ENAGenomicsShowa Medical Universityhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1134211Mus musculusMetastasis remains the leading cause of cancer-related mortality, driven by complex interactions within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) play a pivotal role in metastatic progression, yet molecular diversity and upstream regulators remain poorly defined. Glycoprotein nonmetastatic melanoma protein B (GPNMB), overexpressed in subsets of tumors including triple-negative breast cancer (TNBC), is implicated in epithelial-mesenchymal transition (EMT) and cancer stemness. Recent single-cell RNA-seq studies have identified GPNMB as a marker of immunosuppressive TAMs associated with poor prognosis, but its mechanistic role in TAM polarization in TNBC has remained unclear. Co-culturing monocytic cells with three-dimensional sphere-forming TNBC cells induces their conversion into GPNMB⁺Siglec-9⁺ tumor-associated macrophages (TAMs). Tumor-derived GPNMB promotes monocyte-to-TAM polarization by inducing secondary GPNMB expression in monocytes, establishing a feed-forward amplification loop. Knockdown of GPNMB in TNBC cells significantly inhibits multiple immunosuppressive TAM subtypes, including Siglec-9⁺ TAM and EMT-associated TAM populations, as inferred from scRNA-seq and validated in patient tumors using bulk RNA-seq deconvolution. Distinct sialylation patterns were identified: tumor-derived GPNMB exhibited α2,3-sialylation, whereas macrophage-derived GPNMB exhibited α2,6-sialylation, enabling differential Siglec-9 recognition. Elevated GPNMB and Siglec-9 expression correlated with poor prognosis in TNBC patient cohorts. Importantly, dual inhibition of Siglec-E (murine Siglec-9 ortholog) and PD-1 suppressed IL-6-dependent EMT, reduced tumor stemness, and significantly limited lung metastasis in vivo. The GPNMB–Siglec-9 axis thus represents a critical glyco-immunological checkpoint driving TAM-mediated metastasis, providing a promising therapeutic target in TNBC. Overall design: Female BALB/c mice (8 weeks of age) were injected with 5 × 104 4T1 cells/50 µL of PBS into the left, fourth mammary fat pad. Tumor development was established in 7 days, with tumor volumes of approximately 70 mm3. Then mice were divided into 4 groups as isotype control, anti Siglec-E antibody monotherapy, anti PD-1 antibody monotherapy, and combination therapy of anti Siglec-E antibody and anti PD-1 antibody (n=6 each) were sacrificed. To obtain single cells, the isolated primary tumors (n=2 each) were minced and dissociated using BD HorizonTM Dri Tumor & Tissue Dissociation Reagent (661563 BD Biosciences) following the manufacturer’s instructions. Red blood cells were then lysed using red blood cell lysis buffer (4333 Invitrogen). Dead Cell Removal MicroBead (130090101 Miltenyi Biotec) was applied to the cells. Then separation using CD326 EPCAM (130061101 Miltenyi Biotec) microbead protocol was performed to separate the epithelial and immune cells.ENAfalseSingle-cell RNA-seq of 4T1 breast tumors from mice treated with anti-Siglec-E and/or anti-PD-1 antibodiesSingle-cell RNA-seq of 4T1 breast tumors from mice treated with anti-Siglec-E and/or anti-PD-1 antibodies2025-09-242025-09-08PRJNA1134211GSE27196410090