Dissecting the interplay between cytotoxic T lymphocytes and the pancreatic cancer stroma
Cytotoxic T lymphocytes (CTLs) are key effectors in antitumour immunity, patrolling tissues to detect and destroy cancerous cells by directing cytotoxic granules toward the immunological synapse formed with their target. Although CTL infiltration into solid tumours often correlates with improved patient outcomes, the mechanisms by which CTLs are recruited within tumour sites remain only partially understood. Moreover, it is unclear how the chemical and mechanical features of the solid tumour microenvironment (TME) orchestrate CTL migration and effector functions. Pancreatic ductal adenocarcinoma (PDAC) is characterised by a particularly dense fibrotic stroma shaped predominantly by cancer-associated fibroblasts (CAFs), which restrict T cell access and undermine immunotherapeutic efficacy. To better understand how this stromal architecture shapes CTL behaviour, we developed an in vitro organotypic co-culture model that mimics key features of the PDAC tumour microenvironment, enabling long-term, real-time analysis of CTL interactions with CAFs and PDAC cells. Our findings reveal that CAFs generate extensive tunnel-like networks, proteolytically digested and lined with denatured collagen, structures also found in human PDAC. In early stages of remodelling, CAFs attract CTLs through redundant chemokine secretion (CXCL12 and CCL5); however, as stromal remodelling progresses, these tunnels evolve into mechanical conduits that confine CTL migration, even in the absence of CAFs. Furthermore, CAFs markedly impair the ability of CTLs to recognise and engage PDAC spheroids, preventing effective recruitment to tumour masses and disrupting coordinated swarming toward malignant cells. Strikingly, CTLs that reach the tumour core fail to arrest and recognise cognate targets. We tested whether inhibition of CAF contractility or exposure of CTLs to CAF-secreted factors alone could recapitulate or mitigate these effects, allowing mechanical and soluble contributions to be distinguished. In parallel, bulk RNA sequencing and targeted perturbations were used to identify candidate pathways underlying CTL non-activation and tumour cloaking in the PDAC–CAF co-culture system. Our findings suggest that CAFs drive a progressive shift from chemotactic to mechanical confinement, profoundly altering CTL migration within the stroma. In addition, CAFs suppress CTL clustering and tumour-core engagement, underscoring the need for strategies that overcome stromal barriers in PDAC.
Pancreatic Cancer, Mechanobiology, Cytotoxic T Lymphocytes, Stroma, anzsrc-for: 31 BIOLOGICAL SCIENCES
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