Analysis of N-linked Glycan Alterations in Tissue and Serum Reveals Promising Biomarkers for Intrahepatic Cholangiocarcinoma

Abstract

There is an urgent need for the identification of reliable prognostic biomarkers for patients with intrahepatic cholangiocarcinoma (iCCA) and alterations in N-glycosylation have demonstrated an immense potential to be used as diagnostic strategies for many cancers, including hepatocellular carcinoma (HCC). N-glycosylation is one of the most common post-translational modifications known to be altered based on the status of the cell. N-glycan structures on glycoproteins can be modified based on the addition or removal of specific N-glycan residues, some which have been linked to liver diseases. However, little is known concerning the N-glycan alterations that are associated with iCCA. We characterized the N-glycan modifications quantitatively and qualitatively in three cohorts, consisting of two tissue cohorts: a discovery cohort (n=104 cases) and a validation cohort (n=75), and one independent serum cohort consisting of patients with iCCA, HCC, or benign chronic liver disease (n=67). N-glycan analysis in-situ was correlated to tumor regions annotated on histopathology and revealed that bisected fucosylated N-glycan structures were specific to iCCA tumor regions. These same N-glycan modifications were significantly upregulated in iCCA tissue and serum relative to HCC and bile duct disease, including primary sclerosing cholangitis (PSC) (p<0.0001). N-glycan modifications identified in iCCA tissue and serum were used to generate an algorithm that could be used as a biomarker of iCCA. We demonstrate that this biomarker algorithm quadrupled the sensitivity (at 90% specificity) of iCCA detection as compared to CA19-9, the current “gold standard” biomarker of CCA.

Terminal fucosylation of haptoglobin in cancer-derived exosomes during cholangiocarcinoma progression

“…Tumor glycosylation has been proposed as potential cancer biomarkers (6, 7). Many studies have focused on characterizing the aberrant glycosylation of secreted molecules circulating the blood in their soluble form (35). In this study, we provide new evidence that terminal fucosylation of membrane-bound components correlated EVs correlates with CCA.…”

“…Glycomic analysis revealed that CCA-EV contained more α1-3/4 fucosylated glycans (terminal fucosylation) than normal EVs. Notably however, α1-6 fucose (core fucosylation) was comparable between samples, and bisected fucosylated structures reported to increase in tissues of intrahepatic CCA could not be found ( 36 ). Our current approach did not distinguish the linkage of terminal α-L-fucose attached to tri-antennary structures.…”

Spatial Omics Reveals that Cancer-Associated Glycan Changes Occur Early in Liver Disease Development in a Western Diet Mouse Model of MASLD

“…To characterize the N-linked glycan profile in liver disease progression, we used MALDI-IMS. − , MALDI-IMS allows for qualitative analysis of N-linked glycan structures in situ. We were first interested in the full-tissue N-glycome characterization of MASL/MASH progression based on the type of N-glycans that was identified in all groups.…”

“…Liver tissues were harvested at necropsy at respective time points and processed for formalin-fixed paraffin-embedded (FFPE) sample preparation. Tissue blocks were cut into 5 μm sections and processed for N-glycan matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) following previously published protocols. , In summary, the FFPE liver sections were first heated at 60 °C for 1 h, followed by dewaxing and rehydrating steps including xylene, ethanol, and water washes. Next, the tissues were taken through antigen retrieval for 20 min at 95 °C in citraconic buffer at pH 3.0.…”

Imaging of N-Linked Glycans in Biological Tissue Sections Using Nanospray Desorption Electrospray Ionization (nano-DESI) Mass Spectrometry

“…We selected HCC tissue as a model system that has been previously studied using MALDI MSI. − HCC is the most common form of liver cancer accounting for ∼90% of all liver cancer cases and ranking as the third leading cause of cancer-related deaths in 2020. , N-linked glycan distribution has been identified as potential biomarkers of HCC, making this tissue an important target for understanding glycosylation using MSI. − We used nano-DESI MSI to distinguish regions of interest based on the localization of N-linked glycans in HCC tissue. An image of the H&E-stained tissue shown in Figure A displays distinct regions, including hepatocytes, stroma/extracellular matrix, and tumors highlighted in Figure B with green, red, and blue, respectively.…”