Fig 1.
Biosynthetic pathways of mucin-type O-glycans.
β3Gn-T6 is the only core 3 synthetic enzyme. GalNAc: N-Acetylgalactosamine, GlcNAc: N-Acetylglucosamine, NeuAc: N-Acetylneuraminic acid.
Table 1.
Correlations between Exp-scores of β3Gn-T6 and clinicopathological variables.
Table 2.
Primary antibodies and lectins.
Fig 2.
Representative microscopic images of immunohistochemistry and lectin-histochemistry in PDAC tissues.
Middle-power view of tissues stained with (A) HE, (B) β3Gn-T6, (C) T antigen (PNA), (D) Tn antigen (LU-35), (E) 6-sulfo N-acetyllactosamine on extended core 1 O-glycan (MECA-79), (F) sLeX (CSLEX1), (G) sLeX (HECA-452), (H) sLeX on core 2 O-glycan (ST-439) and (I) MUC5AC. All these sections show the same region of PDAC tissue. In the left half of the panel, intraductal spreading of adenocarcinoma cells is seen, and invasive adenocarcinomas are present in the right half. All these antigens are expressed to various degrees of heterogeneity in the adenocarcinoma cells, even within the same case. Different staining patterns of sLeX are observed depending on the specificity of the antibodies used as described in the main text (F–H).
Fig 3.
Comparison of glycan antigens and β3Gn-T6 expression among PDACs (n = 156), PanINs (n = 26), and normal pancreatic tissues (n = 35).
((A) β3Gn-T6, (B) T antigen (PNA), (C) Tn antigen (LU-35), (D) 6-sulfo N-acetyllactosamine on extended core 1 O-glycan (MECA-79), (E) sLeX (CSLEX1), (F) sLeX (HECA-452), and (G) sLeX on core 2 O-glycan (ST-439). Boxes represent medians and interquartile ranges. Crosses represent mean values. Whiskers represent the minimum and maximum 1.5 interquartile ranges. Circles represent extremes. All glycan antigens, except MECA-79 antigen, are expressed significantly more highly in PDAC cells than in NPDEs. MECA-79 antigen expression in PDAC cells is significantly lower than that in NPDEs. Exp-scores were compared and analyzed using the Friedman’s test.
Table 3.
Summary of glycan and its related antigen expression.
Fig 4.
Double immunohistochemistry for β3Gn-T6 and MUC5AC.
Low-power view of histological staining (A) and double immunohistochemical staining (B) and high-power view of double immunohistochemical staining (C). Dotlike staining of β3Gn-T6 (green) and membranous staining of MUC5AC (brown) are often present in the same adenocarcinoma cells.
Table 4.
Correlations among glycans and related proteins (Spearman's correlation coefficient value).
Fig 5.
MUC5AC has core 3 O-glycan generated by B3GNT6 gene expression in pancreatic cancer cells.
(A) Comparison of expression of genes MUC5AC, B3GNT6, and core 2 O-glycan synthase among PDAC cells. Capan-1 cells express MUC5AC more highly than the other cell lines but do not express B3GNT6. (B) Capan1-B3GnT6 cells (right panel) but not Capan1-mock cells (left panel) express β3Gn-T6 as detected by the immunofluorescence assay (red). Nuclei are stained by 4’,6-diamindino-2-phenylindole (DAPI, blue). (C) MUC5AC immunoprecipitated with the anti-MUC5AC antibody was subjected to SDS-PAGE and transferred to a nitrocellulose membrane, then the membrane was blotted with GS-II, PNA, or the anti-MUC5AC antibody. The level of nonreducing terminal GlcNAc is higher in Capan1-B3GnT6 cells compared to Capan1-mock cells, even though the MUC5AC amount is comparable between these two cell lines. Conversely, the T antigen level is lower in Capan1-B3GnT6 cells compared to Capan1-mock cells.
Fig 6.
Kaplan-Meier survival curves for disease-free survival in patients with PDAC according to (A) β3Gn-T6, (B) T antigen (PNA), (C) Tn antigen (LU-35), (D) 6-sulfo N-acetyllactosamine on extended core 1 O-glycan (MECA-79), (E) sLeX (CSLEX1), (F) sLeX (HECA-452), and (G) sLeX on core 2 O-glycan (ST-439). Patients having PDAC with higher expression of β3Gn-T6 (red line) show a significantly longer survival compared to those with lower expression of β3Gn-T6 (blue line) in A. Patients having PDAC with higher expression of sLeX (CSLEX1) (red line) show a tendency to be shorter survival compared to those with lower expression of sLeX (CSLEX1) (blue line) in E. The other antigens are not significantly associated with patient outcome.
Table 5.
Univariate and multivariate analyses of prognostic factors associated with disease-free survival (A) and and overall survival (B) in patients with pancreatic ductal adenocarcinoma (n = 156).