Multiscale characterization of an inflammatory macrophage state associated with an air pollutant–prioritized prognostic signature in gastric cancer

Abstract

Background Air pollutants are implicated in carcinogenesis, but their relevance to gastric cancer (GC) across prognosis, tumor organization and cell behavior remains unclear. We integrated evidence to evaluate pollutant-prioritized GC candidates. Methods Seven pollutants were integrated with GeneCards/OMIM GC genes using toxicology and candidate-prioritization resources. Robustness analyses used alternative disease definitions, Fisher’s tests, 5,000 degree- and annotation-matched null sets, enrichment and protein–protein interaction networks. TCGA-STAD supported nested evaluation of 117 survival pipelines and a five-gene neural network, with locked external testing in GSE84437 and GSE54129. Single-cell, donor-aware macrophage, CellChat, Monocle2, multiplex immunofluorescence and cell2location-guided Visium analyses resolved tissue context. Docking, 100-ns molecular dynamics and bibliometrics examined GSK-3β-centered hypotheses. AGS cells underwent benzene/toluene dose screening followed by CCK-8, colony formation, Transwell and wound-healing assays. Results Among 176 genes, 63 overlapped with GC beyond matched-null expectations (empirical P = 0.012), with enrichment in inflammation, hypoxia, apoptosis, metabolism and carcinogenesis. APRGSig (DNMT1, GSK3B, HNF4A, MMP9 and TERT) remained associated with overall survival after age/T/N/M adjustment (HR per 1-SD increase, 2.31; 95% CI, 1.98–2.71) and achieved a locked external C-index of 0.701; exploratory GC-ANN achieved an external AUC of 0.962. Score-defined APRGSig-high macrophages showed broader predicted crosstalk, whereas donor-aware analysis identified an inflammatory/stress-associated MALAT1-high state. Multiplex immunofluorescence provided representative compartment-level localization of APRGSig-encoded proteins; four Visium sections showed directionally consistent positive spatial concordance between APRGSig scores and inferred MALAT1-high abundance. Formal GSK-3β–benzene/toluene models yielded Vina scores of −7.1/−7.4 kcal/mol and remained numerically persistent over 100 ns; bibliometrics of 311 publications highlighted proliferation, invasion and metastasis. At 2.5 mM benzene and 20 μM toluene, both exposures increased AGS clonogenic capacity, Matrigel traversal and wound closure (all P < 0.05). Conclusions This framework integrates predictive, tissue-resolved, structural and phenotype-level evidence to prioritize exposure-linked molecular and immune hypotheses in GC.