Identification of sialic acid linkage profiles in primary liver cancers

Cholangiocarcinoma (CCA), also known as bile duct cancer, is the second most common form of primary liver cancer after hepatocellular carcinoma (HCC). Subtypes of CCA are diagnosed according to the anatomic location within the biliary tree, such as intrahepatic (iCCA) or extrahepatic (eCCA). Early diagnosis remains difficult due to the low sensitivity and specificity of current biomarkers. There has been significant interest in finding new biomarkers of CCA and much work has been performed to both discover and validate these new markers. In our previous work, we identified specific alterations in N-linked glycosylation that were associated with iCCA, which differed from non-malignant cholestasis disease and HCC. However, that analysis focused only on N-glycan structures devoid of sialic acid, as these N-glycan residues are not stable and were intentionally removed to minimize variability. Here, we explore the specificity of sialic acid residues in liver cancers, since sialic acid residues have been heavily linked to roles in tumor progression and metastasis by helping evade immunological surveillance. To circumvent the instability of sialic acid, we utilize a novel sialic acid stabilization method coupled with Matrix-Assisted Laser Desorption Ionization (MALDI) Imaging Mass Spectrometry (IMS) for detailed analysis of sialic acid linkages in iCCA tissue. We report a clear association between the linkages type of sialic acid residues with a HCC or CCA, including differentiation between CCA subtypes. Overall, we stabilize N-glycans with sialic acid residues to determine their linkages and elucidate their importance in different types of primary liver cancers and their subtypes by MALDI-IMS.