Quantitative Glycome Analysis of N‑Glycan Patterns in Bladder Cancer vs Normal Bladder Cells Using an Integrated Strategy

Diagnosis
of bladder cancer, one of the most common types of human
cancer, at an early (nonmuscle-invasive) stage is the best way to
reduce the mortality rate. Tumor malignancy in general is closely
associated with alterations of glycan expression. Glycosylation status,
particularly global glycomes, in bladder cancer has not been well
studied. We integrated lectin microarray and mass spectrometry (MS)
methods to quantitatively analyze and compare glycan expression in
four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal
bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed
using lectin microarray analysis and confirmed by lectin staining
and lectin blotting. Associations of glycopatterns with diverging
stages were evaluated by lectin histochemistry on tissue microarrays.
N-Glycans were derivatized by amidation of sialylated glycans with
acetohydrazide and reductive amination with the stable isotope tags
[12C6]- and [13C6]-aniline,
and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight
mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated
proteins were quantitatively analyzed by a stable isotope labeling
by amino acids in cell culture (SILAC) proteomics method, which revealed
significant differences in expression of 13 glycosyltransferases and
4 glycosidases. Our findings indicate that sialyl Lewis X (sLex), terminal GalNAc and Gal, and high mannose-type N-glycans
were more highly expressed in bladder cancer cells and tissues than
in normal cells. Bladder cancer cells showed high expression of core-fucosylated
N-glycans but low expression of terminally fucosylated N-glycans.
Each of these glycome changes may be directly related to bladder cancer
progression.