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Background Chronic pancreatitis (CP) is a progressive fibroinflammatory disease. The core of pancreatic fibrosis is the activation of stromal cells, while the heterogeneity of pancreatic stromal cells has not been fully elucidated. Gli1 + stromal cells, a novel type of stromal cells, drive fibrosis in multiple organs such as the kidney and heart. Nevertheless, the role of Gli1+ stromal cells in CP fibrosis remains unclear.Methods We constructed Gli1 + cell lineage tracing mice (Gli1 CreERT2, Rosa-lsl-tomato) and established CP models via cerulein administration (IDDF2026-ABS-0191 Figure 1(A) Gli1 stromal cells can differentiate into myofibroblasts to promote CP fibrosis). Through immunofluorescence and flow cytometry, we clarified the localization and differentiation of Gli1+ stromal cells (IDDF2026-ABS-0191 Figure 1(B-F) Gli1 stromal cells can differentiate into myofibroblasts to promote CP fibrosis). We also constructed Gli1CreERT2, Rosa26-EGFP-DTA mice to determine the role of Gli1+ cells in CP fibrosis (IDDF2026-ABS-0191 Figure 1(G-L) Gli1 stromal cells can differentiate into myofibroblasts to promote CP fibrosis). Gli1+ and Gli1- stromal cells were sorted by flow cytometry for Smart-Seq sequencing (IDDF2026-ABS-0191 Figure 2(A-K) The EFNB1 Ephb2 axis between ADM epithelial cells and Gli1 stromal cells may serve as an activating factor). Cell-cell interaction were identified using human and mouse single-cell RNA sequencing (scRNA-seq) data (IDDF2026-ABS-0191 Figure 2(J-L) The EFNB1 Ephb2 axis between ADM epithelial cells and Gli1 stromal cells may serve as an activating factor). In vitro experiments on human pancreatic fibroblasts were conducted to validate our findings (IDDF2026-ABS-0191 Figure 3(A-J) Knockdown and overexpression of Ephb2 decrease and increase the expression of ECM and FAK, respectively).Results We found that Gli1+stromal cells were located adjacent to endothelial cells in the normal pancreas, while getting close to CK19 +acinar-to-ductal metaplasia (ADM) epithelial cells in CP (IDDF2026-ABS-0191 Figure 1(A,B) Gli1 stromal cells can differentiate into myofibroblasts to promote CP fibrosis). Moreover, Gli1+ stromal cells were expanded and differentiated into myofibroblasts in CP (IDDF2026-ABS-0191 Figure 1(C-F) Gli1 stromal cells can differentiate into myofibroblasts to promote CP fibrosis). The conditional depletion of Gli1+cells significantly alleviated mouse pancreatic fibrosis (IDDF2026-ABS-0191 Figure 1(G-L) Gli1 stromal cells can differentiate into myofibroblasts to promote CP fibrosis). Next, we sorted Gli1+and Gli1-stromal cells and compared their differentially expressed genes (IDDF2026-ABS-0191 Figure 2(A,B) The EFNB1 Ephb2 axis between ADM epithelial cells and Gli1 stromal cells may serve as an activating factor). The results showed that Gli1+stromal cells exhibited enhanced wound healing capacity and significant upregulation of the Eph signaling pathway, in both of which Ephb2 was enriched (IDDF2026-ABS-0191 Figure 2(C-F) The EFNB1 Ephb2 axis between ADM epithelial cells and Gli1 stromal cells may serve as an activating factor). Protein-protein interaction analysis suggested Ephb2 may promote ECM gene expression via Ptk2(FAK) (IDDF2026-ABS-0191 Figure 2(G) The EFNB1 Ephb2 axis between ADM epithelial cells and Gli1 stromal cells may serve as an activating factor). scRNA-seq analysis identified EFNB1 from ADM epithelial cells as a main ligand of EPHB2 in stromal cells, with their interaction confirmed by immunofluorescence (IDDF2026-ABS-0191 Figure 2(J-L) The EFNB1 Ephb2 axis between ADM epithelial cells and Gli1 stromal cells may serve as an activating factor). In vitro, EFNB1-Fc stimulation enhanced ECM proteins and FAK expression, which were reversed by EPHB2 siRNA knockdown (IDDF2026-ABS-0191 Figure 3(A-H) Knockdown and overexpression of Ephb2 decrease and increase the expression of ECM and FAK respectively). Also, EPHB2 overexpression can significantly upregulate the expression of ECM proteins and FAK.Conclusions Gli1 + stromal cells can differentiate into myofibroblast and specifically upregulate EPHB2 expression in CP. ADM epithelial cells in CP provide EFNB1 signals to promote the activation of Gli1+ stromal cells and the downstream activation of the FAK pathway. Our findings highlight the potential of targeting EPHB2 for the treatment of CP fibrosis.Abstract IDDF2026-ABS-0191 Figure 1Abstract IDDF2026-ABS-0191 Figure 2Abstract IDDF2026-ABS-0191 Figure 3