Search

Simple Summary Changes in glycosylation, such as incomplete synthesis and higher density of O-glycans on the cell surface, are frequently observed in cancer cells. Several types of truncated O-glycan structures, e.g., T/Tn antigens, are suspected to disrupt molecular interactions between tumor microenvironment and immune cells, for instance, facilitating cancer immune-escape. Therefore, numerous exogenous lectins targeting aberrant O-glycans are interesting tools for cancer diagnosis, prognosis, and therapy. However, the ability of exolectins to detect subtle alterations in the glycome of tumor cells and to interfere in tumor/healthy cell interactions remains largely unknown. The present article reports for the first time that the Helix pomatia (HPA) lectin, a well-known T/Tn-specific lectin, currently used as a tool in cancer diagnostics, kills Tn-positive leukemia cells and Tn-negative lymphoma cells but does not affect healthy lymphocytes. Thus, HPA could be used to discriminate between tumor and healthy cells, and detect subtle alterations in the glycosylation profile. Abstract Morniga G is a T/Tn-specific lectin, inducing cell death in Tn-positive leukemias but not in healthy lymphocytes. Helix pomatia lectin (HPA) is another T/Tn-specific lectin, currently used as tool for cancer diagnostics. The HPA-mediated tumor cell death was evaluated on human leukemia and mouse lymphoma cells, and compared to the effect of Morniga G. Both lectins induced an equivalent percentage of cell death in Tn-positive Jurkat human leukemia. In contrast, EL4 mouse lymphoma resisted Morniga G-mediated cytotoxicity but were killed by HPA at concentrations of 2.5 μg/mL (0.032 nM) and higher. In both malignant cells, HPA-mediated cell death showed features compatible with apoptosis (annexin-externalization, caspase-activation, mitochondrial membrane depolarization, and ROS production). Cytometry analysis indicated that EL4 cells are T/Tn-negative. Because previous results showed a high amount of N-acetylgalactosamine (GalNAc, sugar present in Tn antigen) on EL4 cell surface, this GalNAc could be involved in the formation of truncated O-glycans other than the T/Tn residues. When compared to Morniga G, bioinformatic analysis suggested that HPA benefits from an extended carbohydrate-binding site, better adapted than Morniga G to the accommodation of more complex branched and truncated O-glycans (such as core 2). Finally, HPA killed EL4 cells but not healthy lymphocytes in a mixture of lymphoma cells + lymphocytes, suggesting that HPA selectively triggers tumor cell death.

Aberrant expression of Sialyl‐Tn (STn) antigen correlates with poor prognosis and reduced patient survival. We demonstrated that expression of Tn and STn in pancreatic ductal adenocarcinoma (PDAC) is due to hypermethylation of Core 1 synthase specific molecular chaperone (COSMC) and enhanced the malignant properties of PDAC cells with an unknown mechanism. To explore the mechanism, we have genetically deleted COSMC in PDAC cells to express truncated O‐glycans (SimpleCells, SC) which enhanced cell migration and invasion. Since epithelial‐to‐mesenchymal transition (EMT) play a vital role in metastasis, we have analysed the induction of EMT in SC cells. Expressions of the mesenchymal markers were significantly high in SC cells as compared to WT cells. Equally, we found reduced expressions of the epithelial markers in SC cells. Re‐expression of COSMC in SC cells reversed the induction of EMT. In addition to this, we also observed an increased cancer stem cell population in SC cells. Furthermore, orthotopic implantation of T3M4 SC cells into athymic nude mice resulted in significantly larger tumours and reduced animal survival. Altogether, these results suggest that aberrant expression of truncated O‐glycans in PDAC cells enhances the tumour aggressiveness through the induction of EMT and stemness properties.

