Prolonged Exposure to Elevated Hydrostatic Pressure diminishes cell mechanosensitivity

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) alters how they respond to physical stimuli. To investigate, we cultured MDA-MB-231 breast cancer cells and HT-1080 fibrosarcoma cells in a gas-permeable, PDMS-based microfluidic device. We created an environment of high (1 kPa) or low (0.08 kPa) HP by varying medium height (Fig. 1A, B). 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, recapitulating the stiffness levels in the body. To replicate the range of fluid viscosities encountered in the interstitial tissue and circulation, we adjusted the viscosity of media from 0.77 to 8 cP. Given that microvessels and surrounding tissue usually have a pressure difference of 0.1 to 0.3 kPa, we examined cell entry into narrow microchannels simulating the gaps between endothelial cells, under a ΔP of -0.16 kPa (Fig. 1C). To establish Serum Response Factor (SRF) as an intracellular regulator of cell mechanosensitivity, we manipulated its levels or activity and evaluated how cells responded to various physical stimuli. Our study also explored the concept of mechanical memory in cells, initially subjecting them to high HP for four days, followed by low HP. We then assessed their responses to physical stimuli, with a focus on determining if these cells preserved a mechanical memory of their high-pressure exposure

Results, Conclusions, and Discussions: Subjecting cells to low HP led to biphasic pattern of migration in response to increasing stiffness, with peak migration speed occurring at intermediate substrate stiffness (12 kPa) (Fig. 1D). Intriguingly, cells pre-exposed to high HP exhibited steady rise in migration speed as matrix rigidity increased (Fig. 1D). Moreover, viscosity-induced migration speed increase observed in low HP-preconditioned cells was nearly abolished in cells under high HP. Cells pretreated at low HP were more sensitive to ΔP of -0.16 kPa, reducing their entry into confined channels (Fig. 1E). In contrast, cells pretreated at high HP were less sensitive to the same ΔP, increasing their channel entry (Fig. 1E). We also found that transcriptional activity of the ubiquitous transcription factor SRF was reduced in cells subjected to high HP compared to those exposed to low HP. Cells pretreated at high HP regained their mechanosensitivity upon expression of constitutively active SRF (SRF-VP16), while cells under low HP conditions showed significant reduction in sensitivity following SRF knockdown (shSRF). This was confirmed by measuring cell responses to substrate stiffness, fluid viscosity and ΔP (Fig. 1F and G). Because calcium is a second messenger mediating cellular mechanotransduction, we studied how HP preconditioning affected its intracellular levels. While control cells subjected to low pressure exhibited an increase in intracellular calcium levels in response to ΔP (Fig. 1H) or elevated viscosity, low HP, shSRF and high HP cells showed no change (Fig. 1H). Interestingly, treating cells preconditioned at high HP with Ionomycin, which enhances intracellular calcium influx, increased their responsiveness to ΔP, resulting in reduced microchannel entry (Fig. 1I). 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 (Fig. 1J) even after their culture under low-pressure conditions for additional four days. Collectively, we demonstrate that elevated pressure downregulates SRF activity, thereby suppressing calcium-dependent mechanoresponses.

Conclusion: Our study merges bioengineering, materials science, and imaging to explore how pressure affects cell mechanoresponses. We establish pressure as a key regulator of mechanosensitivity, revealing potential therapeutic targets by understanding cell-environment interactions

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