To identify biomarkers associated with vestibular schwannomas (VS) by integrating bioinformatics and machine learning techniques, and to investigate their biological functions and mechanisms within the immune microenvironment.

VS patient data (GSE56597 and GSE39645) were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) analysis and weighted gene co-expression network analysis (WGCNA) were employed to identify feature genes. Least absolute shrinkage and selection operator (LASSO) regression, random forest (RF) and Boruta algorithm were applied to precisely identify VS immune microenvironment biomarkers. Diagnostic value was evaluated using receiver operating characteristic (ROC) curve analysis. Pseudotime trajectory analysis was performed to explore the cellular distribution and functional roles of these biomarkers.

(1) DEGs analysis identified 1331 VS-associated DEGs. (2) WGCNA screened seven gene modules related to VS, yielding 2055 candidate genes. (3) From the overlapping DEGs and module hub genes, 722 genes were selected. LASSO regression, Boruta, and RF algorithms identified four signature genes (C10orf11, NCAM2, MLLT4, PI16). (4) In dataset GSE56597, the area under the ROC curve (AUC) values for NCAM2, PI16, C10orf11, and MLLT4 were 0.982, 0.918, 1, and 1, respectively. In validation set GSE39645, the AUCs were 1, 0.968, 1, and 1. (5) Single-cell RNA sequencing (GSE216783) revealed heterogeneous cell clusters in the VS tumor microenvironment, including myeloid cells, natural killer cells, non-myelinating Schwann cells, fibroblasts, myelinating Schwann cells, circulating cells, and B cells/plasma cells, all contributing to tumor progression. NCAM2 exhibited high expression and density in non-myelinating Schwann cells. (6) Pseudotime analysis of single-cell RNA sequencing data revealed that NCAM2+ Schwann cells exhibit enhanced differentiation capacity compared to NCAM2-counterparts. Further cell-cell communication analysis and pathway enrichment analysis demonstrated that NCAM2+ Schwann cells establish robust interactions with other cell types through MIF-(CD74+CD44) and MIF-(CD74+CXCR4) ligand-receptor pairs, indicating more active intercellular communication.

C10orf11, NCAM2, MLLT4, and PI16 were identified as biomarkers of VS, demonstrating significant roles in the immune microenvironment and high diagnostic value.