The link between aberrant glycosylation and cancer progression has formed the basis for detection of cell surface glycans and glycoproteins in the clinical management of cancer. However, the utility of biomarkers has been challenged in recent years due to low cancer specificity and high false-positive rates. The aim of this study was to use synthetic precursors of sialic acid and N-acetylgalactosamine that had been labelled with an azide group (N-azidoacetylmannosamine (Ac4ManNAz) and N-azidoacetylgalactosamine (Ac4GalNAz)), and which are incorporated biosynthetically into cell surface glycans, to monitor changes in cell surface glycosylation in models of prostate and breast cancer progression, in order to identify novel biomarkers.

A panel of human prostate, breast and pancreatic cancer cell lines was used, which represented the stages of cancer progression. The metastatic potential of the established prostate and breast cancer cell lines used in the study was confirmed in the scratch wound and Boyden chamber assays and by demonstrating a correlation with increased expression of vimentin, loss of PTEN expression, and decreased E-cadherin expression. Collectively, the data validated the panels as suitable in vitro models of prostate and breast cancer progression.

Global azidosugar labelling of glycoproteins and glycolipids was detected by NeutrAvidinTM DyLightTM 650 (NA650) fluorescence using flow cytometry. Azidosugar labelling of glycoproteins was detected by Alkyne-Alexa Fluor 488 fluorescence using 1D gel electrophoresis, and Streptavidin 800 fluorescence using 2D gel electrophoresis. An increase in the overall NA650 fluorescence was seen across the breast cancer cell lines using flow cytometry. An increase in the overall Alexa-488 fluorescence on 1D gels and Streptavidin- 800 fluorescence on 2D gels was seen across the prostate, breast, and pancreatic cancer cell lines. A general increase in azidosugar-specific sialylation (Ac4ManNAz) and N- acetylgalactosamine (Ac4GalNAz) glycosylation was associated with an increase in metastatic potential. Proteomic analysis of the prostate cancer cell lines revealed that Basigin (CD147) glycosylation, which showed a strong increase in Ac4ManNAz and Ac4GalNAz labelling, correlated with an increase in metastatic potential, despite its total expression decreasing across the panel of prostate cancer cell lines.

In summary, we have demonstrated the use of bioorthogonal chemistry for detecting differential sialyation and N-acetylgalactosamine glycosylation of proteins from human cell lines that represent the progression of cancer. Our data suggest that differential glycosylation may be a more sensitive and specific marker for monitoring prostate and breast cancer progression, than increases in total protein expression alone.