Substrate stiffness and cellular microenvironment regulate cell and junction mechanics in iPSC-derived brain microvascular endothelial cells.

The blood-brain barrier (BBB) is a mechanically responsive interface that protects the central nervous system. Brain tissue exhibits region-specific stiffness that evolves throughout development and is altered in aging and various neurological diseases. These stiffness changes are increasingly recognized as key modulators of endothelial cell behavior and BBB integrity. However, the mechanisms by which brain endothelial cells sense and adapt to variations in their mechanical microenvironment remain poorly defined. Moreover, how mechanical cues interact with cellular signals from astrocytes and pericytes to modulate endothelial mechanics and junctional organization has been largely unexplored. Here, we demonstrate spatial regulation of subcellular mechanics in human iPSC-derived brain microvascular endothelial cells (iBMECs) in response to physiologically and pathologically relevant substrate stiffness (1-194 kPa). Using atomic force microscopy, we quantified Young's modulus at three distinct cellular regions-tricellular junctions, bicellular junctions, and cell bodies. iBMECs cultured on compliant substrates (1, 2.5, and 15 kPa) exhibited pronounced mechanical polarization, characterized by significantly elevated stiffness at tricellular regions compared with bicellular regions and cell bodies. This spatial organization was lost on supraphysiological stiffness (194 kPa), which reduced overall cell stiffness and eliminated regional distinctions. Co-culture with astrocytes and pericytes decreased global stiffness but preserved the dominant reinforcement at tricellular regions. In contrast, exposure to metastatic breast cancer cells abolished junction polarization at tricellular regions and suppressed stiffness across all regions, particularly on soft substrates. These findings reveal that BBB endothelial mechanics are regulated by both matrix stiffness and BBB cell context in a region-specific manner. This work provides new insight into how physical and cellular cues shape BBB structure and function, with implications for understanding barrier disruption in neurological disease and metastasis.