ST6GAL1-mediated sialyl linkage switching drives cancer-promoting α2,6-sialo-protrusions radiating from anti-inflammatory TAMs

Tumor-associated macrophages (TAMs) are functionally diverse effector cells in the tumor microenvironment (TME). Pro- and anti-inflammatory TAMs are central to cancer progression by shaping inflammation and immune homeostasis, but it remains unknown if polarization-induced remodeling of the TAM glycocalyx critical for cellular communication and interactions occurs within the TME. Taking a systems glycobiology approach, we first used cell surface-focused glycomics and lectin flow cytometry of ex vivo polarized monocyte-derived macrophages to demonstrate profound sialyl linkage switching in proinflammatory (α2,3-sialo-favored) and anti-inflammatory (α2,6-sialo-dominant) macrophages. ST6GAL1 catalyzing α2,6-sialylation was found to be elevated in anti-inflammatory compared to proinflammatory macrophages, suggesting that this glycoenzyme facilitates sialyl linkage switching, which was supported by ST6GAL1 silencing. Sambucus nigra agglutinin (SNA)-focused lectin cytochemistry of anti-inflammatory macrophages revealed dense networks of dynamic α2,6-sialylated protein-based protrusions forming interconnecting extracellular structures that were absent in proinflammatory macrophages. Temporal ST6GAL1 suppression in anti-inflammatory macrophages disintegrated the cellular protrusions as evidenced by SNA and biotin fluorescence microscopy. Moreover, live cell recordings of anti-inflammatory macrophages cultured with and without colorectal cancer (CRC) cells showed reduced macrophage motility, attenuated intermacrophage and macrophage–CRC cell interactions and diminished CRC cell proliferation upon ST6GAL1 suppression indicating functional roles of the sialo-protrusions. Notably, ST6GAL1 silencing in anti-inflammatory macrophages promoted transition toward proinflammatory-like phenotypes, as supported by altered morphology, phagocytotic capacity, and Siglec and cytokine expression. Finally, sialyl linkage switching was recapitulated in pro- and anti-inflammatory TAMs in tumor tissues of patients with advanced CRC. We report on the mechanistic basis for and functional consequences of glycocalyx remodeling accompanying TAM polarization.