Glycan affinity magnetic nanoplatforms for urinary glycobiomarkers discovery in bladder cancer

abstract

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 broadspectrum 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.

keywords

EXPRESSION; DIAGNOSIS; GLYCOPROTEINS; CYTOSCAPE; PROGNOSIS; CARCINOMA; BIOMARKER; CD44V6; TUMORS; RISK

subject category

Chemistry

authors

Azevedo, R; Soares, J; Gaiteiro, C; Peixoto, A; Lima, L; Ferreira, D; Relvas-Santos, M; Fernandes, E; Tavares, A; Cotton, S; Daniel-Da-Silya, AL; Santos, LL; Vitorino, R; Amado, F; Ferreira, JA

Groups

G1 - Porous Materials and Nanosystems

Projects

Projeto de Investigação Exploratória: Ana Luísa Silva (IF/00405/2014)

CICECO - Aveiro Institute of Materials (UID/CTM/50011/2013)

acknowledgements

The authors wish to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the human resources grants: Ph.D. grant SFRH/BD/105355/2014 (Rita Azevedo), SFRH/BD/111242/2015 (Andreia Peixoto), SFRH/BD/103571/2014 (Elisabete Fernandes), SFRH/BD/127327/2016 Cristiana Gaiteiro and Postdoctoral grants SFRH/BPD/101827/2014 (Luis Lima) and SFRH/BPD/111048/2015 (Jose Alexandre Ferreira) as well as for the contract IF/00405/2014 (Ana Luisa Daniel-da-Silva). 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 CICECO research unit (POCI-01-0145-FEDER-007679 and UID/CTM /50011/2013), the Portuguese Oncology Institute of Porto Research Centre (CI-IPOP-29-2014; CI-IPOP-58-2015) and Ph.D. Programs in Biomedicine and Pathology and Molecular Genetics of ICBAS-University of Porto.