Tumor cells acquire aberrant glycosylation structures at their surface. One of the most abundant glycosylation modifications present in gastric cancer tissues is the increased expression of truncated O-glycans, such as the sialyl-Tn (STn), which are associated with patients’ poor survival. Interestingly, some of the tumor-associated glycans have already been identified in cancer extracellular vesicles (EVs). Important results from our group have also demonstrated the effect of premature truncation of O-glycans (truncated Oglycans) in modulating the cells aggressive phenotypic behaviour. The general aim of this project was to explore the impact of STn positive EVs in cancer communication, namely in the modulation of the migration capacity of recipient cells. It was hypothesized that the EVs released by cancer cells with more aggressive features could influence the phenotypic behaviour of the recipient cells, namely their migration capacity. In this project, we used the genetically modified gastric cancer cell line, MKN45 SimpleCell (SC), that homogeneously overexpresses the truncated Oglycans Tn and STn, and the wild type version of this model, the MKN45 WT. To achieve this goal, it was first necessary to optimize the best cell culture conditions and the appropriate EV isolation protocol. We study the effects of removing the fetal bovine serum (FBS) from the culture medium for 48h and 72h on cell viability and proliferation rate. In addition, we assessed the impact of using a medium concentration step in the EV isolation protocol. EVs were then characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The presence of a panel of known EV markers, as well as the presence of the truncated O-glycan STn, were evaluated by western blotting. Finally, the role of STn positive EVs on the modulation of the migration capacity of recipient cells was also assessed. Results showed that removing the FBS from the culture medium for 48h or 72h did not affect the viability of both MKN45 WT and SC cells. Therefore, we opted to collect the EVs after growing the cells for 72h in medium without FBS. In addition, a better EV yield was achieved when isolating the EVs without the medium concentration step when compared to the protocol with this additional step. TEM images showed the presence and integrity of the EVs isolated from both MKN45 WT and SC cell lines. In addition, western blotting results showed the presence of the specific EV markers HSP70, synthenin-1, actin, Alix, CD9 and CD81 in MKN45 WT and SC derived EVs. Curiously, an enrichment of STn antigen was found in SC EVs when compared to the parental cells. NTA results revealed that MKN45 SC cells secreted more EVs than the MKN45 WT cell line. Additionally, an increased migration capacity of the MKN45 SC cell line was observed when comparing with the MKN45 WT cell line. Interestingly, when coculturing the MKN45 WT cells with STn positive EVs an increase in the migration capacity of these cells was observed when compared with the untreated cells. This project was important to demonstrate that STn positive EVs, secreted from cells with high expression of this truncated O-glycan, could effectively modulate the migration capacity of recipient cells. In the future, it will be interesting to explore the impact of STn positive EVs on tumor growth, invasion and metastasis. It will be important to find which mechanisms are regulating the acquired aggressive phenotype caused by STn positive EVs. These results would be crucial for the better understanding of the impact of EV glycosylation, namely the truncated O-glycans, in cancer communication and may bring numerous benefits to the clinical outcome of gastric cancer patients. As células tumorais adquirem estruturas de glicosilação aberrantes à sua superfície. Uma das modificações de glicanos mais abundante e presente em tecidos de cancro gástrico, é o aumento da expressão de