NFAM1 organizes a lipid raft–restricted DAPP1–PLCG2 module to drive M2-like GAM polarization via Ca²⁺–NFAT1 signaling in glioma

BACKGROUND: Glioma-associated macrophages (GAMs) promote glioma progression, but the membrane-proximal mechanisms sustaining their tumor-supportive states remain poorly defined. METHODS: Bulk and single-cell transcriptomic analyses, human glioma specimens, GAM models, and orthotopic glioma models were used to define the expression and function of NFAM1. Lipid-raft fractionation, co-immunoprecipitation, proximity ligation, domain-mutant rescue, IP3 and Ca²⁺ measurements, and NFAT1 nuclear translocation assays delineated its signaling mechanism. Pharmacological, macrophage-depletion/reconstitution, and inducible genetic models evaluated its therapeutic relevance. RESULTS: NFAM1 was enriched in monocyte-derived, M2-like GAMs and associated with higher glioma grade, IDH-wild-type status, and poor survival. NFAM1 promoted GAM migration and M2-like polarization, thereby enhancing mesenchymal plasticity of glioma stem cells (GSCs), angiogenesis, and glioma progression. Mechanistically, phosphorylated NFAM1 recruited DAPP1 and PLCG2 through ITAM-SH2-dependent interactions within lipid rafts, organizing a spatially coordinated and functionally ordered signaling module. DAPP1 acted upstream of PLCG2 to promote IP3 production, Ca²⁺ signaling, and NFAT1 nuclear translocation. Madecassoside showed ITAM-dependent cellular target engagement with NFAM1, disrupted this signaling module, suppressed tumor-supportive GAM phenotypes, reduced orthotopic tumor growth, and prolonged survival. Its antitumor activity was markedly diminished after peripheral macrophage depletion or reconstitution with NFAM1-deficient bone marrow-derived macrophages. Moreover, inducible NFAM1 knockdown in reconstituted BMDM-derived GAMs after tumor establishment reduced tumor burden and extended survival. CONCLUSIONS: NFAM1 spatially organizes lipid-raft-restricted DAPP1-PLCG2-Ca²⁺-NFAT1 signaling in monocyte-derived GAMs and represents a therapeutically targetable vulnerability in glioma.