Comprehensive metabolomic profiling of FFPE human tissues reveals key metabolic reprogramming in colorectal cancer and associated pathways

Introduction: Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable for retrospective clinical studies due to superior morphological preservation and easy storage, but their use in metabolomics is limited by a lack of established sample preparation protocols and concerns over metabolite stability. Here we optimized a novel FFPE metabolomic workflow and validated findings with matched fresh-frozen (FF) samples to identify metabolic signatures altered in human colorectal cancer (CRC). Methods: We analyzed 12 FFPE human tissue samples, comprising 6 paired tumor and Nearby Adjacent Tissue (NAT) specimens from adenocarcinoma patients, alongside matched FF samples for validation. Global metabolomics assays were performed on both polar and lipid fractions using a next-generation mass spectrometry platform. Results: Metabolic profiling detected 2,564 unique metabolites across 200+ classes from FFPE tissues with low technical variation (median CV < 5%). Unsupervised analyses showed clear tumor-NAT distinctions. Statistical analysis identified 200 differential metabolites (|log2(fc)| > 1, p < 0.05). Pathway analysis revealed 39 altered pathways (p<0.05), with upregulated diacylglycerophosphoinositols and downregulated triacylglycerols in tumors being the most significant findings. Consistent upregulation of central carbon metabolites and amino acids indicated metabolic reprogramming for biosynthesis and oxidative stress buffering. FF sample validation showed concordance across the most significant hits and pathways identified, confirming the robustness and biological relevance of FFPE-derived signatures. Conclusion: Our study confirms FFPE metabolomic profiling reliably identifies significant metabolic perturbations in human cancer, consistent with FF findings. The identified shifts in lipid, central carbon, and amino acid metabolism highlight extensive metabolic reprogramming in CRC. FFPE archives are a valuable resource for large-scale retrospective clinical metabolomics studies, offering a powerful avenue for discovery research in human genetics and disease, and for identifying novel biomarkers.