O-glicanos truncados, como o sialil-Tn (STn), que estão associados a uma baixa sobrevivência dos pacientes. De realçar que, alguns dos glicanos associados ao tumor foram já identificados em vesículas extracelulares (VEs). Resultados importantes do nosso grupo demonstraram também o efeito do truncamento prematuro dos O-glicanos (O-glicanos truncados) na modulação do comportamento fenotípico agressivo das células. O objetivo geral deste projeto foi explorar o impacto das VEs positivas para STn na comunicação em cancro, nomeadamente na modulação da capacidade de migração das células recipientes. Foi hipotetizado que as VEs libertadas pelas células cancerígenas com características mais agressivas poderiam influenciar o comportamento fenotípico das células recipientes, nomeadamente a sua capacidade de migração. Neste projeto, usámos uma linha celular de cancro gástrico geneticamente modificada, MKN45 SimpleCell (SC), que sobre-expressa homogeneamente os O-glicanos truncados Tn e STn, e a versão original deste modelo, a MKN45 WT. Para alcançar este objetivo, primeiro foi necessário otimizar as melhores condições de cultura celular e o protocolo apropriado de isolamento das VEs. Estudámos os efeitos da remoção do soro fetal bovino (SFB) do meio de cultura por 48h e 72h na viabilidade celular e na taxa de proliferação das células. Para além disso, avaliámos o impacto do uso de um passo de concentração de meio durante o protocolo de isolamento das VEs. As VEs foram depois caraterizadas por microscopia eletrónica de transmissão (MET) e pela análise da concentração e tamanho das nanopartículas (NTA). A presença de um painel de marcadores conhecidos de VEs, bem como a presença do O-glicano truncado STn, foram avaliadas por “western blotting”. Por último, o papel das VEs positivas para STn na modulação da capacidade de migração das células recipientes foi também avaliado. Os resultados demonstraram que a remoção do SFB do meio de cultura por 48h e 72h não afetou a viabilidade de ambas as células MKN45 WT e SC. Assim, optámos por colher as VEs após o crescimento das células por 72h em meio sem SFB. Para além disso, um melhor rendimento de VEs foi alcançado ao isolar as VEs sem o passo de concentração de meio em comparação com o protocolo com este passo adicional. As imagens de MET mostraram a presença e integridade das VEs isoladas de ambas as linhas celulares MKN45 WT e SC. De seguida, os resultados de “western blotting” revelaram a presença de marcadores específicos de VEs, tais como HSP70, sintenina-1, actina, Alix, CD9 e CD81 em VEs derivadas de MKN45 WT e SC. Curiosamente, um enriquecimento do antigénio STn foi encontrado nas VEs SC quando comparado com as células parentais. Os resultados de NTA revelaram que as células MKN45 SC secretaram mais VEs do que a linha celular MKN45 WT. Adicionalmente, foi observado um aumento na capacidade de migração da linha celular MKN45 SC quando comparada com a linha celular MKN45 WT. De realçar que, ao cultivar as células MKN45 WT juntamente com VEs positivas para STn, um aumento na capacidade de migração dessas células foi observado quando comparado com as células não tratadas. Este projeto foi importante para demonstrar que VEs positivas para STn, secretadas de células com elevada expressão deste O-glicano truncado, poderiam modular efetivamente a capacidade de migração das células recipientes. No futuro, seria interessante explorar o impacto das VEs positivas para STn no crescimento tumoral, invasão e metástase. Será também importante perceber quais os mecanismos que estão a regular a aquisição do fenótipo agressivo causado pelas VEs positivas para STn. Estes resultados seriam cruciais para uma melhor compreensão do impacto da glicosilação das VEs, nomeadamente dos O-glicanos truncados, na comunicação em cancro e poderão trazer inúmeros benefícios para o desfecho clínico dos pacientes com cancro gástrico. Mestrado em Bioquímica

