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The Cost Action “Innovation with glycans: new frontiers from synthesis to new biological targets” (INNOGLY) hosted the Workshop “Neuroglycoproteins in health and disease”, in Alicante, Spain, on March 2022. This event brought together an european group of scientists that presented novel insights into changes in glycosylation in diseases of the central nervous system and cancer, as well as new techniques to study protein glycosylation. Herein we provide the abstracts of all the presentations., This work has been funded by “Fundació La Marató de TV3” (Grant 201922-30-31) and by Roche Diagnostics (Barcelona, Spain; Grant IDI-20170423). This workshop was funded by the INNOGLY Cost (CA18103).

The clinical performance of the therapeutic monoclonal antibody trastuzumab in the treatment of ErbB2-positive unresectable gastric cancer (GC) is severely hampered by the emergence of molecular resistance. Trastuzumab’s target epitope is localized within the extracellular domain of the oncogenic cell surface receptor tyrosine kinase (RTK) ErbB2, which is known to undergo extensive N-linked glycosylation. However, the site-specific glycan repertoire of ErbB2, as well as the detailed molecular mechanisms through which specific aberrant glycan signatures functionally impact the malignant features of ErbB2-addicted GC cells, including the acquisition of trastuzumab resistance, remain elusive. Here, we demonstrate that ErbB2 is modified with both α2,6- and α2,3-sialylated glycan structures in GC clinical specimens. In-depth mass spectrometry-based glycomic and glycoproteomic analysis of ErbB2’s ectodomain disclosed a site-specific glycosylation profile in GC cells, in which the ST6Gal1 sialyltransferase specifically targets ErbB2 N-glycosylation sites occurring within the receptor’s trastuzumab-binding domain. Abrogation of ST6Gal1 expression reshaped the cellular and ErbB2-specific glycomes, expanded the cellular half-life of the ErbB2 receptor, and sensitized ErbB2-dependent GC cells to trastuzumab-induced cytotoxicity through the stabilization of ErbB dimers at the cell membrane, and the decreased activation of both ErbB2 and EGFR RTKs. Overall, our data demonstrates that ST6Gal1-mediated aberrant α2,6-sialylation actively tunes the resistance of ErbB2-driven GC cells to trastuzumab.

SARS-CoV-2 causes the respiratory syndrome COVID-19 and is responsible for the current pandemic. The S protein of SARS-CoV-2-mediating virus binding to target cells and subsequent viral uptake is extensively glycosylated. Here we focus on how glycosylation of both SARS-CoV-2 and target cells crucially impacts SARS-CoV-2 infection at different levels: (1) virus binding and entry to host cells, with glycosaminoglycans of host cells acting as a necessary co-factor for SARS-CoV-2 infection by interacting with the receptor-binding domain of the SARS-CoV-2 spike glycoprotein, (2) innate and adaptive immune response where glycosylation plays both a protective role and contributes to immune evasion by masking of viral polypeptide epitopes and may add to the cytokine cascade via non-fucosylated IgG, and (3) therapy and vaccination where a monoclonal antibody-neutralizing SARS-CoV-2 was shown to interact also with a distinct glycan epitope on the SARS-CoV-2 spike protein. These evidences highlight the importance of ensuring that glycans are considered when tackling this disease, particularly in the development of vaccines, therapeutic strategies and serological testing.

The present work reports on a 3D model of the tumor microenvironment that contains hyaluronic acid (HA) and alginate, and demonstrates the utility of this model to study the effect of HA size on the crosstalk between cancer cells and mesenchymal stem cells (MSCs). The system incorporates a core that contains HA of specific size (i.e. 6.4, 741 or 1500 kDa) with encapsulated epithelial MKN45 cancer cells and a shell with MSCs that mimic the presence of stem cells next to the tumor site. It was found that short HA (i.e. 6.4 kDa) promotes the invasion of cancer cells from the core to the shell, whereas longer HA (i.e. 741 and 1500 kDa) recruits the MSCs into the core, i.e. the tumor site, where a reduction of the formation of cancer cell aggregates was observed. In summary, the developed 3D model recapitulates some key tumor features related to the effect of HA size on both cancer cell invasiveness and MSC behavior at the tumor site., We acknowledge the financial support from the European Commission and the Horizon 2020 - WIDESPREAD programme, under the grant agreement number #668983-FORECAST. SA acknowledges the Portuguese Foundation for Science and Technology for her PhD grant (SFRH/BD/112075/2015).

With the advent of personalized medicine, there is a movement to develop “smaller” and “smarter” microdevices that are able to distinguish similar cancer subtypes. Tumor cells display major differences when compared to their natural counterparts, due to alterations in fundamental cellular processes such as glycosylation. Glycans are involved in tumor cell biology and they have been considered to be suitable cancer biomarkers. Thus, more selective cancer screening assays can be developed through the detection of specific altered glycans on the surface of circulating cancer cells. Currently, this is only possible through time-consuming assays. In this work, we propose the “intelligent” Lab on Fiber (iLoF) device, that has a high-resolution, and which is a fast and portable method for tumor single-cell type identification and isolation. We apply an Artificial Intelligence approach to the back-scattered signal arising from a trapped cell by a micro-lensed optical fiber. As a proof of concept, we show that iLoF is able to discriminate two human cancer cell models sharing the same genetic background but displaying a different surface glycosylation profile with an accuracy above 90% and a speed rate of 2.3 seconds. We envision the incorporation of the iLoF in an easy-to-operate microchip for cancer identification, which would allow further biological characterization of the captured circulating live cells. This work was partially funded by the projects NanoSTIMA and NORTE-01-0145-FEDER-000029, both supported by the North Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and through the European Regional Development Fund (ERDF); and by the Portuguese Foundation for Science and Technology, within the scope of the PhD grant PD/BD/135023/2017 and the projects: PTDC/BBB-EBI/0567/2014 (to CAR) and UID/BIM/04293/2013. It was also funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; PPBI-POCI-01-0145-FEDER-022122). MB acknowledges the Marie Sklodowska-Curie grant agreement No. 748880.

The incidence of gastrointestinal pathologies (cancer in particular) has increased progressively, with considerable morbidity and mortality, and a high economic impact on the healthcare system. The dietary intake of natural phytochemicals with certain bioactive properties has shown therapeutic and preventive effects on these pathologies. This includes the cruciferous vegetable derivative phenylethyl isothiocyanate (PEITC), a bioactive compound present in some vegetables, such as watercress. Notably, PEITC has antioxidant, anti-inflammatory, bactericidal, and anticarcinogenic properties. This review summarized the current knowledge on the role of PEITC as a potential natural nutraceutical or an adjuvant against oxidative/inflammatory-related disorders in the gastrointestinal tract. We also discussed the safe and recommended dose of PEITC. In addition, we established a framework to guide the research and development of sustainable methodologies for obtaining and stabilizing this natural molecule for industrial use. With PEITC, there is great potential to develop a viable strategy for preventing cancer and other associated diseases of the gastrointestinal tract. However, this topic still needs more scientific studies to help develop new PEITC products for the nutraceutical, pharmaceutical, or food industries.

