An Extravasation-on-Chip Integrating Mechanical Measurements for Post-Extravasation Metastatic Pathways Study

Tumor cells (TCs) typically achieve brain metastasis through two distinct post-extravasation routes: invasion into the parenchyma or migration along the abluminal surface of vessels. However, the biomechanical cues dictating this route selection remain poorly understood. Here, we employed a triparallel extravasation-on-chip that enables independent modulation of key mechanical cues within the brain extravasation microenvironment. Traction force microscopy (TFM) and atomic force microscopy (AFM)-based mechanical characterization were used to quantify tumor-cell mechanical responses. We show that, compared with soft extravascular matrix, stiff extravascular matrix markedly suppresses invasion of TCs into the matrix after extravasation. Strikingly, this inhibitory effect can be reversed when vascular wall compactness is increased and fluid shear stress (FSS) is applied, even under stiff-matrix condition. To elucidate the mechanical basis underlying this adaptive invasion, AFM and TFM measurements revealed that tumor-cell stiffening, accompanied by increased F-actin organization, was associated with enhanced traction generation and matrix deformation, consistent with the increased matrix invasion observed under these conditions. These results suggest that tumor-cell stiffening represents a mechanoadaptive state, highlighting tumor-cell mechanical adaptation as an additional regulator of post-extravasation invasion beyond matrix compliance alone. This study provides a controllable platform to dissect TCs-matrix interfacial mechanics and elucidate how physical microenvironmental cues regulate organ-specific metastatic progression.