The Crosstalk Between Mechanical Forces and Intracellular pH in Driving Cancer Cell Phenotypes

Intracellular pH (pHi) dynamics are central to regulating key cellular behaviors such as migration, proliferation, differentiation, and apoptosis. However, most investigations into pHi-sensitive processes have relied on population-level measurements under non-physiological conditions, limiting our understanding of how external cues influence pHi and the role of pHi in cell behaviors. This gap is particularly relevant in cancer, where cells exhibit dysregulated pHi dynamics which promote cancer cell behaviors, like migration and invasion, but also exist within the abnormal tumor microenvironment, characterized by increased extracellular matrix (ECM) stiffness, compression, and shear stress. These mechanical changes further promote cancer cell behaviors like migration and invasion, yet the interplay between ECM mechanics and pHi regulation remains poorly understood. If mechanical forces directly influence pHi, then pHi may serve as an overlooked driver and potential therapeutic target of progressive cell phenotypes supported by the tumor microenvironment. Addressing this gap requires studying pHi dynamics in physiologically relevant mechanical models. The work presented in this thesis demonstrates that mechanical forces regulate pHi dynamics and identifies the role of pHi in mechanosensitive cell behaviors. By developing in vitro systems that mimic the mechanical forces of the TME and measurement of single-cell pHi in both cancerous and normal cells, we uncover a critical role for pHi in mediating cell responses to mechanical cues. We show that ECM stiffness decreases pHi in metastatic cancer cells, further identifying decreased pHi as both necessary and sufficient to drive vasculogenic mimicry, a progressive cancer phenotype, via regulation of ß-catenin. We determine that prior mechanical exposure (stiffness memory) influences stiffness-mediated pHi regulation in normal epithelial cells. Furthermore, we show that mechanical compression induces a cancer-specific increase in pHi that correlates with an adaptive amoeboid morphology phenotype. By isolating distinct mechanical forces, we identify their individual contributions to pHi dynamics and adaptive cell behavior within the complex tumor microenvironment and lay a framework for future studies to further investigate pHi as a direct and targetable mediator of adaptive and progressive cancer cell behaviors.