Caracterização glicoproteómica de cancro avançado de bexiga direcionado para novas terapias

Mestrado em Biologia Molecular e CelularA heterogenidade da natureza molecular dos tumores de bexiga tem dificultado o estabelecimento de abordagens no campo da medicina de precisão, revelando-se a necessidade de terapias mais eficientes e novas ferramentas de detecção não-invasivas. Contudo, têm-se denotado um desenvolvimento no estudo da carcinogénese de bexiga e na progressão do tumor, acompanhado de profundas alterações na glicosilação de proteínas que, dada a sua superfície celular e a natureza secretada, apresenta um potencial elevado na melhoria da gestão da doença. Segundo esta abordagem foi efectuado um estudo sobre tumores de bexiga de diferentes naturezas clinicopatológicas para O-glicanos de cadeia curta, regularmente encontrados na maioria dos tumores sólidos, recorrendo-se à imunohistoquímica. O estudo incluiu os antígenos Tn e T e os seus homólogos sialilados sialil-Tn (STn) e sialil-T (ST), geralmente associados com um mau prognóstico. Explorou-se ainda a sialilação da natureza dos antigénios T, especificamente as sialoformas sialil-3-T (S3T) e sialil-6-T (S6T), com base em combinações de tratamentos enzimáticos. Observou-se uma predominância de sialoglicanos, em comparação com as glicoformas neutras (antígenos Tn e T) em tumores de bexiga. Em particular, o antigénio STn foi associado ao estado avançado da doença e invasão muscular. Os antígenos S3T e S6T foram detectados pela primeira vez em tumores de bexiga, estando ausentes no urotélio normal, permitindo destacar a natureza específica em tumores. Verificou-se também a sobreexpressão dos glicanos em lesões avançadas, especialmente nos casos com invasão muscular.As análises glicoproteómicas dos tumores avançados de bexiga permitiram identificar diversas glicoproteínas-chave associadas ao cancro (MUC16, CD44, integrinas), denotando uma glicosilação alterada.As glicoformas da MUC16 STN positivas, características do cancro de ovário, encontram-se num subconjunto de tumores de bexiga em estado avançado, com um pior prognóstico. Em suma, os tumores de bexiga apresentam severas alterações no O-glicoma e no Oglicoproteoma devendo ser abordados de forma abrangente com o objectivo de desenvolver ferramentas de diagnóstico não invasivas e terapias dirigidas. As glicoformas aberrantes de MUC16 apresentam potencial como biomarcadores de mau prognóstico. Este trabalho estabeleceu um guia para a descoberta de glicobiomarcadores no cancro de bexiga, que pode ser utilizado para a estratificação dos pacientes e, por fim, levar à descoberta de novos alvos terapêuticos.The heterogeneous molecular nature of bladder tumours has hampered the establishment of precision medicine approaches, more efficient therapeutics and novel non-invasive detection tools. Still, it has been long described that bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation which, given its cell surface and secreted nature, holds tremendous potential for disease management improvement. Therefore, we have screened series of bladder tumours of different clinicopathological natures for short-chain O-glycans, found in most solid tumours, by immunohistochemistry. These included the Tn and T antigens and their sialylated counterparts sialyl-Tn (STn) and sialyl-T(ST), generally associated with poor prognosis. We have also explored the nature of T antigens 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 identified several key cancer-associated glycoproteins (MUC16, CD44, integrins) carrying altered glycosylation. Particular interest was devoted to MUC16 STn+-glycoforms, characteristic of ovarian cancers, which were found in a subset of advanced stage bladder tumours facing worst prognosis. In summary, bladder tumours present severe O-glycome and O-glycoproteome alterations that should be comprehensively addressed envisaging novel non-invasive diagnostic tools and targeted therapeutics. Furthermore, abnormal MUC16 glycoforms holds potential as surrogate biomarkers of poor prognosis. Finally, this work established a roadmap for glycobiomarker discovery in bladder cancer, which may be used for patient stratification and ultimately lead to novel therapeutic targets

Glycan affinity magnetic nanoplatforms for urinary glycobiomarkers discovery in bladder cancer

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.publishe

Target Score-A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness

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

Aqueous batteries as grid scale energy storage solutions

Energy storage technologies are required to make full use of renewable energy sources, and electrochemical cells offer a great deal flexibility in the design of energy systems. For large scale electrochemical storage to be viable, the materials employed and device production methods need to be low cost, devices should be long lasting and safety during operation is of utmost importance. Energy and power densities are of lesser concern. For these reasons, battery chemistries that make use of aqueous electrolytes are favorable candidates where large quantities of energy need to be stored. Herein we describe several different aqueous based battery chemistries and identify some of the research challenges currently hindering their wider adoption. Lead acid batteries represent a mature technology that currently dominates the battery market, however there remain challenges that may prevent their future use at the large scale. Nickel–iron batteries have received a resurgence of interest of late and are known for their long cycle lives and robust nature however improvements in efficiency are needed in order to make them competitive. Other technologies that use aqueous electrolytes and have the potential to be useful in future large-scale applications are briefly introduced. Recent investigations in to the design of nickel–iron cells are reported with it being shown that electrolyte decomposition can be virtually eliminated by employing relatively large concentrations of iron sulfide in the electrode mixture, however this is at the expense of capacity and cycle life

Transcript identification in the BRCA1 candidate region

Chromosome 17q12-21 is known to contain a gene (or genes) which confers susceptibility to early-onset breast cancer and ovarian cancer (BRCA1). Identification and isolation of BRCA1 will likely provide the basis for increased understanding of the pathogenesis of breast and ovarian cancer, the development of targeted diagnostic and therapeutic approaches, and a means of screening women at risk of being BRCA1 mutation carriers. Genetic and physical maps of the BRCA1 candidate region have been largely completed and efforts are being directed at identification of candidate genes from within this region. We have begun the task of identifying transcripts from this region employing three complementary strategies. These include: 1) direct cDNA screening with cosmids derived from the BRCA1 region; 2) exon amplification; and 3) magnetic bead capture. Transcripts identified using these approaches are being characterized for: 1) tissue expression pattern; 2) the presence of genomic rearrangement in DNA derived from affected members of families believed to show linkage between breast cancer and genetic markers in the BRCA1 candidate interval; 3) altered size and/or expression pattern in RNA prepared from such individuals; and 4) homology to known genes or functional motifs. Germline mutations in affected individuals from these families will serve as presumptive evidence of BRCA1 identity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44201/1/10549_2004_Article_BF00682719.pd

Discriminating Epithelial to Mesenchymal Transition Phenotypes in Circulating Tumor Cells Isolated from Advanced Gastrointestinal Cancer Patients

Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes

Target Score—A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness

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