(323b) Hydrostatic Pressure Is a Key Regulator of Cell Mechanosensation

Introduction: Cells can perceive and convert physical stimuli into biochemical signals, acting as mechanosensitive entities. The physical cues of the tissue environment (e.g., hydrostatic pressure, extracellular matrix stiffness, fluid flow, viscosity) activate mechanosensors on the cell surface, such as ion channels and integrins. This activation can alter the structure of the cytoskeleton, thereby triggering a cascade of biochemical signaling. In addition, key effector intracellular molecules, including nuclear envelope proteins and transcription factors, could sense cytoskeletal changes and induce downstream gene expression changes. While it is established that mechanical stimuli play a crucial role in regulating cellular processes like migration, proliferation, and differentiation, the specific mechanisms governing how cells adjust their mechanosensitivity after prolonged exposure to physical stimuli are still elusive.

Materials and Methods: We hypothesized that preconditioning of tumor cells at elevated hydrostatic pressure (HP) could alter how they responded to physical stimuli. To investigate this, we cultured MDA-MB-231 breast cancer cells and HT-1080 fibrosarcoma cells in a gas-permeable, PDMS-based microfluidic device. This setup allowed us to create environments of high (1 kPa) or low (0.08 kPa) HP by varying medium height. After a four-day preconditioning, cells were exposed to various levels of substrate stiffness, fluid viscosities, and pressure differentials (ΔP) to study their migration patterns. To assess how cells react to shifts in substrate stiffness, we varied the rigidity of polyacrylamide gels from 0.2 to 20 kPa, thereby recapitulating the stiffness levels in the body. Moreover, to replicate the range of fluid viscosities encountered in the interstitial tissue and circulation, we adjusted the viscosity of the media from 0.77 to 8 cP. Using a microchannel device, we studied the effects of fluid forces on cells. Given that microvessels and surrounding tissue usually have a pressure difference of 0.1 to 0.3 kPa, we examined cell entry into narrow, collagen-type I-coated microchannels simulating the gaps between endothelial cells, under a ΔP of -0.16 kPa. Our study further delved into the notion of mechanical memory in tumor cells that underwent preconditioning at elevated HP. To achieve this, cells were first subjected to a high HP environment for four days, followed by a period of four days under low HP conditions. We then assessed their responses to the aforementioned physical stimuli, with a focus on determining if these cells preserved a mechanical memory of their high-pressure exposure.

Results and Discussion: Subjecting cells to low HP led to a biphasic pattern of migration in response to increasing stiffness, with peak migration speed occurring at a substrate stiffness of 12 kPa. On the other hand, cells pre-exposed to high HP exhibited a steady rise in migration speed as matrix rigidity increased. Also, the viscosity-induced increase in migration speed of cells preconditioned at low HP was nearly abolished in cells treated under high HP, indicating their diminished response to elevated viscosity conditions. When exposed to a substrate stiffness of 0.2 kPa in a medium with a viscosity of 0.77 cP, cells pre-treated with both low and high hydrostatic pressure (HP) migrated at similar speeds. This led to further investigation on low HP pre-treated cells' response to higher viscosity under the same stiffness. Interestingly, low HP pre-conditioned cells increased their migration speed on a 0.2 kPa substrate when medium viscosity was elevated to 8 cP, unlike cells pre-conditioned with high HP, which showed no change under the same conditions. This suggests a change in the mechanosensitivity of cells preconditioned with high HP. Cells pretreated at low HP showed increased sensitivity to ΔP, leading to decreased entry into confined channels. However, cells pretreated at high HP exhibited increased channel entry compared to those treated at low pressure. Our data further indicated that MDA-MB-231 breast cancer cells pre-exposed to elevated HP retained their diminished responses to changes in substrate stiffness, extracellular viscosity and ΔP even after their culture under low-pressure conditions for an additional four days. It is well established that calcium is a critical second messenger, mediating cellular mechanotransduction. Thus, we explored the impact of HP preconditioning on intracellular calcium levels and calcium-dependent mechanoresponses. While cells subjected to low pressure exhibited elevated intracellular calcium levels in response to mechanical stimuli, those exposed to high pressure showed no such increase. Interestingly, treating cells preconditioned at high HP with Ionomycin, which enhances intracellular calcium influx, increased their responsiveness to fluid forces, resulting in reduced microchannel entry.

Conclusion: In our research, we utilized a multidisciplinary approach encompassing bioengineering, materials science, and imaging to investigate the impact of (patho)physiologically relevant pressures on cellular mechanoresponses. Our findings highlight pressure as a crucial factor in regulating cell mechanosensitivity and pinpoint the mechanisms responsible for this regulation. This study illuminates the complex interplay between migrating cells and their microenvironment, potentially paving the way for the discovery of novel therapeutic targets.