The potential impact of GLS and PDHA1 on tumor immunity and immunotherapy response in LUSC

Background Lung squamous cell carcinoma (LUSC), a therapeutically challenging non-small cell lung cancer (NSCLC) subtype with a poor prognosis, exhibits heterogeneous responses to immunotherapy. Cuproptosis, a recently discovered regulated cell death pathway, has been hypothesised to modulate the tumour immune microenvironment (TIME). Despite the well-established role of PDHA1 as a metabolic regulator, the specific mechanisms by which it interacts with GLS in cuproptosis-mediated immune-metabolic crosstalk remain to be elucidated in LUSC. The present study investigates the manner in which GLS/PDHA1 expression patterns influence TIME composition and contribute to the stratification of immunotherapy responsiveness. Methods It was determined that GLS and PDHA1 were the most significant copper oxidation-related genes, due to their highest absolute correlation with the ESTIMATE immune score. A consensus clustering analysis was conducted on a cohort of 501 TCGA-LUSC patients, with the objective of stratifying patients based on GLS/PDHA1 expression levels. Quantitative analysis of immune infiltration was performed using ESTIMATE, CIBERSORT, and ssGSEA methods. The pathway enrichment analysis was conducted using GSEA and WGCNA. A detailed analysis of 17,050 single-cell RNA sequencing (scRNA-seq) data from two LUSC patients was conducted, which revealed unique gene expression patterns. The validity of these findings was confirmed through the integration of four independent GEO cohorts (GSE181043/37745/43580/115457; n = 278). Results Consensus clustering delineated two subtypes:Cluster 1 (low GLS/high PDHA1) and Cluster 2 (high GLS/low PDHA1). Cluster two showed enhanced immune infiltration, characterized by: Elevated immune checkpoint expression and Enriched T-cell activation pathways. Validation across four GEO cohorts confirmed Cluster two conserved immune-hot phenotypewith elevated ESTIMATE stromal scores, reduced tumor purity, and activated immune subsets. scRNA-seq identified malignant epithelial cells as the hub of divergent GLS/PDHA1 expression (high GLS/low PDHA1), orchestrating cuproptosis-immunometabolic crosstalk. Conclusion GLS and PDHA1 have been proposed as potential prognostic markers for immunotherapy. Targeting cuproptosis has the potential to convert immunologically cold to hot tumours, thereby advancing precision immunotherapy.