High-Mannose N-Glycans as Malignant Progression Markers in Early-Stage Colorectal Cancer

Abstract

The increase incidence of early colorectal cancer (T1 CRC) last years is mainly due to the introduction of population-based screening for CRC. T1 CRC staging based on histological criteria remains challenging and there is high variability among pathologists in the scoring of these criteria. It is crucial to unravel the biology behind the progression of adenoma into T1 CRC. Glycomic studies have reported extensively on alterations of the N-glycomic pattern in CRC; therefore, investigating these alterations may reveal new insights into the development of T1 CRC. We used matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) to spatially profile the N-glycan species in a cohort of pT1 CRC using archival formalin-fixed and paraffin-embedded (FFPE) material. To generate structural information on the observed N-glycans, CE-ESI-MS/MS was used in conjunction with MALDI-MSI. Relative intensities and glycosylation traits were calculated based on a panel of 58 N-glycans. Our analysis showed pronounced differences between normal epithelium, dysplastic, and carcinoma regions. High-mannose-type N-glycans were higher in the dysplastic region than in carcinoma, which correlates to increased proliferation of the cells. We observed changes in the cancer invasive front, including higher expression of α2,3-linked sialic acids which followed the glycosylation pattern of the carcinoma region.

Keywords: MALDI-MSI; N-glycosylation; colorectal cancer; early cancer progression; mass spectrometry imaging; molecular histology.

Figure 1

Morphological and molecular progression of CRC. (A) A schematic representation of the morphological progression from normal colon epithelium to dysplasia and ultimately invasive carcinoma. (B) Examples of the different morphological stages of CRC development (40× magnification) using H&E stained tissues. The scale bars represent 100 µm. (C) The molecular progression of normal colon epithelium to CRC on the glycomic level. The glycome, shown here as different N-glycosylation traits, change throughout the progression to CRC.

Figure 2

High-mannose N-glycans are abundant in proliferative cells. (A) The abundance of high-mannose-type N-glycans changes with progression through the morphological spectrum of T1 CRC development. (B) The changes in number of proliferative cells (Ki-67⁺) cells shows a similar pattern to the changes in high-mannose N-glycans. (C) All individual high-mannose-type N-glycans contribute and change in a similar fashion to the high-mannose trait depicted in (A). * = p-value ≤ 0.05, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.

Figure 3

Glycosylation trait image. H&E staining of three annotated slides are depicted at the left side and the remaining images are three reconstructed derived traits in the example tissues. The scale represents the relative expression of the traits.