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. Competing Interest Statement Z.X.M. is currently employed by AstraZeneca. E.J.F. was on the scientific advisory board of Resistance Bio and a consultant for Mestag Therapeutics. L.M.W. sits on the Scientific Advisory Boards of Celldex Therapeutics, Bobcat Bio, Kuiper and Cytomx Therapeutics, is an advisor for Fortress Biotech and is founder and Chair of the Board of PushCART Therapeutics. Footnotes The current submission represents a conceptually re-envisioned study that diverges substantially from the earlier version in focus, scope, and mechanistic insight. In the prior submission, our work centered on the spatial distribution and epithelial interactions of natural killer (NK) cells within the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Since then, we have fundamentally reframed the study to address a broader and more impactful question: how the malignant ductal-fibrotic interface governs immune exclusion through a CD44-dependent stromal checkpoint. Major conceptual and experimental advances include: Reframed focus from NK cells to pan-leukocyte spatial organization at the malignant ductal regions -We shifted the emphasis from a single immune subset (NK cells) to a comprehensive analysis of all leukocyte populations within PDAC epithelial-ductal regions. -This reframing positions the study within a new conceptual framework, the periductal fibroblast density as a defining determinant of immune accessibility and epithelial proliferation. Identification of a stromal checkpoint governing immune access. Through integrated spatial and single-cell analyses, we defined a "stromal checkpoint" that restricts immune cell access to malignant ducts. Within this framework, collagen-CD44 adhesion emerged as a key mechanistic axis mediating immune cell retention in fibroblast-rich zones. Functional 3D spheroid assays demonstrated that disrupting this interaction enhances immune motility independently of ECM degradation, establishing a new conceptual model in which stromal architecture--not immune cell exhaustion--acts as the rate-limiting barrier to infiltration. Validation of IMC analysis We validated our IMC findings, by performing Vectra multiplex immunofluorescence (mfIHC) on an independent cohort of 17 PDAC tissues, confirming the inverse correlation between periductal fibroblast abundance and pro-inflammatory immune infiltration. Conceptual and structural overhaul of the manuscript -The new version has been retitled, reorganized, and rewritten to reflect this paradigm shift, from a descriptive NK-cell study to a mechanistically grounded analysis of stromal architecture as an immune barrier. - The manuscript now defines a broadly applicable mechanism of immune exclusion relevant to tumor immunology, microenvironmental biology, and translational oncology.