Although the ErbB receptors remain incontrovertible drivers of human neoplastic transformation, the clinical performance of ErbB-directed therapeutics is significantly undermined by the emergence of molecular resistance. The ErbB extracellular region undergoes extensive post-translational glycosylation, which crucially impacts receptor structure, functionality, and therapeutic response, thereby hindering efforts towards the successful translation of such molecular insights into the clinical setting. The unraveling of the ErbB site-specific glycome will allow for the design of more efficient ErbB-directed therapeutic strategies capable of circumventing molecular resistance, the establishment of novel prognostic and predictive clinical biomarkers supporting improved patient stratification, and the rational guidance of therapeutic decisions.

KRAS mutations are one of the most frequent oncogenic mutations of all human cancers, being more prevalent in pancreatic, colorectal, and lung cancers. Intensive efforts have been encouraged in order to understand the effect of KRAS mutations, not only on tumor cells but also on the dynamic network composed by the tumor microenvironment (TME). The relevance of the TME in cancer biology has been increasing due to its impact on the modulation of cancer cell activities, which can dictate the success of tumor progression. Here, we aimed to clarify the pro- and anti-inflammatory role of KRAS mutations over the TME, detailing the context and the signaling pathways involved. In this review, we expect to open new avenues for investigating the potential of KRAS mutations on inflammatory TME modulation, opening a different vision of therapeutic combined approaches to overcome KRAS-associated therapy inefficacy and resistance in cancer., This work was financed by the Portuguese Foundation for Science and Technology (Fundacao para a Ciencia e a Tecnologia, FCT) within the scope of two PhD grants, SFRH/BD/140137/2018 attributed to FP and SFRH/BD/142486/2018 attributed to AF. A.P. acknownledges FCT within the scope of the project PTDC/QUIQIN/28662/2017. A.P. and M.J.S. also acknowledge the support of the CBMA strategic programme "Contrato-Programa" UIDB/04050/2020 funded by national funds through the FCT I.P. C.A.R. acknowledges the project Norte-01-0145-FEDER-000051-"Cancer Research on Therapy Resistance: From Basic Mechanisms to Novel Targets", supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). M.J.O. acknowledges FEDER funds through COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, Portuguese funds through FCT/Ministerio da Ciencia, Tecnologia e do Ensino Superior in the framework of the project POCI-01-0145-FEDER-31859/2017.

Helicobacter pylori infects half of the world population, being associated with several gastric disorders, such as chronic gastritis and gastric carcinoma. The Helicobacter genus also includes other gastric helicobacters, such as H. heilmannii¸ H. ailurogastricus, H. suis, H. felis, H. bizzozeronii, and H. salomonis. These gastric helicobacters colonize both the human and animal stomach. The prevalence of gastric non-Helicobacter pylori Helicobacter (NHPH) species in humans has been described as low, and the in vitro binding to the human gastric mucosa was never assessed. Herein, human gastric tissue sections were used for the evaluation of the tissue glycophenotype and for the binding of gastric NHPH strains belonging to different species. Histopathological evaluation showed that 37.5% of the patients enrolled in our cohort presented chronic gastritis, while the presence of neutrophil or eosinophilic activity (chronic active gastritis) was observed in 62.5% of the patients. The secretor phenotype was observed in 68.8% of the individuals, based on the expression of Lewis B antigen and binding of the UleX lectin. The in vitro binding assay showed that all the NHPH strains evaluated were able to bind, albeit in low frequency, to the human gastric mucosa. The H. heilmannii, H. bizzozeronii, and H. salomonis strains displayed the highest binding ability both to the gastric superficial epithelium and to the deep glands. Interestingly, we observed binding of NHPH to the gastric mucosa of individuals with severe chronic inflammation and intestinal metaplasia, suggesting that NHPH binding may not be restricted to the healthy gastric mucosa or slight chronic gastritis. Furthermore, the in vitro binding of NHPH strains was observed both in secretor and non-secretor individuals in a similar frequency. In conclusion, this study is the first report of the in vitro binding ability of gastric NHPH species to the human gastric mucosa. The results suggest that other glycans, besides the Lewis antigens, could be involved in the bacterial adhesion mechanism; however, the molecular intervenients remain unknown.

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells' glycocalyx and extracellular matrix. By acting as scaffolds for protein-protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell-extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4 cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell–cell communications and in the regulation of immune responses. Through the interaction with glycan‐binding receptors, glycans are able to affect the activation status of antigen‐presenting cells, leading either to induction of pro‐inflammatory responses or to suppression of immunity and instigation of immune tolerance. This unique feature of glycans has attracted the interest and spurred collaborations of glyco‐chemists and glyco‐immunologists to develop glycan‐based tools as potential therapeutic approaches in the fight against diseases such as cancer and autoimmune conditions. In this review, we highlight emerging advances in this field, and in particular, we discuss on how glycan‐modified conjugates or glycoengineered cells can be employed as targeting devices to direct tumor antigens to lectin receptors on antigen‐presenting cells, like dendritic cells. In addition, we address how glycan‐based nanoparticles can act as delivery platforms to enhance immune responses. Finally, we discuss some of the latest developments in glycan‐based therapies, including chimeric antigen receptor (CAR)‐T cells to achieve targeting of tumor‐associated glycan‐specific epitopes, as well as the use of glycan moieties to suppress ongoing immune responses, especially in the context of autoimmunity., Glycans are macromolecules that perform crucial roles in development, in cell–cell communication, and in the regulation of immune responses. In this review, we highlight how glycans can be exploited and empowered as treatment or vaccine modalities, particularly in the fight against cancer and autoimmune diseases.

This review provides insights on the impact of glycosylation in cancer biology and its influence in the current approaches of targeted cancer therapies in the clinical setting. The roles of glycosylation in cancer signaling, tumor progression, and metastasis are reviewed as well as glycans and glycan-binding proteins in tumor immunomodulation. Moreover, the latest reports on glycans influencing targeted therapeutic approaches in cancer are summarized. Finally, we discuss the future challenges of the field, outlining potential applications of glycan-based biomarkers for patient stratification and strategies for improving personalized cancer treatment.

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 ).

Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies. This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors COMPETE 2020 (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 C.A.R 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).

