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

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.