Specific (sialyl-)Lewis core 2 O-glycans differentiate colorectal cancer from healthy colon epithelium

Abstract

Cells are covered with a dense layer of carbohydrates, some of which are solely present on neoplastic cells. The so-called tumor-associated carbohydrate antigens (TACAs) are increasingly recognized as promising targets for immunotherapy. These carbohydrates differ from those of the surrounding non-cancerous tissues and contribute to the malignant phenotype of the cancer cells by promoting proliferation, metastasis, and immunosuppression. However, due to tumor tissue heterogeneity and technological limitations, TACAs are insufficiently explored. Methods: A workflow was established to decode the colorectal cancer (CRC)-associated O-linked glycans from approximately 20,000 cell extracts. Extracts were obtained through laser capture microdissection of formalin fixed paraffin embedded tissues of both primary tumors and metastatic sites, and compared to healthy colon mucosa from the same patients. The released O-glycans were analyzed by porous graphitized carbon liquid chromatography-tandem mass spectrometry in negative ion mode. Results: Distinctive O-glycosylation features were found in cancerous, stromal and normal colon mucosal regions. Over 100 O-linked glycans were detected in cancerous regions with absence in normal mucosa. From those, six core 2 O-glycans were exclusively found in more than 33% of the cancers, carrying the terminal (sialyl-)Lewis^X/A antigen. Moreover, two O-glycans were present in 72% of the analyzed cancers and 94% of the investigated cancers expressed at least one of these two O-glycans. In contrast, normal colon mucosa predominantly expressed core 3 O-glycans, carrying α2-6-linked sialylation, (sulfo-)Lewis^X/A and Sda antigens. Conclusion: In this study, we present a novel panel of highly specific TACAs, based upon differences in the glycomic profiles between CRC and healthy colon mucosa. These TACAs are promising new targets for development of innovative cancer immune target therapies and lay the foundation for the targeted treatment of CRC.

Keywords: Tumor associated carbohydrate antigens (TACAs); glycomics; mass spectrometry; therapeutic target.

Figure 1

Tumor associated carbohydrate antigens identified in colorectal cancer. A subset of the O-glycans overexpressed in cancer which is detected in more than 33% of the cancers and undetectable in normal colon mucosa samples (A, B, C, D, E, F) or detected in maximum one normal mucosa (G). Either one of the two TACAs in (H) is detected in 94% of the cancers. The selected TACAs show higher specificity for invasive cancer (Dukes stages C and D, n = 12) than non-invasive cancer (Dukes stages A and B, n = 6) (right panel). Structures confirmed by synthesized standards (J. Weber et al., manuscript in preparation, 2022) are labeled with an asterisk. Blue square: N-acetylglucosamine, yellow square: N-acetylgalactosamine, yellow circle: galactose, red triangle: deoxyhexose, pink diamond: N-acetylneuraminic acid, S: sulfate modification.

Figure 2

Example of O-glycan chromatographic profile from (a) adenocarcinoma and (b) normal colon mucosa from the same patient (T8 vs C8). A) The adenocarcinoma from patient 8 is characterized by specific expression of core 2 O-glycans, carrying terminal Le^X and sialyl-Lewis^X/A antigens, or just terminal α2-3 sialylation linked to the galactose. Presence of TACAs (Figure 1) are circled with orange background. B) Normal colon mucosa from the same patient shows expression of a diversity of core 3 structures, in most cases carrying an α2-6 linked sialic acid linked to the core GalNAc. Core 3 O-glycans are carrying terminal Sda antigens, as well as Le^X/A and sulfo-Le^X/A epitopes. The TACAs observed in adenocarinoma are not detected in the normal colon mucosa from the same patient. Blue square: N-acetylglucosamine, yellow square: N-acetylgalactosamine, yellow circle: galactose, red triangle: deoxyhexose, pink diamond: N-acetylneuraminic acid, S: sulfate modification.

Figure 3

Structural O-glycan features that differentiate between cancer and normal colon mucosa. Statistically significant upregulation in cancer is found for various O-glycan features; A) Core 2 O-glycans, B) α2-3 sialylation, C) core 2 O-glycans with sLe^X/A and D) core 2 O-glycans with terminal α2-3 sialylation linked to a galactose together with E) one individual O-glycan with composition H2N2S2. Whereas, F) core 3 O-glycans, G) α2-6 sialylation of the core GalNAc, H) core 3 O-glycans with sLe^X/A antigen, I) core 3 O-glycans with terminal α2-3 sialylation, J) core 3 O-glycans with α2-6 sialylation, K) Sda antigen, L) Le^X/A and M) sulfo-Le^X/A antigen show statistically significant downregulation in cancer. Differences between groups were tested using Wilcoxon-Mann-Whitney non-parametric statistical test. Correction for multiple testing was made using the Benjamini-Hochberg method. ^# no p-value is reported as there is no variance in the control group. Blue square: N-acetylglucosamine, yellow square: N-acetylgalactosamine, yellow circle: galactose, red triangle: deoxyhexose, pink diamond: N-acetylneuraminic acid, S: sulfate modification.

Figure 4

Proposed biosynthetic model explaining the differences in glycosylation of colorectal cancer and normal colon mucosa. The most abundant structures in cancer (upper red and yellow panel) and normal colon mucosa (lower green and gray panels) are depicted together with genes encoding for the GTs involved in their biosynthesis. The biosynthetic pathways of different core structures are labelled with different colors. Pathways upregulated in cancer are marked in orange and yellow, whereas pathways downregulated in cancer are marked in light and dark gray. Core 4 O-glycans show no statistically significant difference between normal mucosa and cancer, marked in green. Glycosyltransferase genes upregulated in cancer are labelled with red and those downregulated in cancer are displayed in blue. Blue square: N-acetylglucosamine, yellow square: N-acetylgalactosamine, yellow circle: galactose, red triangle: deoxyhexose, pink diamond: N-acetylneuraminic acid, S: sulfate modification.