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen-specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency virus or to induce cellular T-cell immunity in the fight against cancer. The recent SARS-CoV-2 outbreak has reinforced the relevance of designing smart therapeutic vaccine modalities to ensure public health. Indeed, academic and private companies have ongoing joint efforts to develop novel vaccine prototypes for this virus. Many pathogens are covered by a dense glycan-coat, which form an attractive target for vaccine development. Moreover, many tumor types are characterized by altered glycosylation profiles that are known as “tumor-associated carbohydrate antigens”. Unfortunately, glycans do not provoke a vigorous immune response and generally serve as T-cell-independent antigens, not eliciting protective immunoglobulin G responses nor inducing immunological memory. A close and continuous crosstalk between glycochemists and glycoimmunologists is essential for the successful development of efficient immune modulators. It is clear that this is a key point for the discovery of novel approaches, which could significantly improve our understanding of the immune system. In this review, we discuss the latest advancements in development of vaccines against glycan epitopes to gain selective immune responses and to provide an overview on the role of different immunogenic constructs in improving glycovaccine efficacy.

In an era when cancer glycobiology research is exponentially growing, we are witnessing a progressive translation of the major scientific findings to the clinical practice with the overarching aim of improving cancer patients’ management. Many mechanistic cell biology studies have demonstrated that heparan sulfate (HS) glycosaminoglycans are key molecules responsible for several molecular and biochemical processes, impacting extracellular matrix properties and cellular functions. HS can interact with a myriad of different ligands, and therefore, hold a pleiotropic role in regulating the activity of important cellular receptors and downstream signalling pathways. The aberrant expression of HS glycan chains in tumours determines main malignant features, such as cancer cell proliferation, angiogenesis, invasion and metastasis. In this review, we devote particular attention to HS biological activities, its expression profile and modulation in cancer. Moreover, we highlight HS clinical potential to improve both diagnosis and prognosis of cancer, either as HS-based biomarkers or as therapeutic targets. This work was financed by FEDER-Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalization (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 Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and by the grant POCI-01-0145-FEDER-028489 (to A.M.). J.P. and C.M. are funded by FCT PhD scholarships SFRH/BD/137319/2018 and 2020.06412.BD, respectively. The APC was funded by FCT grant POCI-01-0145-FEDER-028489. The authors acknowledge the support of the COST Action CA18103 INNOGLY.

Aberrant cell surface glycosylation signatures are currently known to actively drive the neoplastic transformation of healthy cells. By disrupting the homeostatic functions of their protein carriers, cancer-associated glycans mechanistically underpin several molecular hallmarks of human malignancy. Furthermore, such aberrant glycan structures play key roles in the acquisition of molecular resistance to targeted therapeutic agents, which compromises their clinical efficacy, by modulating tumour cell aggressiveness and supporting the establishment of an immunosuppressive microenvironment. Recent advances in the study of the tumour cell glycoproteome have unravelled previously elusive molecular mechanisms of therapeutic resistance, guided the rational design of novel personalized therapeutic strategies, and may further improve the clinical performance of currently approved anti-cancer targeted agents. In this review, we highlight the impact of glycosylation in cancer targeted therapy, with particular focus on receptor tyrosine kinase-targeted therapy, immune checkpoints blockade therapy, and current developments on therapeutic strategies directed to glycan-binding proteins and other innovative glycan therapeutic strategies.

Microsatellite instability (MSI) is a molecular phenotype due to a deficient DNA mismatch repair (dMMR). In colorectal cancer (CRC), dMMR/MSI is associated with several clinical and histopathological features, influences prognosis, and is a predictive factor of response to therapy. In daily practice, dMMR/MSI profiles are identified by immunohistochemistry and/or multiplex PCR. The Thomsen&ndash, Friedenreich (TF) antigen was previously found to be a potential single marker to identify MSI-high gastric cancers. Therefore, in this study, we aimed to disclose a possible association between TF expression and MSI status in CRC. Furthermore, we evaluated the relationship between TF expression and other clinicopathological features, including patient survival. We evaluated the expression of the TF antigen in a cohort of 25 MSI-high and 71 microsatellite stable (MSS) CRCs. No association was observed between the expression of the TF antigen and MSI-high status in CRC. The survival analysis revealed that patients with MSI-high CRC showed improved survival when the TF antigen was expressed. This finding holds promise as it indicates the potential use of the TF antigen as a biomarker of better prognosis in MSI-high CRCs that should be validated in an independent and larger CRC cohort.

Thanks are due to the University of Aveiro and FCT - Fundacao para a Ciencia e a Tecnologia/MCT (Portugal) for the financial support for the QOPNA Research Unit (UID/QUI/00062/2019), LAQV-REQUIMTE (UIDB/50006/2020), CICECO - Aveiro Institute of Materials (UIDB/50011/2020+UIDP/50011/2020), CESAM (UIDB/50017/2020+UIDP/50017/2020) and RNEM (LISBOA-01-0145-FEDER-402-022125), and LEPABE (UIDB/00511/2020) through national founds and, where applicable, co-financed by the FEDER - Fundo Europeu de Desenvolvimento Regional, within the PT2020 Partnership Agreement, and to the Portuguese NMR Network; and the Applied Molecular Biosciences UnitUCIBIO which is financed by national funds from FCT (UIDB/04378/2020). This work was also supported by project grants from FCT (EXPL/BBB-BQB/0750/2012 and PTDC/BIA-MIB/31730/2017), a Biomedical Resource grant from Wellcome Trust (WT099197MA); and FCT individual grants to LMS (SFRH/BD/71455/2010), VGC (PD/BD/105727/2014), ASPM (SFRH/BD/80553/2011), AM (FRH/BPD/75871/2011), FCT researcher positions under the Individual Call to Scientific Employment Stimulus 2007 call to JAF (CEECIND/03186/2017) and to RMF (CEECIND/01854/2017), and FCT Investigator (GN12IF/00033/2012 GN212) to ASP. Made available in DSpace on 2021-10-09T04:54:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-02-01 publishersversion published

The epidermal growth factor receptor (EGFR) is a key protein involved in cancer development. Monoclonal antibodies targeting EGFR are approved for the treatment of metastatic colorectal cancer (CRC). Despite the beneficial clinical effects observed in subgroups of patients, the acquisition of resistance to treatment remains a major concern. Protein N-glycosylation of cellular receptors is known to regulate physiological processes leading to activation of downstream signaling pathways. In the present study, the role of EGFR-specific terminal ⍺2,6-sialylation was analyzed in modulation of the malignant phenotype of CRC cells and their resistance to monoclonal antibody Cetuximab-based therapy.Glycoengineered CRC cell models with specific sialyltransferase ST6GAL1 expression levels were applied to evaluate EGFR activation, cell surface glycosylation and therapeutic response to Cetuximab.Glycoproteomic analysis revealed EGFR as a major target of ST6Gal1-mediated ⍺2,6-sialylation in a glycosite-specific manner. Mechanistically, CRC cells with increased ST6Gal1 expression and displaying terminal ⍺2,6-sialylation showed a marked resistance to Cetuximab-induced cytotoxicity. Moreover, we found that this resistance was accompanied by downregulation of EGFR expression and its activation.Our data indicate that EGFR ⍺2,6-sialylation is a key factor in modulating the susceptibility of CRC cells to antibody targeted therapy, thereby disclosing a potential novel biomarker and providing key molecular information for tailor made anti-cancer strategies.

