Aberrant Glycosylation in Cancer: A Novel Molecular Mechanism Controlling Metastasis

Glycosylation alterations are involved in several steps of human cancer pathogenesis. In this issue of Cancer Cell, Agrawal et al. identified the glycosyltransferase FUT8 as a previously unrecognized mediator of melanoma metastasis, establishing core fucosylation as a potential therapeutic target for prevention and treatment of metastatic tumors.The authors acknowledge funding by FEDER, COMPETE and FCT: POCI-01-0145-FEDER-007274 (UID/BIM/04293/2013), POCI-01-0145-FEDER-016585 (PTDC/BBB-EBI/0567/2014), NORTE 2020 (NORTE-01-0145-FEDER-000029), and EU 7th Framework Programme ITN 316929

Glycomic Approaches for the Discovery of Targets in Gastrointestinal Cancer

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

Heparan sulfate glycosaminoglycans: (un)expected allies in cancer clinical management

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

Different isolation approaches lead to diverse glycosylated extracellular vesicle populations

Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter-cellular communication and mediating a broad spectrum of biological functions. EVs cargo iscomposed of a large repertoire of molecules, including glycoconjugates. Herein, we report the firststudy on the impact of the isolation strategy on the EV populations’glycosylation profile. The use ofdifferent state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isola-tion (TEI), OptiPrepTMdensity gradient (ODG) and size exclusion chromatography (SEC) resulted in EVpopulations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODGand SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated themost distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EVglycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn(STn) were identified as packaged cargo into EVs independently of the isolation methodology. STncarrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-relatedproteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of usingdifferent isolation methodologies in the populations of EVs that are obtained, with consequences inthe glycosylation profile of the isolated population. Furthermore, our results highlight the importanceof selecting adequate EV isolation protocols and cell culture conditions to determine the structuraland functional complexity of the EV glycoconjugates.This work was funded by FEDER funds through theOperational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; POCI-01-0145-FEDER-028489) and NationalFunds through the Foundation for Science and Technology(FCT), under the projects: PTDC/BBB-EBI/0567/2014 (toCAR), 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 PartnershipAgreement, through the European Regional DevelopmentFund (ERDF). DF acknowledges the FCT PhD Programmesand Programa Operacional Potencial Humano (POPH), speci-fically the Biotech Health Programme (Doctoral Programme onCellular and Molecular Biotechnology Applied to HealthSciences), with the reference PD/0016/2012 funded by FCTand the grant SFRH/BD/110636/2015 from FCT, POPH andFSE (Fundo Social Europeu); MB acknowledges the EuropeanUnion’s Horizon 2020 research and innovation programmeunder the Marie Sklodowska-Curie grant agreement No.748880; and JP acknowledges FCT (SFRH/BD/137319/2018).The authors acknowledge Rede Nacional de Espectrometria deMassa, ROTEIRO/0028/2013, ref. LISBOA-01-0145-FEDER-022125, supported by COMPETE and North PortugalRegional Operational Programme (Norte2020), under thePORTUGAL 2020 Partnership Agreement, through theEuropean Regional Development Fund (ERDF). SV acknowl-edges the Danish National Research Foundation (DNRF107)

Spatial distribution and populations at risk of A. lumbricoides and T. trichiura co-infections and infection intensity classes: an ecological study.

BACKGROUND: Soil-transmitted helminth (STH) infections are highly prevalent in the Philippines. Mapping the prevalence and high-intensity of STH co-infections can help guide targeted intervention programmes to reduce morbidity, especially among vulnerable school-aged children. In this study, we aimed to predict the spatial distribution of the prevalence of Ascaris lumbricoides and Trichuris trichiura co-infection and infection intensity classes in the Philippines to identify populations most in need of interventions. METHODS: Data on STH infections from 29,919 individuals during the nationwide parasitological survey in 2005 to 2007 were included in the analysis. To geographically predict the prevalence of A. lumbricoides and T. trichiura co-infections and infection intensity classes, Bayesian multinomial geostatistical models were built including age, sex, environmental variables and a geostatistical random effect. The number of individuals co-infected and belonging to each of the infection intensity classes in 2017 was forecast by combining our predictive prevalence maps with population density maps. RESULTS: Our models showed that school-aged children (5-19 years) are most at risk of A. lumbricoides and T. trichiura co-infections and of moderate/high infection intensity compared to other age groups. We identified target provinces where the likelihood of STH-associated morbidity was highest: Luzon (Bulacan, Benguet, Cavite, Sorsogon, Metropolitan Manila, Pampanga and Rizal), the Visayas (Cebu, Iloilo, Leyte and Negros Occidental), and in Mindanao (Agusan Del Norte, Davao Del Sur, Davao Oriental, Lanao Del Sur, Maguindanao, Misamis Oriental, Sulu and Zamboanga Del Sur). Luzon had the highest estimated number of school-aged children with A. lumbricoides and T. trichiura co-infections (estimated total 89,400), followed by the Visayas (38,300) and Mindanao (20,200). CONCLUSIONS: Our study provided epidemiological evidence to highlight national priority areas for controlling co-infections and high intensity infections in the Philippines. Our maps could assist more geographically targeted interventions to reduce the risk of STH-associated morbidity in the Philippines

