Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive solid tumor, closely associated with its unique tumor microenvironment (TME), which is characterized by a dense desmoplastic stroma. Abundant stromal cells, primarily fibroblasts, constitute the majority of cells in the tumor mass and exhibit pronounced spatial heterogeneity. Importantly, the spatial distribution of tumors and fibroblasts is vital for shaping the TME and critically influencing therapeutic responses. Here, we present a facile microarray chip for generating architecturally defined 3D PDAC heterospheroids. This platform enables us to mimic the dynamic interactions between tumor and stromal cells and to investigate how spatial organization influences stroma heterogeneity, tumor invasion and chemoresistance. The chip incorporates square concave microstructure array allowing controllable and reproducible production of uniform-sized spheroids. By simply altering the cell seeding sequence, we successfully constructed heterospheroids with distinct spatial distributions of cancer cells and fibroblasts. We further demonstrated that these organizational patterns modulate tumor-stroma crosstalk and ultimately regulate tumor invasive behavior. Furthermore, the heterospheroids with defined patterns exhibited distinct drug responses, and the potential for combination therapy evaluation was also verified. Beyond providing a robust platform for engineering heterospheroids with controllable tumor-stroma architectures, this system offers a robust 3D co-cultured model for advancing cancer research and drug screening.