OP11Multi-omics Analysis Reveals Polyamine-Driven Fibroblast Dysfunction in Perianal Fistulizing Crohn’s Disease

Abstract Background Perianal fistulizing Crohn’s disease (PFCD) represents a distinct, inflammation driven subtype of perianal fistula that differs fundamentally from idiopathic perianal fistula (IPF), which typically arises from cryptoglandular infection and follows a predominantly infectious and mechanical pathogenesis. PFCD is characterized by chronic inflammation, complex fistula tracts, and poor healing despite combined surgical and medical therapy. In contrast, IPF often responds well to drainage and local interventions. The biological basis underlying these divergent clinical behaviors remains poorly defined. We aim to elucidate the unique pathological features of PFCD and identify potential therapeutic targets. Methods We enrolled 26 patients with PFCD, 26 patients with IPF, and 3 hemorrhoid patients as non-fistulous controls. Surgical specimens were collected for bulk RNA sequencing, single-cell RNA sequencing, and untargeted metabolomic analysis. Fibroblast phenotype was assessed in the CCD-18co cell line in vitro by using cell counting kit-8 (CCK-8) assays, apoptosis detection assays, and wound healing scratch assays. Results Single-cell RNA analyses revealed that PFCD exhibited distinct cellular composition and molecular features (Figure.1A, B). Compared to non-fistulous controls and IPF, PFCD showed a more pronounced inflammatory response and impaired tissue-reparative capacity (Figure.1C). While both PFCD and IPF displayed activation of NF-κB and TNF signaling pathways, CD-associated fistulas demonstrated stronger activation of IFN-γ and JAK/STAT signaling (Figure.1D, E). PFCD also showed decreased proportions of reparative fibroblasts and myofibroblasts (Figure.2A, B), accompanied by upregulation of pro-inflammatory genes and disruption of polyamine metabolism in these cells (Figure.2C, D). Untargeted metabolomic profiling confirmed elevated spermidine levels in PFCD (Figure.2E). In vitro, spermine and spermidine inhibited fibroblast proliferation (P 0.05), while spermidine and putrescine suppressed the migration of CCD-18co cells (P 0.05, Figure.2F-H), suggesting that local accumulation of polyamines may impair fibroblast-mediated tissue repair. Conclusion Our study provides a comprehensive molecular and cellular landscape of PFCD, highlighting its distinct immune and stromal microenvironment compared to idiopathic perianal fistulas and non-fistulous controls. Local polyamine accumulation may contribute to defective fibroblast function and impaired tissue repair, highlighting a potential metabolic target to promote fistula healing in PFCD. Conflict of interest: Tian, Chuwen: No conflict of interest Wu, Lexi: No conflict of interest Liu, Wei: No conflict of interest Huang, Lingjie: No conflict of interest Cao, Qian: No conflict of interest Figure 1. Single-cell RNA profiling and inflammatory expression patterns.