MSACL 2022 Abstract

INTRODUCTION
Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide with over 900,000 deaths in 2020[1]. New specific molecular targets are needed that can be utilized for the development for new targeted therapies to provide a better treatment for a larger group of CRC patients. A potential target might be the altered glycome of proteins as these are found to be a hallmark of many diseases[2]. For example, heavily glycosylated proteins mucins play a very important role in the gut homeostasis, however, little attention was given to their O-glycosylation. The characteristic glycan alterations in cancer include specific aberrant expression of incomplete carbohydrate structures or de novo expression of carbohydrate antigens also known as tumor-associated carbohydrate antigens (TACAs) which show a great potential for the development of new immunotherapeutic strategies for CRC. Here we present an in-depth study of CRC glycosylation, with a focus on decoding the tumor specific N- and O-linked glycan signatures of CRC that are derived from epithelial regions of both primary tumors and metastatic sites compared to healthy colon mucosa from the same patients.

METHODS
We established a workflow that sequentially releases the N-and O-glycans from laser capture microdissected regions of formalin fixed paraffin embedded (FFPE) tissues followed by their analysis with porous graphitized carbon (PGC)-LC-MS/MS in negative ion mode. This platform enables a powerful separation of isomeric species as well as in-depth structural characterization of potential TACAs.

RESULTS
Distinctive N- and O-glycosylation features were found in cancer, cancer stroma as well as in normal colon mucosa. Over 100 O-linked glycosylation signatures were detected in the cancer regions which did not show any expression in normal mucosa. From those, seven core 2 O-glycans were exclusively found in more than 33% of the cancers which carried the terminal sialyl-LewisX/A antigen or terminal α2-3-linked sialylation. Of these, two glycans were found in 72% of the analyzed cancers and 94% of the investigated cancers expressed either one of the two O-glycans with the composition H2N2F1S1 and H2N2S1 demonstrating great specificity. In contrast, normal colon mucosa predominantly expressed core 3 O-glycans, carrying α2-6-linked sialylation, (sulpho-)LewisX/A and Sda antigens. Additionally, preliminary results revealed that 26 N-linked glycosylation signatures were found only in the cancer samples, and 11 individual N-glycans showed statistically significant upregulation in cancer compared to normal colon mucosa.
CONCLUSION
In this study we present a novel panel of highly specific TACA’s, based upon changes in the glycomic profiles between CRC and healthy colon mucosa. Our data provides strong evidence that specific glycans as well as glycomic traits, such as epitope expression and different core structures, are significantly changed between CRC and normal colon mucosa tissue. These TACA’s are promising new targets for development of innovative cancer immunotherapeutics and lay the foundation for the targeted treatment of CRC.

1. Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 71, 209–249 (2021).
2. Pinho, S. S. & Reis, C. A. Glycosylation in cancer: mechanisms and clinical implications. Nat. Rev. Cancer 15, 540–555 (2015).