Multiomics analyses revealed the roles of the epithelial-mesenchymal transition and novel early candidate diagnostic biomarkers in neonatal necrotising enterocolitis.

Abstract

Background: Necrotizing enterocolitis (NEC) is a severe neonatal gastrointestinal disease with incompletely understood pathogenesis and a lack of reliable early biomarkers. We investigated intestinal molecular alterations associated with NEC and explored candidate diagnostic biomarkers.

Methods: Bulk proteomics of formalin-fixed paraffin-embedded intestinal tissues was integrated with spatial proteomics of an index NEC case, single-cell RNA sequencing, immunohistochemical validation, serum enzyme-linked immunosorbent assays, and a murine NEC model with pharmacological inhibition of Snail-mediated epithelial-mesenchymal transition (EMT).

Results: Multiomics analyses indicated layer-associated epithelial injury, inflammatory activation, and EMT-like fibroblast/myofibroblast remodelling during NEC. Single-cell sequencing of six intestinal samples showed reduced epithelial-cell abundance with increased fibroblast and smooth-muscle-cell proportions in NEC. In the murine model, CYD19 treatment was associated with improved intestinal pathology, survival, inflammatory cytokine profiles, and barrier-related readouts. ANXA2, RPL13, RPL23A, and RPS28 were identified as tissue candidate biomarkers and validated by immunohistochemistry. Serum ANXA2 was higher in NEC than in available non-NEC controls (AUC, 0.944) and showed exploratory discrimination between medical NEC and highly suspected cow's-milk protein allergy; the adjusted AUC was 0.899.

Conclusions: NEC is associated with inflammation-linked EMT-like intestinal remodelling and epithelial injury. ANXA2 and selected ribosomal proteins are candidate tissue biomarkers, while serum ANXA2 warrants prospective multicentre validation as a blood-based diagnostic and risk-stratification biomarker.

Key points: NEC was associated with epithelial injury, excessive inflammation, and EMT-like fibroblast/myofibroblast remodelling, as revealed by integrated bulk proteomics, spatial proteomics, and single-cell sequencing. Pharmacological inhibition of Snail-mediated EMT in a murine NEC model was associated with improved intestinal injury, inflammatory responses, barrier-related readouts, and survival. ANXA2, RPL13, RPL23A, and RPS28 were identified as candidate tissue biomarkers, while serum ANXA2 showed exploratory potential for NEC diagnosis and risk stratification.