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. Although fibrosis has been studied globally and at the region-of-interest level, its impact on stromal–ductal architecture and immune cell localization remains unknown. In this study, 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 in which 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 uncover that activated, proinflammatory leukocytes preferentially localize near malignant ducts in regions with low fibroblast density. Stratifying epithelial ductal regions by CAF abundance reveals a graded constraint: increasing fibroblast content corresponds to reduced leukocyte–epithelial proximity and elevated collagen I deposition. Despite their exclusion in high-CAF ducts, leukocytes in low-CAF ducts retain functional competence. Mechanistically, ligand–receptor inference implicates collagen–CD44 signaling as an adhesion axis anchoring immune cells within fibroblast-rich zones. CD44 blockade augments natural killer cell motility in vitro, whereas enhancing lymphocyte collagenase activity through matrix metalloproteinase 14 (MMP14) overexpression promotes infiltration by overcoming alpha smooth muscle actin (αSMA+) CAF-mediated stromal barriers in vivo. Thus, by establishing ductal regions as critical spatial units of immune exclusion, these findings provide a framework for dissecting stromal–immune interactions, reveal targetable “stromal checkpoints,” and provide complementary strategies to overcome CAF-driven barriers to leukocyte motility and infiltration in PDAC.