A bitter sweetness

Colorectal cancer (CRC) is a complex, heterogeneous disease that displays an aberrant glycosylation profile. The multi-step development of CRC, its distinct molecular subtypes, immune landscape, and the relevance of glycosylation are described in more detail in Chapter 1. In this thesis, we investigated the role of tumor glycosylation in CRC progression, focusing on how tumor-associated glycans modulate the immune system’s ability to recognize and eliminate cancer cells. One of the glycan structures frequently overexpressed in cancer are sialic acids, which can alter immune cell activity through interaction with Siglec receptors. In Chapter 2, we examined the effect of complete desiaylation on tumor growth and anti-tumor immunity in the CT26 CRC mouse model. Using CRISPR/Cas9, we knocked out the Cmas gene, a key component of the sialic acid biosynthesis pathway. We demonstrate that complete removal of tumor sialylation significantly reduces tumor growth in vivo, and profoundly reshapes the immune landscape toward a more immune-responsive TME. In Chapter 3, we unraveled that sialylation-related genes are significantly higher expressed in peritoneal metastases compare to primary CRC tumors or liver metastases. We further investigated how sialylation affects tumor formation in the peritoneal cavity and influences immunosurveillance. Intraperitoneal injection of CT26 cells lacking sialic acids led to significantly reduced tumor burden and lower peritoneal cancer index (PCI) score. We identified immune cell correlations unique to desialylated tumors, suggesting a profound reshaping of the immune networks in desialylated tumors. In Chapter 4, we summarized the immunomodulatory role of gangliosides in shaping the tumor microenvironment. During malignant transformation, cancer cells overexpress gangliosides and either present them on the cell surface or shed them into the tumor micromilieu. Certain gangliosides play a dual role in immunosurveillance which is highly dependent on the tumor type. Nevertheless, many gangliosides have been shown to contribute to immune evasion and are now being regarded as promising therapeutic targets for novel immunotherapy regimens. In Chapter 5, we explored the immunomodulatory role of ST3Gal5, the sialyltransferase responsible for catalyzing the addition of α2-3 sialic acids to glycosphingolipids. Deletion of the St3gal5 gene did not alter tumor growth in either of the models; however, CT26 tumors lacking ST3Gal5 showed an increase in regulatory T cell numbers, while loss of ST3Gal5 in MC38 tumors resulted in changes in blood vessel size and density. Given that both MC38 and CT26 cell lines predominantly express α2-3 sialic acids, and that α2-6 sialylation is typically upregulated in CRC—where it contributes to cell growth, angiogenesis, therapy resistance, and immune evasion—we investigated its impact on tumor development in Chapter 6. To this end, we introduced α2-6 sialic acids by inducing expression of the sialyltransferase ST6Gal1. Surprisingly, overexpression of ST6Gal1 in MC38 and CT26 cells had no effect on tumor growth in vivo. Another hallmark of cancer glycosylation is the aberrant expression of fucosylated epitopes, synthesized by specific fucosyltransferases (FUTs). In Chapter 7, we identified key gene regulatory networks linking FUT9 expression to cancer stemness. In addition, MC38 cells expressing FUT9 exhibited several phenotypic and functional features characteristic of cancer stem-like cells, along with enhanced tumor growth in vivo. Given that cancer stem cells can evade immune recognition and elimination, targeting FUT9 may offer a strategy to counteract their immunosuppressive effects and improve anti-tumor immunity. In conclusion, this thesis highlights the pivotal role of glycosylation in tumor progression and its influence on the anti-tumor immune response. Future research should focus on the comprehensive characterization of the tumor “glyco-code” to enable the development of novel glycan-based therapies, which could be employed as individual treatments or in combination with already existing therapeutic strategies.