With the advent of personalized medicine, there is a movement to develop "smaller" and "smarter" microdevices that are able to distinguish similar cancer subtypes. Tumor cells display major differences when compared to their natural counterparts, due to alterations in fundamental cellular processes such as glycosylation. Glycans are involved in tumor cell biology and they have been considered to be suitable cancer biomarkers. Thus, more selective cancer screening assays can be developed through the detection of specific altered glycans on the surface of circulating cancer cells. Currently, this is only possible through time-consuming assays. In this work, we propose the "intelligent" Lab on Fiber (iLoF) device, that has a high-resolution, and which is a fast and portable method for tumor single-cell type identification and isolation. We apply an Artificial Intelligence approach to the back-scattered signal arising from a trapped cell by a micro-lensed optical fiber. As a proof of concept, we show that iLoF is able to discriminate two human cancer cell models sharing the same genetic background but displaying a different surface glycosylation profile with an accuracy above 90% and a speed rate of 2.3 seconds. We envision the incorporation of the iLoF in an easy-to-operate microchip for cancer identification, which would allow further biological characterization of the captured circulating live cells.

Hyaluronic acid (HA) has a key role in cancer progression. The HA's molecular weight (Mw) is altered in this pathological state: increased concentration of shorter fragments due to the overexpressed hyaluronidases and ROS. Aiming to mimic this microenvironment, we developed a Layer-by-Layer (LbL) platform presenting HA of different Mws, namely 6.4, 752 and 1500 kDa, to study the influence of HA Mw on the formation of focal adhesion sites (FAs), and the involvement of paxillin and CD44 in this process. High paxillin expression and formation of FAs, via CD44, is observed for MKN45 cells seeded on LbLs presenting HA 6.4 kDa, with the activation of the ERK1/2 pathway, responsible for cell motility and tumour progression. In contrast, activation of p38 pathway, usually related with cancer latency, is observed for cells seeded on LbLs with high Mw HA, i.e. 1500 kDa. Overall, we demonstrate the suitability of the developed platform to study cancer invasiveness.

Cancer is a deadly disease that encompasses numerous cellular modifications. Among them, alterations in glycosylation are a proven reliable hallmark of cancer, with most biomarkers used in the clinic detecting cancer-associated glycans. Despite their clear potential as therapy targets, glycans have been overlooked in drug discovery strategies. The complexity associated with the glycosylation process, and lack of specific methodologies to study it, have long hampered progress. However, recent advances in new methodologies, such as glycoengineering of cells and high-throughput screening (HTS), have opened new avenues of discovery. We envision that glycan-based targeting has the potential to start a new era of cancer therapy. In this article, we discuss the promise of cancer-associated glycosylation for the discovery of effective cancer drugs.

We report on the development of layer-by-layer (LbL) constructs whose viscoelastic properties and bioactivity can be finely tuned by using polyanions of different size and/or crosslinking. As a polyanion we used hyaluronic acid (HA) â a multi-signaling biomolecule whose bioactivity depends on its molecular weight. We investigated the interplay between the mechanical properties of the LbL systems built using HA of different sizes and the specific HA-mediated biochemical interactions. We characterized the assembled materials and their interactions with CD44, the main HA receptor, by Quartz Crystal Microbalance with Dissipation (QCM-D), Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM). We observed that the presence of CD44 resulted in the disruption of the non-crosslinked multilayers, while crosslinked films remain stable and bind CD44 in a HA molecular weight and charge specific fashion., The authors acknowledge the financial support from the European Commission’s H2020 programme, under grant agreements H2020- WIDESPREAD-2014-668983-FORECAST and H2020-WIDESPREAD01-2016-2017-739572-THE DISCOVERIES CTR. S. A. acknowledges the Portuguese Foundation for Science and Technology (FCT) for her PhD grant (SFRH/BD/112075/2015)

CD44 variant isoforms are often upregulated in cancer and associated with increased aggressive tumor phenotypes. The CD44v9 is one of the major protein splice variant isoforms expressed in human gastrointestinal cancer cells. Immunodetection of CD44 isoforms like CD44v9 in tumor tissue is almost exclusively performed by using specific monoclonal antibodies. However, the structural variability conferred by both the alternative splicing and CD44 protein glycosylation is disregarded. In the present work, we have evaluated the role of O-glycosylation using glycoengineered gastric cancer models in the detection of CD44v9 by monoclonal antibodies. We demonstrated, using different technical approaches, that the presence of immature O-glycan structures, such as Tn and STn, enhance CD44v9 protein detection. These findings can have significant implications in clinical applications mainly at the detection and targeting of this cancer-related CD44v9 isoform and highlight the utmost importance of considering glycan structures in cancer biomarker detection and in therapy targeting. This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE, grant numbers POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; OCI-01-0145-FEDER-031028; and national funds through the Foundation for Science and Technology (FCT), grant numbers PTDC/BBB-EBI/0567/2014 (to CAR), UID/BIM/04293/2013, and PTDC/MED-QUI/29780/2017; 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). F. Pinto received a fellowship from FCT (SFRH/BPD/115730/2016).

Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.

Triple negative breast cancer (TNBC) is a major global public health problem. The lack of targeted therapy and the elevated mortality evidence the need for better knowledge of the tumor biology. Hypoxia and aberrant glycosylation are associated with advanced stages of malignancy, tumor progression and treatment resistance. Importantly, serum deprivation regulates the invasive phenotype and favors TNBC cell survival. However, in TNBC, the role of hypoxia and serum deprivation in the regulation of glycosylation remains largely unknown. The effects of hypoxia and serum deprivation on the expression of glycosyltransferases and glycan profile were evaluated in the MDA-MB-231 cell line. We showed that the overexpression of HIF-1α was accompanied by acquisition of epithelial-mesenchimal transition features. Significant upregulation of fucosyl- and sialyltransferases involved in the synthesis of tumor-associated carbohydrate antigens was observed together with changes in fucosylation and sialylation detected by Aleuria aurantia lectin and Sambucus nigra agglutinin lectin blots. Bioinformatic analysis further indicated a mechanism by which HIF-1α can regulate ST3GAL6 expression and the relationship within the intrinsic characteristics of TNBC tumors. In conclusion, our results showed the involvement of hypoxia and serum deprivation in glycosylation profile regulation of TNBC cells triggering breast cancer aggressive features and suggesting glycosylation as a potential diagnostic and therapeutic target. We thank the following funding agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Ciência e Tecnologia de Pernambuco (FACEPE). This work was also 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); and 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 by EU 7th framework programme ITN 316929, and by the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 748880. F. Pinto received a fellowship from FCT (SFRH/BPD/115730/2016).

