PPBI ( PPBI-POCI-01-0145-FEDER-022122 ).

Here, we introduce a novel scFv antibody, G2-D11, specific for two adjacent Tn-antigens (GalNAc- Ser/Thr) binding equally to three dimeric forms of the epitope, Ser-Thr, Thr-Thr and Thr-Ser. Compared to other anti-Tn reagents, the binding of G2-D11 is minimally influenced by the peptide structure, which indicates a high degree of carbohydrate epitope dominance and a low influence from the protein backbone. With a high affinity (K Dapp = 1.3 × 10 -8 M) and no cross-reactivity to either sialyl-Tn epitope or blood group A antigens, scFv G2-D11 is an excellent candidate for a well-defined anti-Tn-antigen reagent. Detailed immunohistochemical evaluation of tissue sections from a cohort of 80 patients with gastric carcinoma showed in all cases positive tumor cells. The observed staining was localized to the cytoplasm and in some cases to the membrane, whereas the surrounding tissue was completely negative demonstrating the usefulness of the novel Tn-antigen binding antibody. ; This work was supported by the European Union, Seventh Framework Programme, GastricGlycoExplorer (GGE) initial training network [grant number 316929 to OB]; The Danish Research Council [grant number DFF-4005-00285 to OB]; Innovationsfonden in the framework of the EU-ERAsynBio-SynGlycTis project to OB; The EU-IMI 2 Joint Undertaking project EbolaMoDRAD [grant number 115843 to OB]; grants from Mrs. Berta Kamprad's Foundation to CW; FEDER funds through the Operational Programme for Competitiveness Factors COMPETE [grant number FCOMP-01-0124-FEDER028188 to CR] and National Funds through FCT projects [grant numbers PEst-C/SAU/LA0003/2013, PTDC/BBB-EBI/0567/2014 to CR].

Background Terminal a2-3 and a2-6 sialylation of glycans precludes further chain elongation, leading to the biosynthesis of cancer relevant epitopes such as sialyl-Lewis X (SLe X ). SLe X overexpression is associated with tumor aggressive phenotype and patients' poor prognosis. Methods MKN45 gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry. We further validated an identified target expression by proximity ligation assay in gastric tumors. Results Our results showed that ST3GAL4 overexpression leads to several glycosylation alterations, including reduced O-glycan extension and decreased bisected and increased branched N-glycans. A shift from a2-6 towards a2-3 linked sialylated N-glycans was also observed. Sialoproteomic analysis further identified 47 proteins with significantly increased sialylated N-glycans. These included integrins, insulin receptor, carcinoembryonic antigens and RON receptor tyrosine kinase, which are proteins known to be key players in malignancy. Further analysis of RON confirmed its modification with SLe X and the concomitant activation. SLe X and RON co-expression was validated in gastric tumors. Conclusion The overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. General significance This study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer patients' stratification. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. ; We acknowledge the support from the European Union, Seventh Framework Programme, Gastric Glyco Explorer initial training network: grant number 316929. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (FCOMP-01-0124-FEDER028188) and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/LA0003/2013, PTDC/BBB-EBI/0786/2012, and PTDC/BBB-EBI/0567/2014 (to CAR). This work was also supported by"Glycoproteomics" project grant number PCIG09-GA-2011-293847(to DK) and the Danish Natural Science Research Council and a generous grant from the VILLUM Foundation to the VILLUM Center for Bioanalytical Sciences at the University of Southern Denmark (to MRL). Grants were received from FCT, POPH (Programa Operacional Potencial Humano) and FSE (Fundo Social Europeu): SFRH/BPD/75871/2011 to AM; SFRH/BPD/111048/2015 to JAF; SFRH/BPD/96510/2013 to CG. The UPLC instrument was obtained with a grant from the Ingabritt and Arne Lundbergs Research Foundation (to NK). C.J. was supported by the Knut and Alice Wallenberg Foundation. The mass spectrometer (LTQ) was obtained by a grant from the Swedish Research Council (342-2004-4434) (to NK).