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Background Muscle invasive bladder cancer (MIBC) remains amongst the deadliest genitourinary malignancies due to treatment failure and extensive molecular heterogeneity, delaying effective targeted therapeutics. Hypoxia and nutrient deprivation, oversialylation and O-glycans shortening are salient features of aggressive tumours, creating cell surface glycoproteome fingerprints with theranostics potential. Methods A glycomics guided glycoproteomics workflow was employed to identify potentially targetable biomarkers using invasive bladder cancer cell models. The 5637 and T24 cells O-glycome was characterized by mass spectrometry (MS), and the obtained information was used to guide glycoproteomics experiments, combining sialidase, lectin affinity and bottom-up protein identification by nanoLC-ESI-MS/MS. Data was curated by a bioinformatics approach developed in-house, sorting clinically relevant molecular signatures based on Human Protein Atlas insights. Top-ranked targets and glycoforms were validated in cell models, bladder tumours and metastases by MS and immunoassays. Cells grown under hypoxia and glucose deprivation disclosed the contribution of tumour microenvironment to the expression of relevant biomarkers. Cancer-specificity was validated in healthy tissues by immunohistochemistry and MS in 20 types of tissues/cells of different individuals. Results Sialylated T (ST) antigens were found to be the most abundant glycans in cell lines and over 900 glycoproteins were identified potentially carrying these glycans. HOMER3, typically a cytosolic protein, emerged as a top-ranked targetable glycoprotein at the cell surface carrying short-chain O-glycans. Plasma membrane HOMER3 was observed in more aggressive primary tumours and distant metastases, being an independent predictor of worst prognosis. This phenotype was triggered by nutrient deprivation and concomitant to increased cellular invasion. T24 HOMER3 knockdown significantly decreased proliferation and, to some extent, invasion in normoxia and hypoxia; whereas HOMER3 knock-in increased its membrane expression, which was more pronounced under glucose deprivation. HOMER3 overexpression was associated with increased cell proliferation in normoxia and potentiated invasion under hypoxia. Finally, the mapping of HOMER3-glycosites by EThcD-MS/MS in bladder tumours revealed potentially targetable domains not detected in healthy tissues. Conclusion HOMER3-glycoforms allow the identification of patients’ subsets facing worst prognosis, holding potential to address more aggressive hypoxic cells with limited off-target effects. The molecular rationale for identifying novel bladder cancer molecular targets has been established. Graphical abstract

Circulating tumour cells (CTC) are rare cells that actively detach or are shed from primary tumours into the lymph and blood. Some CTC subpopulations gain the capacity to survive, home and colonize distant locations, forming metastasis. This results from a multifactorial process in which cancer cells optimize motility, invasion, immune escape and cooperative relationships with microenvironmental cues. Here we present evidences of a self-fuelling molecular crosstalk between cancer cells and the tumour stroma supporting the main milestones leading to metastasis. We discuss how the tumour microenvironment supports pre-metastatic niches and CTC development and ultimately dictates CTC fate in targeted organs. Finally, we highlight the key role played by protein glycosylation in metastasis development, its prompt response to microenvironmental stimuli and the tremendous potential of glycan-based molecular signatures for liquid biopsies and targeted therapeutics.

Background: Gastric cancer (GC) is a major health burden worldwide, with half of patients developing metastases within 5 years after treatment, urging novel biomarkers for diagnosis and efficient therapeutic targeting. Sialyl-Lewis A (SLeA), a terminal glycoepitope of glycoproteins and glycolipids, offers tremendous potential towards this objective. It is rarely expressed in healthy tissues and blood cells, while it is present in highly metastatic cell lines and metastases. SLeA is also involved in E-selectin mediated metastasis, making it an ideal target to control disease dissemination. Methods and Results: To improve cancer specificity, we have explored the SLeA-glycoproteome of six GC cell models, with emphasis on glycoproteins showing affinity for E-selectin. A novel bioinformatics-assisted algorithm identified nucleolin (NCL), a nuclear protein, as a potential targetable biomarker potentially involved in metastasis. Several immunoassays, including Western blot and in situ proximity ligation reinforced the existence of cell surface NCL-SLeA glycoforms in GC. The NCL-SLeA glycophenotype was associated with decreased survival and was not reflected in relevant healthy tissues. Conclusions: NCL-SLeA is a biomarker of poor prognosis in GC holding potential for precise cancer targeting. This is the first report describing SLeA in preferentially nuclear protein, setting a new paradigm for cancer biomarkers discovery and targeted therapies. The authors wish to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the human resources grants: PhD grant SFRH/BD/103571/2014 (EF), SFRH/BD/111242/2015 (AP), SFRH/BD/146500/2019 (MRS), SFRH/BD/142479/2018 (JS), SFRH/BD/105355/2014 (RA), SFRH/BD/127327/2016 (CG); and assistant researcher grant CEECIND/03186/2017 (JAF). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge FCT funds for CI-IPOP research unit (PEst-OE/SAU/UI0776/201), CI-IPOP funds (CI-IPOP-29-2014; CI-IPOP-58-2015; CI-IPOP-Proj.70-bolsa2019-GPTE), FCT/MCTES funds for the LAQV research unit (UIDB/50006/2020) and PhD Programs in Biomedical Sciences and Pathology and Molecular Genetics of ICBAS-University of Porto. The authors EF, DF and MRS also acknowledge the financial support of the Research Group of Digestive Cancers (GICD), the ?Early stage cancer treatment, driven by context of molecular imaging (ESTIMA)? framework (NORTE-01-0145-FEDER-000027), IPO-Score (DSAIPA/DS/0042/2018) and ?Towards a single therapy with a synergistic combination against triple negative breast cancer and neuroblastoma by nucleolin mediated multicellular targeting? (EU joint call ENMed/0006/2015). This article is also a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work was financed by FEDER funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by FCT/MCTES funds in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274).