Helicobacter pylori infection is one of the major risk factors for gastric cancer development. Available antibiotic-based treatments not only fail in around 20% of patients but also have a severe negative impact on the gut microbiota. Recently, we demonstrated that nanostructured lipid carriers (NLC), even without any drug loaded, are bactericidal against H. pylori at low concentrations. This work aims to clarify NLC mode of action and to evaluate if their bactericidal effect is specific to H. pylori without affecting bacteria from microbiota. NLC were produced by hot homogenization followed by ultrasonication method, using Precirol®ATO5 and Miglyol®812 as lipids and Tween®60 as a surfactant. NLC were able to eradicate H. pylori without affecting the other tested bacteria (Lactobacillus, E. coli, S. epidermidis and S. aureus). Bioimaging assays demonstrated that NLC rapidly bind to and cross the H. pylori bacterial membrane, destabilizing and disrupting it, which leads to leakage of the cytoplasmic contents and consequent bacterial death. In an era where efficient alternatives to antibiotics are urgent, NLC are an interesting route to be explored in the quest for new antibiotic-free therapies to fight H. pylori infection.

International audience; Persistent Helicobacter pylori (H. pylori) infection is related to 90% of gastric cancers. With bacterial resistance rising and treatment inefficiency affecting 15% of the patients, alternative treatments urge. Chitosan microspheres (ChMics) have been proposed as an H. pylori-binding system. This work evaluates ChMics biocompatibility, mucopenetration and capacity to treat H. pylori infection in mice after oral administration. ChMics of different size (XL, ∼120 µm and XS, ∼40 µm) and degree of acetylation (6% and 16%) were developed and revealed to be able to adhere both human and mouse-adapted H. pylori strains without cytotoxicity towards human gastric cells. Ex vivo studies showed that smaller (XS) microspheres penetrate further within the gastric foveolae, suggesting their ability to reach deeply adherent bacteria. In vivo assays showed 88% reduction of infection when H. pylori-infected mice (C57BL/6) were treated with more mucoadhesive XL6 and XS6 ChMics. Overall, ChMics clearly demonstrate ability to reduce H. pylori gastric infection in mice, with chitosan degree of acetylation being a dominant factor over microspheres' size on H. pylori removal efficiency. These results evidence the strong potential of this strategy as an antibiotic-free approach to fight H. pylori infection, where microspheres are orally administered, bind H. pylori in the stomach, and remove them through the gastrointestinal tract. STATEMENT OF SIGNIFICANCE: Approximately 90% of gastric cancers are caused by the carcinogenic agent Helicobacter pylori, which infects >50% of the world population. Bacterial resistance, reduced antibiotic bioavailability, and the intricate distribution of bacteria in mucus and within gastric foveolae hamper the success of most strategies to fight H. pylori. We demonstrate that an antibiotic-free therapy based on bare chitosan microspheres that bind and remove H. pylori from stomach can achieve 88% reduction of infection from H. pylori-infected mice. Changing size and mucoadhesive properties, microspheres can reach different areas of gastric mucosa: smaller and less mucoadhesive can penetrate deeper into the foveolae. This promising, simple and inexpensive strategy paves the way for a faster bench-to-bedside transition, therefore holding great potential for clinical application.

The importance of sugars to protein function is real and is of significant clinical relevance. Technology advances enable large population studies to be carried out, shedding light on individual sugar variation and variations with time. Three-dimensional mass spectroscopy on solid pathological specimens is going to open up a whole new world of pathology visualisation. The door is now open to exploit carbohydrate recognition in new therapeutics by identifying novel biomarkers in cancer to aid diagnosis, and also providing therapeutic targets for treatment. Glycan age correlates with biological age. This means we can map the reversal of biological age with exercise and diet.

CD44 isoforms are often upregulated in gastric cancer and have been associated with increased metastatic potential and poor survival. To evaluate the functional impact of O-glycan truncation on CD44 we have analysed glyco-engineered cancer cell models displaying shortened O-glycans. Here, we demonstrate that induction of aberrant O-glycan termination through various molecular mechanisms affects CD44 molecular features. We show that CD44 is a major carrier of truncated O-glycans and that this truncation is accompanied by an increased hyaluronan binding capacity and affects extracellular shedding. In addition, short O-glycans promoted the colocalization of CD44v6 with the receptor tyrosine kinase RON and concomitantly increased activation. Our in vitro findings were validated in gastric cancer clinical samples. We This 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), UID/BIM/04293/2013 and CEECIND/02760/2017 (to SM); 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). We acknowledge the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 748880 (to MB). The authors acknowledge the support by Gastric Glyco Explorer Initial Training Network (European Union Seventh Framework Programme GastricGlycoExplorer project, grant number 316929). The authors declare noconflict of interest.

O-glycosylation is a highly frequent post-translation modification of proteins, with important functional implications in both physiological and disease contexts. The biosynthesis of O-glycans depends on several layers of regulation of the cellular glycosylation machinery, being organ-, tissue- and cell-specific. This review provides insights on the molecular mechanism underlying O-glycan biosynthesis and modification, and highlights illustrative examples of diseases that are triggered or modulated by aberrant cellular O-glycosylation. Particular relevance is given to genetic disorders of glycosylation, infectious diseases and cancer. Finally, we address the potential of O-glycans and their biosynthetic pathways as targets for novel therapeutic strategies.

Cadherins are key components in tissue morphogenesis and architecture, contributing to the establishment of cohesive cell adhesion. Reduced cellular adhesiveness as a result of cadherin dysfunction is a defining feature of cancer. During tumor development and progression, major changes in the glycan repertoire of cancer cells take place, affecting the stability, trafficking, and cell-adhesion properties of cadherins. Importantly, the different glycoforms of cadherins are promising biomarkers, with potential clinical application to improve the management of patients, and constitute targets for the development of new therapies. This review discusses the most recent insights on the impact of glycan structure on the regulation of cadherin function in cancer, and provides a perspective on how cadherin glycans constitute tumor biomarkers and potential therapeutic targets. IPATIMUP integrates the I3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação). This work was financed by FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through the FCT in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274), PTDC/DTP-PIC/0560/2014, and PTDC/BBB-EBI/0567/2014. S.C. also acknowledges funding from the FCT (SFRH/BD/77386/2011).

