Lipotoxic fingerprints in clinically relevant postoperative pancreatic fistula: fatty acid–driven cytotoxicity targets cells involved in anastomotic healing

Abstract Background & Aims Clinically relevant postoperative pancreatic fistula (CR-POPF) remains a major cause of morbidity following pancreatic surgery, potentially due to fatty acid release by lipase activity. This study investigated how the biochemical composition of CR-POPF effluents drives cellular injury and transcriptional stress responses. Methods Drain effluents from 14 patients undergoing pancreatoduodenectomy (7 with CR-POPF and 7 controls) were analyzed using gas chromatography–mass spectrometry. Candidate lipids were tested on human foreskin fibroblasts, mesothelial cells, and pancreatic epithelial cells using viability and cytotoxicity assays. Effluents were applied directly to cultures, and RNA sequencing was performed on cells exposed to the two most cytotoxic CR-POPF samples. Results Metabolomic profiling revealed lipolytic traits characterized by long-chain saturated fatty acids, including palmitic and stearic acid, and the palmitic acid monoacylglycerol monopalmitin, in drain effluents. These fatty acids accounted for over 70% of the variance in multivariate metabolomic analyses between CR-POPF and control groups. Dose–response assays confirmed concentration-dependent cytotoxicity (p < 0.0001), with a subtoxic threshold of 0.2 mM. Two effluents (AES1448 and GR1479) consistently reduced cell viability across models (F > 19, p < 0.0001). Transcriptomic profiling showed enrichment of inflammatory, unfolded-protein, and stress-response pathways, along with suppression of proliferation modules. GR1479 induced metabolic adaptation, whereas AES1448 and monopalmitin triggered overt lipotoxic stress. Conclusions Lipolysis-derived lipids may mediate stromal and mesothelial injury in CR-POPF. Integrating metabolomic, functional, and transcriptomic data uncovers a spectrum of cellular responses, spanning from adaptive remodeling to lipolysis-driven proteotoxic stress. These findings support lipid toxicity as a biochemical property of CR-POPF and a potential target for prevention. Synopsis This study identifies long-chain saturated fatty acids in postoperative pancreatic effluents as key mediators of cytotoxic and inflammatory stress. Integrating metabolomic and transcriptomic analyses link effluent composition directly to cellular injury and impaired healing after pancreatic surgery. Abstract Figure Graphical abstract