Extracellular matrix stiffness affects microbubble-assisted endothelial permeabilization under flow

Cancer immunotherapy has faced challenges in the treatment of solid cancers due to the complex tumor microenvironment (TME), including physical barriers that prohibit immune cell infiltration. Ultrasound (US) stimulated microbubbles present a novel way to potentiate cancer immunotherapy in tumors by permeabilizing the tumor vasculature, however the effect of individual TME parameters on treatment efficacy has not yet been elucidated. Here, we focus on one biophysical parameter, showing that an increase in matrix stiffness increases US-assisted membrane permeabilization. Using a novel setup that allows for real-time visualization under flow, HUVECs seeded on polyacrylamide hydrogels of different stiffnesses (800 and 1600 Pa) showed an increase in sonoporation rates. Collagen models corroborate this trend at two different flow rates for a range of stiffnesses (5, 25, and 75 Pa): for both 5 ml/min and 30 ml/min, there is a relative increase in sonoporation from the stiffer substrate. For a given collagen substrate, there is a significant increase in sonoporation efficiency with increasing flow rate. These data can be used to fine-tune treatments according to different TMEs; further, our findings have applications in designing US parameters for targeted drug delivery and other clinical contexts that consider a variety of tissue stiffnesses.