// Sandra Carvalho 1, 2, * , Tiago Oliveira 1, * , Markus F. Bartels 3 , Eiji Miyoshi 4 , Michael Pierce 5 , Naoyuki Taniguchi 6 , Fatima Carneiro 1, 7, 8 , Raquel Seruca 1, 8 , Celso A. Reis 1, 2, 8 , Sabine Strahl 3 , Salome S. Pinho 1, 7 1 Instituto de Investigacao e Inovacao em Saude (I3S) / Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465 Porto, Portugal 2 Institute of Biomedical Sciences of Abel Salazar (ICBAS), University of Porto, 4050-313 Porto, Portugal 3 Centre for Organismal Studies (COS) Heidelberg, Cell Chemistry, University of Heidelberg, 69120 Heidelberg, Germany 4 Department of Molecular Biochemistry and Clinical Investigation, Osaka University Graduate School of Medicine, 565-0871 Osaka, Japan 5 Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA 6 Department of Biochemistry, Graduate School of Medicine, Osaka University, 565-0871 Osaka, Japan 7 Medical Faculty, University of Porto, 4200-319 Porto, Portugal 8 Department of Pathology, Hospital S. Joao, 4200-319 Porto, Portugal * These authors have contributed equally to this work Correspondence to: Salome S. Pinho, email: salomep@ipatimup.pt Keywords: E-cadherin, O -mannosylation, N -glycosylation, gastric cancer Received: May 09, 2016 Accepted: August 01, 2016 Published: August 12, 2016 ABSTRACT Dysregulation of tumor suppressor protein E-cadherin is an early molecular event in cancer. O -mannosylation profile of E-cadherin is a newly-described post-translational modification crucial for its adhesive functions in homeostasis. However, the role of O -mannosyl glycans in E-cadherin-mediated cell adhesion in cancer and their interplay with N -glycans remains largely unknown. We herein demonstrated that human gastric carcinomas exhibiting a non-functional E-cadherin display a reduced expression of O -mannosyl glycans concomitantly with increased modification with branched complex N -glycans. Accordingly, overexpression of MGAT5 -mediated branched N -glycans both in gastric cancer cells and transgenic mice models led to a significant decrease of O -mannosyl glycans attached to E-cadherin that was associated with impairment of its tumour suppressive functions. Importantly, overexpression of protein O -mannosyltransferase 2 ( POMT2 ) induced a reduced expression of branched N -glycans which led to a protective effect of E-cadherin biological functions. Overall, our results reveal a newly identified mechanism of (dys)regulation of E-cadherin that occur through the interplay between O -mannosylation and N -glycosylation pathway.

Cisplatin-based chemotherapeutic regimens are the most frequently used (neo)adjuvant treatments for the majority of solid tumors. While platinum-based chemotherapeutic regimens have proven effective against highly proliferative malignant tumors, significant relapse and progression rates as well as decreased overall survival are still observed. Currently, it is known that sub-populations of chemoresistant cells share biological properties with cancer stem cells (CSC), which are believed to be responsible for tumor relapse, invasion and ultimately disease dissemination through acquisition of mesenchymal cell traits. In spite of concentrated efforts devoted to decipher the mechanisms underlying CSC chemoresistance and to design targeted therapeutics to these cells, proteomics has failed to unveil molecular signatures capable of distinguishing between malignant and non-malignant stem cells. This has hampered substantial developments in this complex field. Envisaging a novel rationale for an effective therapy, the current review summarizes the main cellular and molecular mechanisms underlying cisplatin resistance and the impact of chemotherapy challenge in CSC selection and clinical outcome. It further emphasizes the growing amount of data supporting a role for protein glycosylation in drug resistance. The dynamic and context-dependent nature of protein glycosylation is also comprehensively discussed, hence highlighting its potentially important role as a biomarker of CSC. As the paradigm of cancer therapeutics shifts towards precision medicine and patient-tailored therapeutics, we bring into focus the need to introduce glycomics and glycoproteomics in holistic pan-omics models, in order to integrate diverse, multimodal and clinically relevant information towards more effective cancer therapeutics. This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT, the Portuguese Foundation for Science and Technology) under the projects with the references FCOMP-01-0124-FEDER 028188 (PTDC/BBB-EBI/0786/2012) and PTDC/BBB-EBI/0567/2014. C.R. acknowledges the support by Gastric Glyco Explorer Initial Training Network (Seventh Framework Programme grant no. 316929). IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, (PEst-C/SAU/LA0003/2013). Grants were received from FCT: SFRH/BPD/111048/2015 to J.A.F and SFRH/BD/111242/2015 to A.P. FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF).

The overproduction and deposition of hyaluronic acid (HA) of different sizes in the tumor microenvironment is associated with cancer metastasis. Here, the development of layerâ byâ layer (LbL) constructs containing HA of different molecular weights (i.e., 5.6, 618, and 1450Â kDa) that mimic the HAâ rich cancer extracellular matrix is described to study the effect of the HA's size on the behavior of gastric cancer cells (AGS). The results demonstrate that LbL constructs with short HA, i.e., 5.6Â kDa, activate the cytoskeleton rearrangement leading to the â hummingbirdâ morphology, promote high cellular motility, and activate signaling pathways with increased expression of pâ ERK1/2 and pâ AKT. In addition, it is demonstrated that this malignant transformation involves an active participation of the HA coreceptor RHAMM in AGS cells., The authors acknowledge the financial support from the European Commission’s H2020 Programme, under grant agreements H2020-WIDESPREAD-2014-668983-FORECAST and H2020-MSCA-RISE-2019-872648-MEPHOS. S.A. acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD grant (SFRH/BD/112075/2015). The authors also thank Ramon Novoa-Caballal for performing the GPC experiments and data analysis.

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 FCT for the PhD fellowship awarded to FD (SFRH/BD/137896/2018), for the IF research position IF/00296/2015 to CCB and the CEECIND/02760/2017 to S.M. Acknowledgments: The authors would like to thank Rossana Correia (HEMS, i3S Scientific platform) for support on cells processing. The authors acknowledge the support of the ALM i3S Scientific Platform, member of the PPBI (PPBI-POCI-01-0145-FEDER-022122).

Circulating tumour cells (CTCs) originating from a primary tumour, lymph nodes and distant metastases hold great potential for liquid biopsies by providing a molecular fingerprint for disease dissemination and its temporal evolution through the course of disease management. CTC enumeration, classically defined on the basis of surface expression of Epithelial Cell Adhesion Molecule (EpCAM) and absence of the pan-leukocyte marker CD45, has been shown to correlate with clinical outcome. However, existing approaches introduce bias into the subsets of captured CTCs, which may exclude biologically and clinically relevant subpopulations. Here we explore the overexpression of the membrane protein O-glycan sialyl-Tn (STn) antigen in advanced bladder and colorectal tumours, but not in blood cells, to propose a novel CTC isolation technology. Using a size-based microfluidic device, we show that the majority (>90%) of CTCs isolated from the blood of patients with metastatic bladder and colorectal cancers express the STn antigen, supporting a link with metastasis. STn+ CTC counts were significantly higher than EpCAM-based detection in colorectal cancer, providing a more efficient cell-surface biomarker for CTC isolation. Exploring this concept, we constructed a glycan affinity-based microfluidic device for selective isolation of STn+ CTCs and propose an enzyme-based strategy for the recovery of viable cancer cells for downstream investigations. Finally, clinically relevant cancer biomarkers (transcripts and mutations) in bladder and colorectal tumours, were identified in cells isolated by microfluidics, confirming their malignant origin and highlighting the potential of this technology in the context of precision oncology.

