Proximity Labeling and Genetic Screening Reveals the Role of DSG2/Siglec-9 Axis in Suppressing Macrophage Phagocytosis

In current study, we have established a combined strategy using proximity labeling and genetic screening to identify the cellular ligands of Siglec-9. Our use of proximity labeling using APEX2 fusion proteins followed by quantitative proteomics allowed us to efficiently label and enrich cell surface protein ligands that directly interact with Siglec-9 via sialic acid moieties. By combining with CRISPR knockout screening, we were able to identify functionally significant ligands. We confirmed at the cellular level that DSG2 expressed on cell surfaces directly interacts with Siglec-9, and this interaction is mediated by site-specific N-glycans on the ECD of DGS2. We also demonstrated the binding between DSG2 on A375 melanoma cells and Siglec-9 on macrophages contributes significantly to Siglec-9 signaling and blocking the DSG2-Siglec-9 axis by either DSG2 knockdown or anti-Siglec-9 antibody, releases immune-suppression and increases phagocytosis. Our findings revealed an important ligand in cancer cells for the immunosuppressive receptor Siglec-9, and suggest the potential of disrupting DSG2-Siglec-9 signaling axis in anti-cancer therapy.