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).

Optimal research results rely on the selection of cellular models capable of recapitulating the characteristics of primary tumours from which they originate. The expression of mucins (MUC16 and MUC1) and truncated O-glycans (Tn, STn and T) represents a characteristic footprint of serous ovarian carcinomas (SOCs). Therefore, selecting ovarian cancer (OVCA) cell lines that reflect this phenotype is crucial to explore the putative biological role of these biomarkers in the SOC setting. Here, we investigated a panel of OVCA cell lines commonly used as SOC models, and tested whether, when cultured in 2D and 3D conditions, these recapitulate the mucin and O-glycan expression profiles of SOCs. We further explored the role of truncating the O-glycosylation capacity in OVCAR3 cells through knockout of the COSMC chaperone, using in vitro and in vivo assays. We found that the majority of OVCA cell lines of serous origin do not share the mucin and truncated O-glycan footprint of SOCs, although 3D cultures showed a higher resemblance. We also found that genetic truncation of the O-glycosylation capacity of OVCAR3 cells did not enhance oncogenic features either in vitro or in vivo. This study underscores the importance of well-characterized cellular models to study specific features of ovarian cancer. Funding: IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and partially supported by Fundação para a Ciência e a Tecnologia (FCT, I.P.). This research was funded by NORTE 2020 grant numbers NORTE-01-0145-FEDER-000029 and NORTE-01-0145-FEDER-000003. Ricardo Coelho acknowledges FCT, I.P. for financial support through a PhD fellowship number SFRH/BD/111885/2015. Acknowledgments: We acknowledge Tatiana Martins for all the support regarding 3D cultures and projects: KFS-3841-02-2016 from Krebsliga Schweiz, DNRF107 from the Danish National Research Foundation, UID/Multi/04462/2013 from iNOVA4Health (program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement).

Optimal research results rely on the selection of cellular models capable of recapitulating the characteristics of primary tumours from which they originate. The expression of mucins (MUC16 and MUC1) and truncated O-glycans (Tn, STn and T) represents a characteristic footprint of serous ovarian carcinomas (SOCs). Therefore, selecting ovarian cancer (OVCA) cell lines that reflect this phenotype is crucial to explore the putative biological role of these biomarkers in the SOC setting. Here, we investigated a panel of OVCA cell lines commonly used as SOC models, and tested whether, when cultured in 2D and 3D conditions, these recapitulate the mucin and O-glycan expression profiles of SOCs. We further explored the role of truncating the O-glycosylation capacity in OVCAR3 cells through knockout of the COSMC chaperone, using in vitro and in vivo assays. We found that the majority of OVCA cell lines of serous origin do not share the mucin and truncated O-glycan footprint of SOCs, although 3D cultures showed a higher resemblance. We also found that genetic truncation of the O-glycosylation capacity of OVCAR3 cells did not enhance oncogenic features either in vitro or in vivo. This study underscores the importance of well-characterized cellular models to study specific features of ovarian cancer.

// SonTung NguyenHoang 1,* , Yidong Liu 2,* , Le Xu 3,* , Lin Zhou 1 , Yuan Chang 1 , Qiang Fu 2 , Zheng Liu 2 , Zongming Lin 1 and Jiejie Xu 2 1 Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China 2 Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China 3 Department of Urology, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China * These authors have contributed equally to this work Correspondence to: Zongming Lin, email: // Jiejie Xu, email: // Keywords : localized clear-cell renal cell carcinoma, truncated O-glycans, overall survival, recurrence-free survival, prognosticator Received : September 07, 2016 Accepted : February 20, 2017 Published : March 04, 2017 Abstract Truncated O-glycans, including Tn-antigen, sTn-antigen, T-antigen, sT-antigen, are incomplete glycosylated structures and their expression occur frequently in tumor tissue. The study aims to evaluate the abundance of each truncated O-glycans and its clinical significance in postoperative patients with localized clear-cell renal cell carcinoma (ccRCC). We used immunohistochemical testing to analyze the expression of truncated O-glycans in tumor specimens from 401 patients with localized ccRCC. Truncated-O-glycan score was built by integrating the expression level of Tn-, sTn- and sT-antigen. Kaplan-Meier survival and Cox regression analysis were done to compare clinical outcomes in subgroups. Receiver operating characteristic (ROC) was applied to assess the impact of prognostic factors on overall survival (OS) and recurrence-free survival (RFS). The results identified Tn-, sTn-, sT-antigen as independent prognosticators. The OS and RFS were shortened among the 198 (49.4%) patients with high Truncated-O-glycan score than among the 203 (50.6%) patients with low score (hazard ratio for OS, 7.060; 95% confidence interval [CI]: 2.765 to 18.027; p