Abstract
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, largely due to its extensive desmoplastic stroma and an intensely immunosuppressive tumor microenvironment (TME) that facilitates early metastatic dissemination. While single-cell RNA sequencing has illuminated cellular heterogeneity in PDAC, it destroys the spatial context required to understand localized microenvironmental interactions. In this study, we integrated high-resolution spatial transcriptomics with matched single-cell RNA sequencing to reconstruct the spatial architecture of primary PDAC tumors and matched liver metastases. Spatial mapping revealed distinct microenvironmental zoning within metastatic lesions, characterized by a spatially constrained desmoplastic boundary enriched in periostin-expressing myofibroblastic cancer-associated fibroblasts (myCAFs). Within the core metastatic regions, we identified a spatial co-localization of SPP1-positive macrophages and exhausted CD8-positive T lymphocytes, forming specialized immunosuppressive niches. Cell-cell interaction analysis identified localized TGFB1 and CXCL12 signaling axes emanating from stromal barriers that enforce physical and chemical T cell exclusion. Ex vivo functional validation using patient-derived 3D organoid co-cultures demonstrated that knocking down stromal SPP1 expression restored CD8-positive T cell motility and cytotoxic activity. Our findings provide a high-definition spatial atlas of metastatic PDAC microenvironments, identifying spatially anchored immunomodulatory targets to overcome immune evasion in advanced pancreatic cancer.