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, with half of patients developing metastases within 5 years after treatment, urging the need for novel biomarkers for diagnosis and more efficient therapeutic targeting. The sialyl-Lewis A (SLeA) antigen, a terminal glycoepitope of glycoproteins and glycolipids, offers tremendous potential towards this objective. This glycan is rarely expressed by healthy tissues and blood cells but is often present in cancer cells of high metastatic potential, as well as in the metastases. The SLeA antigen is also directly involved in E-selectin mediated metastasis, making it an ideal target to control disease dissemination. To improve further on cancer specificity, we have zoomed in on the SLeA-glycoproteome of six distinct GC cell models. Emphasis was set on glycoproteins showing affinity for E-selectin. A bioinformatics-assisted algorithm, used for the first time in this study, identified nucleolin (NCL), a nuclear protein frequently observed at the cell-surface in cancer cells, as a potential targetable biomarker potentially involved in metastasis development. Several immunoassays, including western blot and in situ proximity ligation reinforced the existence of NCL-SLeA glycoforms at the cancer cells surface and in GC. The NCL-SLeA glycophenotype was associated with decreased survival and was not reflected in relevant healthy tissues, supporting its tremendous potential for precise cancer targeting with limited off-target effects. To our knowledge, this is also the first report describing SLeA in a protein preferentially found in the nucleus, setting a new paradigm for cancer biomarkers discovery and targeted therapies. Funding Statement: Portuguese Foundation for Science and Technology (FCT) for the human resources grants: PhD grant BD/103571/2014 (EF), SFRH/BD/111242/2015 (AP), SFRH/BD/146500/2019 (MRS), SFRH/BD/142479/2018 (JS), SFRH/BD/105355/2014 (RA), SFRH/BD/127327/2016 (CG); and FCT assistant researcher grant CEECIND/03186/2017 (JAF). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge FCT the funding for CI-IPOP research unit (PEst-OE/SAU/UI0776/201), the Portuguese Oncology Institute of Porto Research Centre (CI-IPOP-29-2014; CI-IPOP-58-2015; CI-IPOPProj.70-bolsa2019-GPTE) and PhD Programs in Biomedical Sciences and Pathology and Molecular Genetics of ICBAS-University of Porto. The authors EF, DF and MRS also acknowledge the financial support of the Research Group of Digestive Cancers (GICD) and the “Early stage cancer treatment, driven by context of molecular imaging (ESTIMA)” framework (NORTE-01-0145-FEDER-000027), IPO-Score (DSAIPA/DS/0042/2018) and “Towards a single therapy with a synergistic combination against triple negative breast cancer and neuroblastoma by nucleolin mediated multicellular targeting” (EU joint call ENMed/0006/2015) for financial support. This article is also a result of the project NORTE-01-0145-FEDER-000012, supported by Norte Portugal Regional Operational 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 the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundacao para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia e Ensino Superior in the framework of the project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274). Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: All procedures concerning the inclusion of patients were approved by the institutional Ethics Committee after patient’s informed consent.

Bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation on the cell surface, which may be explored for improving disease management. In a search for prognosis biomarkers and novel therapeutic targets we have screened, using immunohistochemistry, a series of bladder tumours with differing clinicopathology for short-chain O-glycans commonly found in glycoproteins of human solid tumours. These included the Tn and T antigens and their sialylated counterparts sialyl-Tn(STn) and sialyl-T(ST), which are generally associated with poor prognosis. We have also explored the nature of T antigen sialylation, namely the sialyl-3-T(S3T) and sialyl-6-T(S6T) sialoforms, based on combinations of enzymatic treatments. We observed a predominance of sialoglycans over neutral glycoforms (Tn and T antigens) in bladder tumours. In particular, the STn antigen was associated with high-grade disease and muscle invasion, in accordance with our previous observations. The S3T and S6T antigens were detected for the first time in bladder tumours, but not in healthy urothelia, highlighting their cancer-specific nature. These glycans were also overexpressed in advanced lesions, especially in cases showing muscle invasion. Glycoproteomic analyses of advanced bladder tumours based on enzymatic treatments, Vicia villosa lectin-affinity chromatography enrichment and nanoLC-ESI-MS/MS analysis resulted in the identification of several key cancer-associated glycoproteins (MUC16, CD44, integrins) carrying altered glycosylation. Of particular interest were MUC16 STn+ -glycoforms, characteristic of ovarian cancers, which were found in a subset of advanced-stage bladder tumours facing the worst prognosis. In summary, significant alterations in the O-glycome and O-glycoproteome of bladder tumours hold promise for the development of novel noninvasive diagnostic tools and targeted therapeutics. Furthermore, abnormal MUC16 glycoforms hold potential as surrogate biomarkers of poor prognosis and unique molecular signatures for designing highly specific targeted therapeutics.

Esophageal cancer (EC) is a life-threatening disease, demanding the discovery of new biomarkers and molecular targets for precision oncology. Aberrantly glycosylated proteins hold tremendous potential towards this objective. In the current study, a series of esophageal squamous cell carcinomas (ESCC) and EC-derived circulating tumor cells (CTCs) were screened by immunoassays for the sialyl-Tn (STn) antigen, a glycan rarely expressed in healthy tissues and widely observed in aggressive gastrointestinal cancers. An ESCC cell model was glycoengineered to express STn and characterized in relation to cell proliferation and invasion in vitro. STn was found to be widely present in ESCC (70% of tumors) and in CTCs in 20% of patients, being associated with general recurrence and reduced survival. Furthermore, STn expression in ESCC cells increased invasion in vitro, while reducing cancer cells proliferation. In parallel, an ESCC mass spectrometry-based proteomics dataset, obtained from the PRIDE database, was comprehensively interrogated for abnormally glycosylated proteins. Data integration with the Target Score, an algorithm developed in-house, pinpointed the glucose transporter type 1 (GLUT1) as a biomarker of poor prognosis. GLUT1-STn glycoproteoforms were latter identified in tumor tissues in patients facing worst prognosis. Furthermore, healthy human tissues analysis suggested that STn glycosylation provided cancer specificity to GLUT1. In conclusion, STn is a biomarker of worst prognosis in EC and GLUT1-STn glycoforms may be used to increase its specificity on the stratification and targeting of aggressive ESCC forms. The authors wish to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the human resources grants: PhD grant SFRH/BD/111242/2015 (AP), SFRH/BD/146500/2019 (MRS), SFRH/BD/142479/2018 (JS), SFRH/BD/105355/2014 (RA) and FCT assistant researcher grant CEECIND/03186/2017 (JAF). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The authors also acknowledge FCT the funding for CI-IPOP research unit (PEst-OE/SAU/UI0776/201) and LAQV-REQUIMTE research unit (UIDB/50006/2020), the Portuguese Oncology Institute of Porto Research Centre (CI-IPOP-29-2016-2020; CI-IPOP-58-2016-2020; CI-IPOP-Proj.70-bolsa2019-GPTE) and PhD Program in Biomedical Sciences of ICBAS-University of Porto. The author also thanks “Early stage cancer treatment, driven by context of molecular imaging (ESTIMA)” framework (NORTE-01-0145-FEDER-000027) and IPO-Score (DSAIPA/DS/0042/2018) for financial support. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020 and by Portuguese funds through FCT/MCTES—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior. The authors also acknowledge support from the Portuguese League against Cancer grant LPCC-NRN-2020 (DF).

