Multi-Omics Integrative Analysis of the Aspirin-Gut-Brain-Glioma Axis: Transcriptomic, Proteomic, Epigenetic, Mendelian Randomization, and Single-Cell Transcriptomic Evidence Converges on NEO1/Hepcidin Iron Reprogramming and Ferroptosis Vulnerability

Background: Despite epidemiological interest in aspirin's chemopreventive potential against glioma, the underlying multilayered molecular mechanisms spanning gut microbial ecology, COX-2/PGE2 signaling, inter-organ iron homeostasis via the NEO1/hepcidin regulatory axis, epigenetic reprogramming, and ferroptosis have not been systematically characterized at the multi-omics level. Methods: We conducted an integrative multi-omics analysis leveraging TCGA-GBM (n = 172) and TCGA-LGG (n = 534) transcriptomes, CPTAC GBM proteomics (n = 99), TCGA HM450K DNA methylation data (GBM n = 140, LGG n = 516), GEO aspirin perturbation datasets, IEU OpenGWAS summary statistics, and independent single-cell RNA-seq data (GSE131928, 28 GBM patients). Nine analytical tracks were executed: (1) COX-2/PGE2 pathway profiling, (2) BBB tight junction characterization, (3) GEO-derived aspirin response signature projection, (4) BBB integrity evaluation, (5) Mendelian randomization (MR) using PTGS2 cis-SNPs, (6) iron metabolism and ferroptosis pathway analysis, (7) NEO1/HFE2/BMP6/HAMP regulatory axis characterization with multi-omics validation, (8) single-cell transcriptomic validation across GBM malignant cell states, and (9) multi-pathway PCD crosstalk analysis with Visium spatial transcriptomic validation. Results: Transcriptomic analysis revealed profound reprogramming of the NEO1/hepcidin iron regulatory axis in GBM: HAMP (hepcidin) was massively upregulated (log2FC = +2.92, P = 5.0 x 10^-31), accompanied by TFRC upregulation (log2FC = +1.38, HR = 2.30, P = 3.6 x 10^-42) and NEO1 downregulation (log2FC = -0.57, HR = 0.59, P = 4.6 x 10^-10). De novo HM450K methylation analysis revealed HAMP as the dominant epigenetic target in the iron network, exhibiting the strongest hypomethylation signal (Delta beta = -0.265; Bonferroni-corrected P = 2.5 x 10^-40), while NEO1 and TFRC showed constitutively low baseline methylation (beta < 0.05). Gene set enrichment analysis identified ferroptosis driver genes (NES = +1.861, P = 0.030) and the iron deficiency response pathway (NES = +1.698, P = 0.010) as the most significantly enriched pathways in GBM. Molecular subtype analysis revealed that the mesenchymal GBM subtype exhibits the highest iron metabolism gene expression. CPTAC protein-level estimation confirmed directionally concordant changes, and Mendelian randomization established a causal relationship between PTGS2 expression and glioma risk (IVW OR = 1.31, P = 1.1 x 10^-8). The COX risk score demonstrated superior prognostic power (HR = 1.93, P = 5.3 x 10^-9, C-index = 0.80). Single-cell RNA-seq analysis of GBM (GSE131928, 28 patients) validated that iron metabolism gene expression is heterogeneously distributed across malignant cell states, with the mesenchymal (MES) state exhibiting the highest HAMP expression (0.338 vs. 0.029-0.044 in other states, P < 0.001) and elevated ferroptosis vulnerability. PTGS2 and ACSL4 were selectively enriched in the MES state, consistent with the GSEA-identified ferroptosis driver pathway activation. GPX4 was universally highly expressed across all cell states (mean 1.46-1.68), indicating pan-GBM dependence on GPX4-mediated ferroptosis suppression. Multi-pathway PCD analysis revealed coordinated ferroptosis-PANoptosis activation with reciprocal pyroptosis suppression in GBM (P = 2.0 x 10^-30). Independent validation in the CGGA cohort (693 samples) confirmed 100% direction concordance for all 13 iron metabolism genes and reproduced the NEO1 protective survival effect (HR = 0.75, P = 0.016), demonstrating that these findings are generalizable across populations. Visium spatial transcriptomic analysis independently validated the NEO1 edge-high expression gradient, GPX4 pan-tumor expression, and TFRC core-high pattern at the tissue architecture level. TCGA microbiome profiling of 161 GBM patients identified intratumoral bacterial signatures correlated with NEO1/HAMP axis gene expression, providing the first integrative evidence linking the tumor microbiome to iron metabolism reprogramming in glioma. Transcription factor analysis identified CEBPB as the dominant regulator of HAMP and HMOX1, SMAD3 as the top NEO1-associated TF, and SP1/MAFG as negative regulators of GPX4; virtual NEO1 knockout simulation predicted network-wide shifts toward ferroptosis vulnerability. Conclusions: This multi-omics investigation reveals an inter-organ signaling cascade in which the NEO1/hepcidin iron regulatory axis is epigenetically reprogrammed in glioma, driving iron-dependent vulnerability that bridges COX-2 signaling with ferroptosis susceptibility. The convergent evidence from transcriptomics, proteomics, epigenomics, PCD multi-pathway analysis, and spatial transcriptomic validation provides a comprehensive mechanistic framework for aspirin's protective effects against glioma and identifies the NEO1/HAMP/TFRC axis as a promising therapeutic target.