(E-163) Tumor Cell Exposure to Elevated Hydrostatic Pressure Alters the Mechanosensitivity of Cell Migration

Introduction:: Introduction: Metastatic spread has been the scourge of tumor treatment, making cancer one of the leading causes of death worldwide. In vivo, leaky microvessels and/or compromised lymphatic drainage raise the interstitial fluid pressure within the tumor to ~1 - 5 kPa. While at this high fluid pressure, a small fraction of cancer cells split away from the primary tumor, enter the bloodstream and exit the circulation to form a secondary tumor. During their journey, migrating tumor cells encounter and respond to diverse physical signals, including pressure-generated flow fields, extracellular viscosity and stiffness. Although the widely held view is that cancer cells can adapt to these signals, the specific cues that control cell mechanosensitivity remain unknown

Materials and Methods:: Materials and Methods: We hypothesized that tumor cell preconditioning at elevated hydrostatic pressure (HP) alters the migratory responses to physical signals. To test this hypothesis, we cultured MDA-MB-231 breast cancer cells or HT-1080 fibrosarcoma cells under high (1 kPa) or low (0.08 kPa) HP in gas permeable, PDMS-based μ-fluidic devices (Fig. 1 A, B). After four days of preconditioning, cells were exposed to different stiffness, viscosities or pressure differentials (ΔP). The migratory responses of cells to substrate stiffness and extracellular viscosity were examined by tuning the rigidity of polyacrylamide gels (0.2-20 kPa) and altering media viscosity (0.77 or 8 cP), respectively, to cover the magnitude of these two physical cues encountered by cells during metastasis. The impact of ΔP on the migratory behavior of cells was evaluated through a microchannel device. Specifically, we quantified cell entry into collagen type I-coated confining (Width x Height = 10 x 3 μm²) microchannels that were similar in size to openings in the endothelial layer while subjecting cells to a pressure drop of ΔP=P₂-P₁=‒0.16 kPa (Fig. 1 C). This pressure drop was chosen to simulate the pressure difference between microvessels and surrounding tissue, which typically ranges from 0.1-0.3 kPa. We also investigated whether tumor cells preconditioned at elevated HP acquired mechanical memory. Cells were subjected to high HP for four days, followed by an additional four days of culture under low HP. Then, their responses to the aforementioned physical signals were evaluated

Results, Conclusions, and Discussions:: Results and Discussion: Preconditioning cells at low HP triggered a biphasic migratory response to substrate stiffness, with the highest cell speed observed at intermediate stiffnesses (12 kPa) (Fig. 1 D). Strikingly, for cells treated under high HP, cell migration efficiency increased monotonically with matrix rigidity (Fig. 1 D). While elevated viscosity doubled the migration speed of cells pre-exposed to low HP, it only slightly enhanced the motility of cells pre-treated under high HP (Fig. 1 E). We also found that ΔP=‒0.16 kPa markedly suppressed entry of cells pre-exposed to low HP into confining microchannels (Fig. 1 F). The effect of ΔP was less pronounced for cells preconditioned at high HP (Fig. 1 F). These results held true for both HT-1080 fibrosarcoma cells and MDA-MB-231breast cancer cells and suggest that high HP altered the mechanosensitivity of cell migration. Furthermore, we examined if the effects of high HP on cell mechanosensitivity persisted after the discontinuation of this signal. Our results showed that MDA-MB-231 breast cancer cells, pre-treated at high HP for four days, retained diminished migratory responses to substrate stiffness, extracellular viscosity and pressure-driven flow after their culture under low HP (Fig. 1 G). RNA-seq analysis revealed genes differentially expressed under high vs. low HP. Using this knowledge, our future work will focus on identifying pathways involved in mediating mechanical memory and migratory mechanoresponses.
Conclusion: In sum, by integrating state-of-the-art bioengineering and materials with imaging tools, we identified HP as a key regulator of cell mechanosensitivity. Our work has broader implications in cancer metastasis since it generates new fundamental knowledge on how migrating tumor cells sense and responds to physical signals of the local microenvironment.

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