Stiffness-Tunable Hydrogel Microfluidic Chip Reveals the Role of Stiffness in Cholangiocarcinoma Invasion and Pre-Metastatic Niche Formation

Cholangiocarcinoma (CCA), particularly intrahepatic cholangiocarcinoma (ICC), is a highly aggressive malignancy with extensive stromal fibrosis and frequent pulmonary metastasis. Existing in vitro models, including 2D cultures, animal systems, and Matrigel-based organoids with fixed stiffness, fail to replicate physiologically relevant cell–matrix interactions. Here, we developed a stiffness-tunable microfluidic chip integrating sodium alginate–decellularized ECM (SA–dECM) hydrogels, enabling dynamic modulation of stiffness (2.90–13.31 kPa) to mimic clinical tumor conditions (∼7.94 kPa). Increased stiffness promotes chemoresistance, with gemcitabine IC 50 rising from 0.139 to 0.282 µ m and cisplatin from 2.49 to 4.23 µ m , alongside epithelial-mesenchymal transition (EMT), as evidenced by a 64% reduction in E-cadherin and a 6.35 fold increase in MMP2 expression. Co-culture of ICC organoids and lung fibroblasts on-chip further reveals that stiff matrices activate fibroblasts via TGF-β signaling, increasing α-SMA and collagen deposition by 7.5 and 5.4 fold, respectively. These changes contribute to a pre-metastatic niche, as confirmed by a 4.11 fold increase in invasive cell counts in PET membrane invasion assays. This dynamic stiffness-tunable platform provides a robust in vitro model for studying stiffness-driven invasion and pre-metastatic niche formation in ICC and offers a valuable tool for personalized screening of anti-fibrotic and chemosensitizing therapies.