Abstract B63: Bittersweet symphony: How tumor-associated glycan structures orchestrate immune evasion
Authors: Cornelissen Lenneke A.M. , Blanas Athanasios , Horst Joost C. Van der , Kruijssen Laura , Kooyk Yvette Van , Vliet Sandra J. Van 
Published: February 2019

The tumor microenvironment is immune suppressive, allowing tumor cells to escape from immune attack. By relieving this immune suppression, immune cells might be able to effectively eliminate the tumor. Therefore, a detailed understanding on how tumor cells induce an immunosuppressive tumor microenvironment is required. Glycosylation is the most abundant post-translational modification of proteins. It is highly diverse on a cellular level and strongly affected by oncogenesis. Tumor cells generally display an aberrant glycosylation profile and this is now well accepted as a new hallmark of cancer. Overexpression of glycan structures containing sialic acids (Sia) is frequently observed on tumors. Glycan structures can be recognized by lectin receptors expressed on immune cells, whereby sialic acids specifically bind to the Siglec receptor family. The majority of Siglec receptors has an immunoreceptor tyrosine-based inhibition motif and therefore, sialylated glycan structures are known to suppress ongoing immune responses. Hence, we hypothesize that tumor cells overexpress sialylated glycan structures in order to communicate with the immune system and to dampen immune attack.

We have previously demonstrated that a genetically engineered Sialow glycovariant of the mouse melanoma B16 model displayed delayed growth in vivo compared to wild-type B16 Siahigh tumors (1). Moreover, higher frequencies of IFNγ-producing effector T-cells were detected in B16 Sialow tumors. This was paralleled by a 50% reduction in FoxP3+CD4+ regulatory T-cell frequencies in Sialow tumors (1), together indicating that tumor-associated sialic acids indeed play, indeed, a crucial role in suppressing the anti-tumor immune response.

To check whether the inhibitory effect of sialic acids on the effector T cell response is tumor type specific, we investigated the role of sialic acids in a mouse colorectal cancer model (MC38). With the use of Crispr-Cas9 we successfully knocked out the N-acylneuraminate cytidylyltransferase (CMAS) gene, which is responsible for the sialylation pathway and thus the generation of sialylated glycans on the cell surface. In contrast to the B16 Sialow tumors, the Sianeg MC38 CMAS KO tumors displayed an increased tumor growth in vivo compared to the Siahigh MC38 Mock tumors. Moreover, less viable leukocytes could be recovered from the Sianeg MC38 CMAS KO tumors compared to the Siahigh MC38 Mock tumors.

With the use of mass spectrometry, we analyzed the complete glycosylation profile of our Sianeg MC38 CMAS KO and Siahigh MC38 Mock cells and observed, in addition to the absence of sialylation, an increase in LacNAc (Galβ1-4GlcNAc) structures on the Sianeg MC38 CMAS KO cells. This glycan structure is a ligand for carbohydrate-binding Galectin receptors. Galectins are well known for their potential to induce leukocyte apoptosis after binding specific glycoproteins on the leukocyte surface. Using qPCR we observed higher mRNA levels of Galectin-1 in our MC38 CMAS KO cells. The enhanced production of Galectin-1 and the elevated expression of LacNAc together suggest that MC38 CMAS KO cells are able to recruit Galectin-1, thereby facilitating the enhanced immune suppression in these tumors. Currently, we are confirming the role of Galectins in our MC38 CMAS KO tumors.

In conclusion, tumor cells alter their glycosylation profile and based on our data, we propose that tumor cells use these glycan structures as a mechanism to influence antitumor immunity and that this phenomenon might be tumor-type specific. Their immune inhibitory nature and their tumor-specific expression identifies tumor-associated glycans as potent candidates for future immune checkpoint blockade therapy.

Reference:

1. Perdicchio M. et al. Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells. Oncotarget 2016;7(8):8771-8.

Citation Format: Lenneke A.M. Cornelissen, Athanasios Blanas, Joost C. Van der Horst, Laura Kruijssen, Yvette Van Kooyk, Sandra J. Van Vliet. Bittersweet symphony: How tumor-associated glycan structures orchestrate immune evasion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B63.