Single-Cell Analysis Delineates Glutathione Metabolism-Related Gene Signatures in the Glioblastoma Microenvironment and Identifies GSTA4 as a Regulator of Malignant Behaviors.

Background and Objective: The cellular distribution characteristics and functional mechanisms of glutathione (GSH) metabolism within the tumor microenvironment (TME) of glioblastoma multiforme (GBM) remain poorly understood. This study aims to elucidate the cellular landscape and GSH metabolic features of the GBM microenvironment and to clarify the role of GSH S-transferase alpha 4 (GSTA4) in GBM progression. Methods: ScRNA-seq was performed on 104,789 GBM cells to systematically identify cell subpopulations and construct cell interaction networks. The pseudotime analysis was conducted on 50 GSH metabolism-related genes. Differential expression of GSTA4 in GBM cell lines (U87 MG, LN229, and U251) and normal human astrocytes (SVG p12) was validated by quantitative PCR (qPCR) and Western blotting. Subsequently, siRNA and plasmid vectors were employed to establish cellular models with GSTA4 overexpression or knockdown. The functional impact of GSTA4 expression on cell proliferation, invasion, and migration was systematically evaluated. Finally, rescue experiments confirmed that GSTA4 modulates GBM cell function via the Wnt/β-catenin signaling pathway. Results: ScRNA-seq analysis identified 10 major cell types, including tumor cells, proliferating cells, immune cells, and stromal cells. Elevated GSH metabolic activity was predominantly observed in proliferating cells. The pseudotime analysis indicates that GSTA4 exhibits high expression levels in the intermediate state of tumor cells. Functional experiments confirmed significant upregulation of GSTA4 in GBM cells. Silencing GSTA4 expression suppressed cell proliferation, invasion, and migration, whereas its overexpression enhanced these malignant phenotypes. Moreover, this study is the first to demonstrate that GSTA4 promotes tumorigenicity and aggressive phenotypes in GBM cells through the Wnt/β-catenin signaling pathway. Conclusion: This study reveals the reprogramming characteristics of GSH metabolism in the GBM microenvironment at the single-cell level. Furthermore, functional experiments demonstrate that GSTA4 acts as an oncogenic driver in GBM. Collectively, these findings advance the mechanistic understanding of GBM and identify GSTA4 as a promising therapeutic target for precision intervention.