Periductal Fibroblast Density Defines Lymphocyte Exclusion via a CD44-Dependent Stromal Checkpoint in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) exhibits dense fibrosis and immune exclusion. While fibrosis has been studied globally and at the region-of-interest level, its impact on stromal-ductal architecture and immune cell localization remains unknown. Here, we establish cancer-associated fibroblast (CAF)-stratified ductal spatial architecture as a fundamental determinant of immune exclusion in PDAC. Focusing on malignant PDAC epithelial ductal regions, the critical interface where immune cells must access tumor epithelium, we demonstrate that periductal fibroblast organization dictates leukocyte proximity. Through integrative analysis of treatment-naïve patient samples from three independent cohorts – including imaging mass cytometry, multiplex immunohistochemistry, and single-cell RNA sequencing – we uncovered that activated, pro-inflammatory leukocytes preferentially localized near malignant ducts in regions with low fibroblast density. Stratifying epithelial-ductal regions by CAF abundance revealed a graded constraint: increasing fibroblast content corresponded to reduced leukocyte–epithelial proximity and elevated collagen I deposition. Despite their exclusion in high-CAF ducts, leukocytes in low-CAF ducts retained functional competence. Mechanistically, ligand–receptor inference implicated collagen–CD44 signaling as an adhesion axis anchoring immune cells within fibroblast-rich zones, with CD44 blockade enhancing natural killer cell invasion and motility in fibrotic spheroid models. Thus, by establishing ductal regions as critical spatial units of immune exclusion, these findings provide a framework for dissecting stromal–immune interactions and reveal targetable “stromal checkpoints” that can be leveraged to overcome CAF-driven barriers to leukocyte motility and infiltration in PDAC. Graphical Abstract