Differential regulation of EMT-related pathways by inflammatory microenvironment in 3D prostate spheroids and tumor-stroma interaction models.

Chronic inflammation plays a major role in the initiation, progression, and metastasis of prostate cancer (PCa) by driving tumorigenic processes such as Epithelial-Mesenchymal Transition (EMT). To investigate these mechanisms in physiologically relevant settings, we optimized two complementary in vitro tumor models: 3D prostate spheroids that mimics multicellular interactions and an epithelial-myofibroblast co-culture system that reflects key tumor-stroma dynamics under inflammatory conditions. Our optimization revealed that successful spheroid formation depends on cell line-specific molecular features rather than a universal spheroid size. Compared with conventional 2D cultures, inflammatory stimuli modulated EMT mediators significantly in a cell line-dependent manner; notably, fibronectin, vimentin, and TWIST1 were differentially regulated in 3D spheroids, indicating enhanced invasive features driven by inflammatory cues. The co-culture model successfully represented reactive stroma formation, and stromal influence dramatically shaped inflammation-induced anoikis resistance and cell migration. Overall, our findings demonstrated that tumor-stroma interactions critically contribute to the impact of inflammation on EMT in PCa. Our optimized 3D spheroid and co-culture models provide a robust and powerful platform to unravel the functional consequences of inflammation and offer mechanistic insight into cellular processes underlying PCa progression, supporting the development of targeted therapeutic strategies.