Mucosal T lymphocytes from patients with ulcerative colitis (UC) were previously shown to display a deficiency in branched N-glycosylation associated with disease severity. However, whether this glycosylation pathway shapes the course of the T cell response constituting a targeted-specific mechanism in UC remains largely unknown. In this study, we demonstrated that metabolic supplementation of ex vivo mucosal T cells from patients with active UC with N-acetylglucosamine (GlcNAc) resulted in enhancement of branched N-glycosylation in the T cell receptor (TCR), leading to suppression of T cell growth, inhibition of the T helper 1 (Th1)/Th17 immune response, and controlled T cell activity. We further demonstrated that mouse models displaying a deficiency in the branched N-glycosylation pathway (MGAT5−/−, MGAT5+/−) exhibited increased susceptibility to severe forms of colitis and early-onset disease. Importantly, the treatment of these mice with GlcNAc reduced disease severity and suppressed disease progression due to a controlled T cell-mediated immune response at the intestinal mucosa. In conclusion, our human ex vivo and preclinical results demonstrate the targeted-specific immunomodulatory properties of this simple glycan, proposing a therapeutic approach for patients with UC., We thank Dr. Hiroaki Korekane and Fumi (RIKEN) for support in preparation of the fluorescent oligosaccharide acceptor substrate. We thank Dr. Michael Pierce for kindly providing the MGAT5 knockout mice. We also thank Paula Paíga (REQUIMTE/LAQV) for technical support with the HPLC system. The Institute of Molecular Pathology and Immunology of the University of Porto integrates the i3S research unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT). This article is a result of the project NORTE-01-0145-FEDER-000029, supported by the Norte Portugal Regional Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund. This work was also funded by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through the FCT in the framework of the project (POCI-01/0145-FEDER-016601 and PTDC/DTP-PIC/0560/2014). S.S.P. acknowledges the European Crohn’s and Colitis Organization (ECCO) for ECCO Grant 2017, the Broad Medical Research Program at the Crohn’s and Colitis Foundation of America, and the Portuguese Group of Study in IBD (GEDII) for funding. A.M.D. [PD/BD/105982/2014], A.C. [SFRH/BPD/91623/2012], and M.S.P. [SFRH/BD/110148/2015] received funding from the FCT. M. Lima thanks the CHP for the research support.

Pancreatic adenocarcinoma (PDAC) lacks efficient biomarkers. Mucins are glycoproteins that can carry aberrant glycosylation in cancer. Our objective was to identify cancer-related glycan epitopes on MUC1 and MUC5AC mucins in PDAC as potential biomarkers. We have analysed the tumour-associated carbohydrate antigens sialyl-Lewis x (SLex) and sialyl-Tn (STn) on MUC1 and MUC5AC in PDAC tissues. The selected cohort for this study consisted of twenty-one PDAC tissues positive for SLex antigen and three normal pancreas specimens as controls. STn expression was shown in 76% of the PDAC tissues. MUC1 and MUC5AC were detected in 90% of PDAC tissues. We performed in situ proximity ligation assay combining antibodies against mucins and glycan epitopes to identify specific mucin glycoforms. MUC1-SLex and MUC5AC-SLex were found in 68% and 84% respectively, of the mucin expressing PDAC tissues, while STn hardly colocalized with any of the evaluated mucins. Further analysis by Western blot of MUC5AC and SLex in eight PDAC tissue lysates showed that six out of eight cases were positive for both markers. Moreover, immunoprecipitation of MUC5AC from positive PDAC tissues and subsequent SLex immunodetection confirmed the presence of SLex on MUC5AC. Altogether, MUC5AC-SLex glycoform is present in PDAC and can be regarded as potential biomarker. M.B. acknowledges University of Girona for a pre-doctoral fellowship and a mobility grant. We also thank David Carreras for part of the IHC analysis. The authors also thank Dr. Carme de Bolós from Gastroesophagic Cancer Research Group, Hospital del Mar Medical Research Institute (IMIM), for kindly providing anti-MUC5AC rabbit polyclonal antibody Lum5.1. This work was supported by Spanish Ministry of Science and Innovation (grant BIO 2015-66356-R, awarded to R.P.). C.A.R. acknowledges the support by Gastric Glyco Explorer Initial Training Network (Seventh Framework Programme, project GastricGlycoExplorer, grant number 316929), and Fundação para a Ciência e Tecnologia, project PTDC/BBBEBI/0567/2014 (POCI-010145-FEDER-016585) and NORTE 2020 (NORTE-01-0145-FEDER-000029).

The physiological importance of the interactions between hyaluronic acid (HA) and its main membrane receptor, CD44, in pathological processes, e.g. cancer, is well recognized. However, these interactions are mainly studied in solution, whereas HA in the extracellular matrix (ECM) is partially immobilized via its interactions with other ECM components. We therefore, developed substrates in which HA is presented in an ECM-relevant manner. We immobilized HA with different molecular weights (Mw) in a Layer-by-Layer (LbL) fashion and studied the interactions of the substrates with CD44 and two human gastric cancer cell lines that overexpress this receptor, namely AGS and MKN45. We demonstrate that MKN45 cells are more sensitive to the LbL substrates as compared with AGS. This difference is due to different CD44 expression: while CD44 is detected mainly in the cytoplasm of AGS, MKN45 express CD44 predominantly at the cell membrane where it is involved in the recognition and binding of HA. The invasiveness of the studied cell lines was also evaluated as a function of HA Mw. Invasive profile characterized by low cell adhesion, high cell motility, high expression of cortactin, formation of invadopodia and cell clusters was observed for MKN45 cells when they are in contact with substrates presenting HA of high Mw., Te authors acknowledge the fnancial support from the European Commission’s H2020 programme, under grant agreements H2020-TWINN-2015-692333-CHEM2NATURE, H2020-WIDESPREAD-2014-668983- FORECAST and H2020-WIDESPREAD-01-2016-2017-739572-THE DISCOVERIES CTR. S.A., D.S.C. and I.P. also acknowledge the Portuguese Foundation for Science and Technology (FCT) for fnancial support under grants SFRH/BD/112075/2015, SFRH/BPD/85790/2012 and IF/00032/2013, respectively., info:eu-repo/semantics/publishedVersion

Despite recent progress in understanding the cancer genome, there is still a relative delay in understanding the full aspects of the glycome and glycoproteome of cancer. Glycobiology has been instrumental in relevant discoveries in various biological and medical fields, and has contributed to the deciphering of several human diseases. Glycans are involved in fundamental molecular and cell biology processes occurring in cancer, such as cell signalling and communication, tumour cell dissociation and invasion, cell-matrix interactions, tumour angiogenesis, immune modulation and metastasis formation. The roles of glycans in cancer have been highlighted by the fact that alterations in glycosylation regulate the development and progression of cancer, serving as important biomarkers and providing a set of specific targets for therapeutic intervention. This Review discusses the role of glycans in fundamental mechanisms controlling cancer development and progression, and their applications in oncology. The Institute of Molecular Pathology and Immunology of the University of Porto integrates the Institute for Research and Innovation in Health, which is partially supported by the Portuguese Foundation for Science and Technology (FCT). This work is funded by the European Regional Development Fund (FEDER) through the Operational Programme for Competitiveness Factors (COMPETE) and by national funds through the FCT, under the projects PEst‑C/SAU/ LA0003/2013, PTDC/BBB-EBI/0786/2012 and EXPL/BIM-MEC/0149/2012. S.S.P. acknowledges a grant from the FCT (number SFRH/BPD/63094/2009). C.A.R. acknowledges sup­port from the European Union Seventh Framework Programme GastricGlycoExplorer (grant number 316929). The authors apologize that they cannot include all the relevant studies on glycosylation in cancer in this article owing to limitation of space. The authors thank Tiago Fontes- Oliveira for support in figures preparations.

