Objective:

To investigate the immunosuppressive mechanisms driving T-cell dysfunction in pancreatic ductal adenocarcinoma (PDAC) and evaluate the effects of CXCR4 and PD-1 inhibition.

Approach:

• 3D PDAC Spheroids: Utilized 3D PDAC spheroids generated from PANC-1 cells and primary pancreatic stellate cells (PSC) to analyze T-cell infiltration, activation, and checkpoint regulation.
• Patient-Derived Spheroids: Infiltrated patient-derived spheroids (PDS) with autologous T-cells to assess patient-specific T-cell suppression patterns.

Key Findings:

• Infiltrated T-cells exhibited a pronounced exhaustion signature, including strong upregulation of PD-1, LAG-3, and CTLA-4.
• Incorporation of pancreatic stellate cells (PSC) generated a fibrotic barrier that markedly restricted T-cell infiltration, modeling the desmoplastic TME characteristic of PDAC.
• Pharmacological CXCR4 blockade with AMD3100 significantly enhanced T-cell infiltration into PSC-containing spheroids.
• Treatment with the anti-PD-1 monoclonal antibody pembrolizumab partially restored the effector cell function of T-cells within the 3D system.

Interpretation:

The study demonstrates that the 3D model effectively captures the immunosuppressive environment of PDAC, highlighting the roles of stroma and checkpoint molecules in T-cell dysfunction.

Limitations:

• The study primarily focuses on in vitro models, which may not fully replicate in vivo conditions.
• Further validation in clinical settings is needed to confirm findings.

Conclusion:

Together, our findings identify critical determinants of T-cell dysfunction in PDAC and introduce a versatile, animal-free 3D model that captures hallmark immune-evasion mechanisms in PDAC.