Multi-omics profiling reveals the molecular network underlying bladder cancer associated with cyclophosphamide toxic metabolites

This study aims to generate a hypothesis regarding bladder cancer associated with toxic metabolites of cyclophosphamide through integrative multi-omics analysis, and to identify potential key molecular targets and signaling pathways. The ADMETlab 2.0 platform was employed to predict the toxicological and pharmacokinetic profiles of cyclophosphamide and its primary toxic metabolites (acrolein and chloroacetaldehyde). Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed on public datasets. Machine learning algorithms were integrated with network toxicology approaches to screen and construct a potential regulatory network underlying cyclophosphamide metabolite-associated bladder cancer. Immune infiltration analysis and immune correlation analysis of core genes were conducted to characterize the immune microenvironment features of the candidate targets. Single-cell sequencing and spatial transcriptomics were further utilized to delineate the expression and distribution patterns of the core targets within bladder cancer tissues. Finally, molecular docking was performed to preliminarily assess the binding potential between the candidate targets and acrolein/chloroacetaldehyde. A total of four core regulatory genes were identified. Single-cell and spatial transcriptomic analyses delineated their expression and distribution profiles across specific cell subpopulations and tissue regions. Molecular docking indicated a “weak reversible binding” mode between the metabolites and target proteins, supporting the mechanistic hypothesis that these metabolites exert persistent toxicity via covalent modification. Through integrative multi-omics analysis, this study identified four core regulatory genes and preliminarily constructed a potential molecular network of bladder cancer associated with toxic cyclophosphamide metabolites. These findings provide well-defined candidate targets and a testable pathogenic hypothesis for subsequent experimental validation.