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

ABSTRACT 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.