“A Good Death” - Palliative Surgery in Trisomy 18

A trissomia 18 caracteriza-se por múltiplas anomalias, incluindo doença cardíaca em 60 a 90% dos casos e elevada mortalidade. O mau prognóstico global, conduz habitualmente a uma politica de “cuidados mínimos” mas, paliar, é também nestas situações, um imperativo ético. Descreve-se o caso de uma recém-nascida sem diagnóstico pré natal, mas com confirmação por cariotipo, com cardiopatia, que condicionou insuficiência cardíaca congestiva e angústia respiratória crescente, inviabilizando alta hospitalar, como era desejo da família. Após consenso entre os pais e o corpo clínico responsável, foi decidida intervenção cirúrgica cardíaca paliativa, que possibilitou melhoria clínica e alta para o domicílio. Os autores defendem que a cirurgia cardíaca pode ser uma atitude a considerar em casos de trissomia 18, pois pode aliviar o sofrimento

A comparison of Helicobacter pylori and non-Helicobacter pylori Helicobacter spp. Binding to Canine Gastric Mucosa with Defined Gastric Glycophenotype

Background: The gastric mucosa of dogs is often colonized by non-Helicobacter pylori helicobacters (NHPH), while H. pylori is the predominant gastric Helicobacter species in humans. The colonization of the human gastric mucosa by H. pylori is highly dependent on the recognition of host glycan receptors. Our goal was to define the canine gastric mucosa glycophenotype and to evaluate the capacity of different gastric Helicobacter species to adhere to the canine gastric mucosa. Materials and Methods: The glycosylation profile in body and antral compartments of the canine gastric mucosa, with focus on the expression of histo-blood group antigens was evaluated. The in vitro binding capacity of FITC-labeled H. pylori and NHPH to the canine gastric mucosa was assessed in cases representative of the canine glycosylation pattern. Results: The canine gastric mucosa lacks expression of type 1 Lewis antigens and presents a broad expression of type 2 structures and A antigen, both in the surface and glandular epithelium. Regarding the canine antral mucosa, H. heilmannii s.s. presented the highest adhesion score whereas in the body region the SabA-positive H. pylori strain was the strain that adhered more. Conclusions: The canine gastric mucosa showed a glycosylation profile different from the human gastric mucosa suggesting that alternative glycan receptors may be involved in Helicobacter spp. binding. Helicobacter pylori and NHPH strains differ in their ability to adhere to canine gastric mucosa. Among the NHPH, H. heilmannii s.s. presented the highest adhesion capacity in agreement with its reported colonization of the canine stomach.We kindly thank Prof. Thomas Boren from the Department of Medical Biochemistry and Biophysics, Umea University, Sweden for providing the 17875/Leb and 17875babA1A2H. pylori strains. The authors thank Dr. Fernando Rodrigues, Dr. Ana Laura Saraiva, and Cristina Bacelar who kindly provided technical support. I. Amorim (SFRH/BD/76237/2011) and A. Magalhães (SFRH/BPD/75871/2011) acknowledge FCT for financial support. This study was partially funded by the Portuguese Foundation for Science and Technology (PTDC/CTM-BPC/121149/2010; PTDC/CVT/117610/2010; PTDC/BBB-EBI/0786/2012). The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT

Initial experience with targeted axillary dissection after neoadjuvant therapy in breast cancer patients

Background: Targeted axillary dissection (TAD) combines sentinel node biopsy (SNB) with the removal of the previously marked metastatic node. TAD is a promising concept for axillary restaging in node-positive breast cancer patients with pathological complete response (pCR) to neoadjuvant therapy (NAT). We aimed to evaluate TAD feasibility in this context. Methods: A prospective observational study was conducted in biopsy-confirmed cN1 patients. The removal of the clipped node (CN) was guided by intraoperative ultrasound. SNB used indocyanine green and patent blue V dye. If the CN or sentinel lymph nodes (SLN) had any metastatic foci, or the TAD procedure was unsuccessful, the patient underwent axillary lymph node dissection (ALND). Results: Thirty-seven patients were included. TAD and SNB identification rates were 97.3%. Every retrieved CN was also a SLN. At the individual level, SNB identification rate was 89.2% with indocyanine green and 85.5% with patent blue V dye. The CN identification rate was 81.1%, being higher when the CN was localized on the intraoperative ultrasound (84.4% vs 60.0%). Nodal pCR was achieved by 54.1% of our patients and was more frequent in HER2-positive and triple-negative tumors (p = 0.039). Nineteen patients were spared from ALND. Conclusion: TAD with intraoperative ultrasound-guided excision of the CN and SNB with indocyanine green and patent blue V dye is a feasible concept to identify patients without axillary residual disease after NAT, that can be spared from ALND, although the need for marking the biopsied node should be further investigated. © 2022, The Author(s), under exclusive licence to The Japanese Breast Cancer Society

Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery

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