Purpose Eastern Africa was recently described as a high-incidence geographic area for esophageal cancer. Mozambique is included in this region. This study aimed to characterize this malignant disease at Maputo Central Hospital (MCH) to develop a global program for esophageal cancer management in Mozambique. Methods MCH records from between 2012 and 2016 were used to assess the clinical, pathologic, and outcome profiles of esophageal tumors. A descriptive analysis of data collected was performed. Overall survival was evaluated using Kaplan-Meier curves. Results In the study, 522 consecutive patient cases of esophageal cancer were recorded. The median patient age was 56.1 years (range, 27 to 97 years); 291 (55.7%) patients were women, and 230 (44.1%) were men. Regarding tumor site, 113 patients (21.6%) had a tumor in the lower third, 154 (29.5%) in the middle, and 50 (9.6%) in the upper third of the esophagus; in the remaining 196 (37.5%), tumor site was unknown. Squamous cell carcinoma comprised 94.4% of cases with documented histopathology (74.9% of the sample). Surgical treatment was possible in 32 patients (6.1%). Disease stage was documented only in these 32 surgical patients; 28.1%, 53.1%, and 18.8% had stage I, II, and III disease, respectively. The remaining patient cases seemed to involve clinically advanced tumors. The median follow-up time was of 1.6 months. The median survival time was of 3.5 months for all patients; for patients treated with curative intent, it was of 8.7 months. Conclusion Esophageal carcinoma is a common malignant tumor at MCH and is diagnosed in the advanced stages resulting in poor prognosis. Therefore, implementation of an Esophageal Cancer Program in Mozambique is essential.

Bladder Cancer (BC) presents one of the highest recurrence rates amongst solid tumours and constitutes the second deadliest disease of the genitourinary track. Non-invasive identification of patients facing disease recurrence and/or progression remains one of the most critical and challenging aspects in disease management. To contribute to this goal, we demonstrate the potential of glycan-affinity glycoproteomics nanoplatforms for urinary biomarkers discovery in bladder cancer. Briefly, magnetic nanoprobes (MNP) coated with three broad-spectrum lectins, namely Concanavalin A (ConA; MNP@ConA), Wheat Germ Agglutinin (WGA; MNP@WGA), and Sambucus nigra (SNA; MNP@SNA), were used to selectively capture glycoproteins from the urine of low-grade and high-grade non-muscle invasive as well as muscle-invasive BC patients. Proteins were identified by nano-LC MALDI-TOF/TOF and data was curated using bioinformatics tools (UniProt, NetOGlyc, NetNGlyc, ClueGO app for Cytoscape and Oncomine) to highlight clinically relevant species. Accordingly, 63 glycoproteins were exclusively identified in cancer samples compared with healthy controls matching in age and gender. Specific glycoprotein sets exclusively found in low-grade non-muscle invasive bladder tumours may aid early diagnosis, while those only found in high-grade non-invasive and muscle-invasive tumours hold potential for accessing progression. Amongst these proteins is bladder cancer stem-cell marker CD44, which has been associated with poor prognosis. Orthogonal validation studies by slot-blotting demonstrated an elevation in urine CD44 levels of high-grade patients, which became more pronounced upon muscle-invasion, in mimicry of the primary tumour. These observations demonstrate the potential of MNP@lectins for identification of clinically relevant glycoproteomics signatures in bladder cancer. Future clinical validation in a larger and well characterized patient subset is required envisaging clinical translation of the results. published

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.

Context Circulating tumor cells (CTC) in peripheral blood of cancer patients provide an opportunity for real-time liquid biopsies capable of aiding early intervention, therapeutic decision, response to therapy, and prognostication. Nevertheless, the rare and potentially heterogeneous molecular nature of CTC has delayed the standardization of robust high-throughput capture/enrichment and characterization technologies. Objective This review aims to systematize emerging solutions for CTC analysis in bladder cancer (BC), their opportunities and limitations, while providing key insights on specific technologic aspects that may ultimately guide molecular studies and clinical implementation. Evidence acquisition State-of-the-art screening for CTC technologies and clinical applications in BC was conducted in MEDLINE through PubMed. Evidence synthesis From 200 records identified by the search query, 25 original studies and 1 meta-analysis met the full criteria for selection. A significant myriad of CTC technological platforms, including immunoaffinity, biophysical, and direct CTC detection by molecular methods have been presented. Despite their preliminary nature and irrespective of the applied technology, most studies concluded that CTC counts in peripheral blood correlated with metastasis. Associations with advanced tumor stage and grade and worst prognosis have been suggested. However, the unspecific nature, low sensitivity, and the lack of standardization of current methods still constitutes a major drawback. Moreover, few comprehensive molecular studies have been conducted on these poorly known class of malignant cells. Conclusion The current rationale supports the importance of moving the CTC field beyond proof of concept studies toward molecular-based solutions capable of improving disease management. The road has been paved for identification of highly specific CTC biomarkers and novel targeted approaches, foreseeing successful clinical applications.

Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may offer potential to treat tumour hypoxic niches harbouring more malignant cells.