A pancreatic cancer organoid-macrophage co-culture using starPEG-heparin hydrogel deciphers tumor-immune cell interactions.

Macrophages are among the most abundant immune cells in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) and play a key role in regulating the immunosuppressive niche that facilitates tumor growth. Although recent three-dimensional (3D) culture systems using patient-derived materials have advanced our understanding of tumor biology, most models lack key cellular TME components and thus fail to capture tumor-immune cell interactions. To address this gap, we developed an in-vitro 3D co-culture model incorporating PDAC patient-derived organoids (PDOs) and macrophages within a synthetic hydrogel matrix. We optimized culture conditions by tuning medium and matrix conditions to support both cell lineages. Flow cytometry and transcriptomic analyses revealed that initially undifferentiated macrophages adopt an M2-like profile upon exposure to PDAC PDOs in starPEG-heparin hydrogels, mirroring the macrophage phenotypes observed by multiplex immunohistochemistry in the matched primary PDAC tissues. Cytokine secretome profiling revealed PDO-specific differences, indicating distinct underlying macrophage polarization subtypes. Collectively, our starPEG-heparin hydrogel-based 3D co-culture enables hypothesis-driven and physiologically relevant studies of tumor-macrophage interactions and may advance immune-modulatory treatment strategies in patients with PDAC.