Targeting stroma-mediated T-cell exclusion and functional exhaustion in pancreatic ductal adenocarcinoma through CXCR4 and PD-1 blockade.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies to date and characterized by a unique immunosuppressive and highly desmoplastic tumor microenvironment (TME). These features drive profound T-cell dysfunction and maintain high resistance to current immunotherapy. By recapitulating the complex 3D architecture of human PDAC, we demonstrate the key immunosuppressive mechanisms that drive T-cell dysfunction within the tumor microenvironment. Method: 3D PDAC spheroids-generated from PANC-1 cells alone or together with primary pancreatic stellate cells (PSC)-were infiltrated with primary human T-cells from healthy donors, allowing controlled analysis of T-cell infiltration, activation, and checkpoint regulation. Furthermore, patient-derived spheroids (PDS) composed of primary tumor cells and cancer-associated fibroblasts were infiltrated with autologous T-cells. Results: Infiltrated T-cells exhibited a pronounced exhaustion signature, including strong upregulation of PD-1, LAG-3, and CTLA-4, closely mirroring the phenotype of tumor-infiltrating lymphocytes (TIL) isolated from PDAC patient samples. Incorporation of pancreatic stellate cells (PSC) generated a fibrotic barrier around the tumor cells that markedly restricted T-cell infiltration, modeling the desmoplastic TME characteristic of PDAC. From a mechanistic perspective, stromal CXCL12-CXCR4 enhanced T-cell exclusion: pharmacological CXCR4 blockade with AMD3100 significantly enhanced T-cell infiltration into PSC-containing spheroids. Furthermore, treatment with the anti-PD-1 monoclonal antibody pembrolizumab partially restored the effector cell function of T-cells within the 3D system. These results demonstrate that this minimalistic platform is capable of capturing complex, cytokine- and stroma-driven immunomodulation typically observed only in advanced organoid or Conclusion: Together, our findings identify critical determinants of T-cell dysfunction in PDAC and introduce a versatile, animal-free 3D model that powerfully captures hallmark immune-evasion mechanisms in PDAC. This system provides a scalable and mechanistically faithful tool for dissecting TME-driven immune suppression and for accelerating the functional evaluation of immunotherapeutic strategies, including patient-tailored approaches.