Disruption of sialic acid metabolism drives tumor growth by augmenting cd8+ t cell apoptosis Lenneke A.M. Cornelissen, Athanasios Blanas, Joost C. van der Horst, Laura Kruijssen, Anouk Zaal, Yvette van Kooyk and Sandra J. van Vliet Amsterdam UMC, Vrije Universiteit Amsterdam, department of Molecular Cell Biology and Immunology, Cancer Center Amsterdam, Amsterdam Infection and Immunity Institute, Amsterdam, Netherlands Malignant transformation is often accompanied by dramatic changes in the metabolism of cellular glycosylation, resulting amongst others in an elevated expression of sialylated epitopes. Sialoglycans can interact with specific lectins on immune cells , such as the sialic acid recognizing Ig-like lectins (Siglecs). Most Siglecs contain tyrosine-based inhibitory motifs in their cytoplasmic tail, which, upon recognition of endogenous levels of sialic acids, dampen unwanted immune activation to healthy cells. Therefore, the current dogma states that tumors exhibit hypersialylation as a form of enhanced-self to initiate immune evasion. Indeed, prior work from our group demonstrated that a partial loss of sialic acids in melanoma reduced tumor growth through augmented tumor destruction by NK and CD8+ T cells and inhibition of regulatory T cell differentiation. To study whether the same principle would uphold in other tumor types, we generated a panel of glyco-engineered colorectal cancer (CRC) cell lines to decipher the in vivo role of sialylated glycans during CRC development. Using CRISPR/Cas9 technology we created Sianull variants of the murine CRC cell line MC38 by abrogating sialic acid metabolism through knock out of the N-acylneuraminate cytidylyltransferase (CMAS) gene, encoding the enzyme responsible for CMP-sialic acid synthesis. Surprisingly and in contrast to the melanoma tumors, MC38-Sianull tumors displayed enhanced growth rates in vivo, which we could attribute to a lack of viable cytotoxic CD8+ T cells in the tumor microenvironment. As systemic CD8+ T cell numbers were normal, this hinted to a local and tumor site-specific phenomenon. Indeed, we could identify a soluble factor secreted by MC38-Sianull cells, that was able to induce CD8+ T cell apoptosis in an antigen-independent manner. Our in vivo findings were recapitulated in human CRC, where low levels of the CMAS gene are associated with a reduced recurrence-free survival. In summary, we are the first to show the detrimental consequences of complete tumor desialylation, indicating that the influence of sialic acid metabolism on cancer immunity is more complex than originally thought. Moreover, our results have strong implications for ongoing efforts aimed at abrogating tumor sialyation to promote anti-tumor immunity.