E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N-glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N-acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N-glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with ß1,6 GlcNAc-branched N-glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis-dimer formation, molecular assembly and stability of the adherens junctions and cell-cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression. 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— and National Funds through the FCT—Foundation for Science and Technology, under the projects: PTDC/CVT/111358/2009; EXPL/BIM-MEC/0149/2012; and PTDC/BBB-EBI/0786/2012. SC (SFRH/BD/77386/2011), AMD (SFRH/BI/52380/2013) BiotechHealth Doctoral Programme, and SSP (SFRH/BPD/63094/2009) thank FCT and the Luso-American Foundation (FLAD) for funding. JMS acknowledges FCT (UID/EEA/50009/2013). DK acknowledges support by the Max Planck Society and European Union (Seventh Framework Programme 'Glycoproteomics', grant number PCIG09-GA-2011-293847. CAR and DK acknowledge GastricGlycoExplorer project, grant number 316929). We thank Ola Soderberg and Gaëlle Cane (Department of Genetics and Pathology, University of Uppsala, Uppsala, Sweden) for providing the Streptavidin PLA probe and to Márcia Pereira and Sara Campos for the support in PLA technique and statistical analyses.

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "Simple- Cell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer. This work was supported by The Danish Research Councils, The Mizutani Foundation, The Danish National Research Foundation (DNRF107) and Fundacão para a Ciência e a Tecnologia (FCT) and COMPETE (Programa Operacional Temático Factores de Competitividade, comparticipado pelo fundo comunitário europeu FEDER) in the framework of the projects: PTDC/BBB-EBI/0786/2012; EXPL/CTM-BIO/0762/2013. Grants were received from FCT (SFRH/BD/73717/2010 to DC), (SFRH/BPD/75871/2011 to AM), (SFRH/BPD/96510/2013 to CG) and (SFRH/BPD/66288/2009 to JAF). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT.

Glycosylation alterations are frequently observed in cancer cells and shape tumor progression. In this issue of Cancer Cell, Nguyen et al. show that GALNT1 relocation from Golgi to endoplasmic reticulum drives liver tumor growth and invasion, due to enhanced glycosylation and consequential activation of the extracellular matrix-degrading metalloproteinase MMP14.

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.

Association of aberrant glycosylation with melanoma progression is based mainly on analyses of cell lines. Here we present a systems-based study of glycomic changes and corresponding enzymes associated with melanoma metastasis in patient samples. Upregulation of core fucosylation (FUT8) and downregulation of α-1,2 fucosylation (FUT1, FUT2) were identified as features of metastatic melanoma. Using both in vitro and in vivo studies, we demonstrate FUT8 is a driver of melanoma metastasis which, when silenced, suppresses invasion and tumor dissemination. Glycoprotein targets of FUT8 were enriched in cell migration proteins including the adhesion molecule L1CAM. Core fucosylation impacted L1CAM cleavage and the ability of L1CAM to support melanoma invasion. FUT8 and its targets represent therapeutic targets in melanoma metastasis.

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid present in fish oil, has been described as a promising molecule to the treatment of Helicobacter pylori gastric infection. However, due to its highly unsaturated structure, DHA can be easily oxidized loosing part of its bioactivity. This work aims the nanoencapsulation of DHA to improve its bactericidal efficacy against H. pylori. DHA was loaded into nanostructured lipid carriers (NLC) produced by hot homogenization and ultrasonication using a blend of lipids (Precirol AT05 (R), Miglyol-812 (R)) and a surfactant (Tween 60 (R)). Homogeneous NLC with 302 +/- 14 nm diameter,-28 +/- 3 mV surface charge (dynamic and electrophoretic light scattering) and containing 66 +/- 7% DHA (UV/VIS spectroscopy) were successfully produced. Bacterial growth curves, performed over 24 h in the presence of different DHA concentrations (free or loaded into NLC), demonstrated that nanoencapsulation enhanced DHA bactericidal effect, since DHA-loaded NLC were able to inhibit H. pylori growth in a much lower concentrations (25 mu M) than free DHA (>100 mu M). Bioimaging studies, using scanning and transmission electron microscopy and also imaging flow cytometry, demonstrated that DHA-loaded NLC interact with H. pylori membrane, increasing their periplasmic space and disrupting membrane and allowing the leakage of cytoplasmic content. Furthermore, the developed nanoparticles are not cytotoxic to human gastric adenocarcinoma cells at bactericidal concentrations. DHA-loaded NLC should, therefore, be envisaged as an alternative to the current treatments for H. pylori infection. (C) 2017 Elsevier B.V. All rights reserved. FCT/MCTES [POCI-01-0145-FEDER-007274, PTDC/CTM-BIO/4043/2014, PTDC/CTM-BPC/121149/2010, NORTE-01-0145-FEDER-000012] FEDER funds through the COMPETE (POCI), Portugal FCT grant [SFRH/BD/89001/2012] info:eu-repo/semantics/publishedVersion

Glycosylation is one of the most common and dynamic post-translational modification of cell surface and secreted proteins. Cancer cells display unique glycosylation patterns that decisively contribute to drive oncogenic behavior, including disease progression and dissemination. Moreover, alterations in glycosylation are often responsible for the creation of protein signatures holding significant biomarker value and potential for targeted therapeutics. Accordingly, many analytical protocols have been outlined for the identification of abnormally glycosylated proteins by mass spectrometry. Nevertheless, very few studies undergo a comprehensive mining of the generated data. Herein, we build on bladder cancer O-glycoproteomics datasets resulting from a hyphenated technique comprising enrichment by Vicia villosa agglutinin (VVA) lectin and nanoLC-ESI-MS/MS to propose an in silico step-by-step tutorial (Panther, UniProtKB, NetOGlyc, NetNGlyc, Oncomine, Cytoscape) for biomarker discovery in cancer. We envisage that this approach may be generalized to other mass spectrometry-based analytical approaches, including N-glycoproteomics studies, and different types of cancers. Significance The glycoproteome is an important source of cancer biomarkers holding tremendous potential for targeted therapeutics. We now present an in silico roadmap for comprehensive interpretation of big data generated by mass spectrometry-based glycoproteomics envisaging the identification of clinically relevant glycobiomarkers.