A five-gene TAM and MYCN signature predicts prognosis and identifies MTHFD2 as a therapeutic target in neuroblastoma

BACKGROUND: Tumor-associated macrophages (TAMs) are key components of the tumor immune-suppressive microenvironment. However, studies focusing on the modulation of TAM-specific gene signatures to improve neuroblastoma (NB) prognosis are limited. MYCN amplification (MNA) is known to influence the immune landscape in neuroblastoma. METHODS: Single-cell sequencing of 19 NB samples and transcriptome data from 498 NB samples were analyzed to identify macrophage-specific genes associated with poor NB prognosis. A risk signature was constructed by integrating differentially expressed genes (DEGs) related to TAMs and MYCN using univariate Cox regression, LASSO regression, and Kaplan-Meier survival analysis. Single-factor and multi-factor Cox regression analyses were conducted to explore the relationship between clinical characteristics and prognosis, and a nomogram was constructed. The impact of downregulating TAM target genes in macrophages was explored using CCK8, EdU incorporation, and Transwell assays. RESULTS: A signature based on five macrophage-related genes associated with NB prognosis (FCGR3A, ATF5, MTHFD2, HMGA1, and LY6E) was successfully established and validated for its strong prognostic predictive performance (AUC: 0.829). A clinically applicable Nomogram incorporating these genes was also developed. CCK-8 assays revealed a time-dependent inhibitory effect on neuroblastoma cell proliferation upon co-culture with MTHFD2-knockdown macrophages. EdU and Transwell assays showed that downregulation of MTHFD2 in macrophages significantly reduced the invasive and migratory capabilities of NB cells, particularly in MYCN-amplified cells. CONCLUSION: We developed and validated a five-gene signature based on TAM and MYCN-related genes with strong prognostic performance. Downregulation of MTHFD2 in macrophages reduced neuroblastoma cell proliferation, invasion, and migration, particularly in MYCN-amplified cells, highlighting MTHFD2 as a potential therapeutic target in high-risk patients.