Pancreatic ductal adenocarcinoma (PDAC) is frequently classified as immune-excluded, where T cells accumulate in stroma-rich, peritumoural regions without significant infiltration of the tumour core, negating immunotherapeutic strategies. A defining feature of PDAC is its extensive desmoplastic stroma, primarily engendered by cancer-associated fibroblasts (CAFs), which aberrantly deposit, contract, and stiffen the stromal extracellular matrix, and modulate leukocyte function through cytokine secretion. However, it remains unclear how CAFs impact T cell dynamics throughout disease progression. Here, we introduce an ex vivo organotypic co-culture model that replicates key features of the PDAC tumour stroma, allowing for extended, real-time observation of the interplay between CAFs, extracellular matrix, and cytotoxic T lymphocytes. We show that CAFs create extensive interconnected ‘tunnel’ networks, proteolytically digested and lined with denatured collagen, also observable in advanced-stage tumours in vivo. During early phases of stromal remodelling, CAFs chemoattract T cells into the tunnel networks, which at advanced stages of fibrosis constitute paths of least resistance, confining T cell movement and restricting their infiltration of the tumour, even upon CAF elimination. Thus, CAFs progressively switch from chemotactic to mechanical confinement of T cell motility patterns in the pancreatic tumour stroma, requiring novel, dual-targeting approaches to overcome their sequestration of T cells.
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