Single-cell RNA-seq of 4T1 breast tumors from mice treated with anti-Siglec-E and/or anti-PD-1 antibodies

Metastasis 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. 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.