Bioengineering Approach to Elucidate How Cells Sense and Respond to Diverse Physical Cues

Cell migration is essential for a variety of (patho)physiological processes, such as immune surveillance, tissue morpho-dynamics and homeostasis and cancer metastasis. In vivo, metastasizing cancer cells migrate through complex, confining tissue microenvironments and interact with a variety of physical forces which can alter their migratory phenotype. How cells sense such mechanical cues and exert forces in their three-dimensional (3D) surroundings are still not completely known. We herein integrate microfabrication, live-cell microscopy, super-resolution imaging, molecular biology and optogenetic tools along with mathematical and in vivo models to identify how specific physical forces affect the process of cell migration and metastasis. We decipher the mechano-sensing mechanisms and downstream pathways involved in each of the processes. We also develop a novel biomimetic platform to study cell migration in high throughput manner under independently tuned stiffness and degree of confinement. We innovate 3D traction force measurements (TFM) around spatially constrained single cells using our platform. Following a discussion of our current understanding of physical cues in cancer metastasis and confined cell migration platforms, we investigate the effect of shear stress during intravasation. Using microfluidic devices, we mimic the entry of cells into shear flow inside blood vessels and demonstrate that normal fibroblasts avoid shear stress, similar to what happens physiologically. Our data reveal a mechanism where shear stress upregulates RhoA activity by inducing calcium influx via the mechanosensitive transient receptor potential cation channel, subfamily M, member 7 (TRPM7). In distinct contrast, cancer cells derived from fibroblasts (fibrosarcoma), have lower shear sensitivity due to reduced expression of TRPM7. When fibrosarcoma cells are engineered to overexpress TRPM7-YFP, they display increased shear sensitivity and significantly reduced intravasation in vivo, compared to YFP control cells. Next, we evaluate the effect of physiologically relevant fluid viscosity on cell migration. The viscosity of the interstitial fluid is a critical physical parameter that varies under both physiological and pathological conditions, such as cancer. However, its impact on cell biology and the mechanism by which cells sense and respond to changes in extracellular viscosity are unknown. We demonstrate that elevated viscosity counterintuitively increases the motility of various cell types in confinement, on two-dimensional (2D) surfaces, and cell dissemination from 3D tumor spheroids. Increased mechanical loading imposed on the cell leading edge by elevated viscosity induces an Actin Related Protein 2/3 (Arp2/3) complex-dependent dense actin network, which enhances Na+/H+ exchanger 1 (NHE1) polarization via its actin-binding partner ezrin. NHE1 together with aquaporin 5 promote cell swelling which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and enhances calcium influx that leads to increased RhoA-dependent cell contractility. The coordinated action of actin remodeling/dynamics, NHE1-mediated swelling and RhoA-based contractility facilitates enhanced motility at elevated viscosities. Moreover, breast cancer cells exposed to elevated viscosity exhibit increased migration in zebrafish and lung metastasis in mice, indicating an ability to memorize prior mechanical stress. Cumulatively, extracellular viscosity is a physical cue that can regulate key (patho)physiological processes like metastasis. Finally, we apply our soft-lithography expertise and sulfo-N-hydroxysuccinimide (NHS) esters as crosslinkers to develop a novel polyacrylamide (PA) based microfluidic device with an array of microchannels which are surrounded by physiologically relevant compliant walls on four sides. The design enables us to subject cells to confinement with anisotropic stiffnesses on the apico-lateral versus basal surfaces. Ultimately by embedding nanobeads in the PA gel, we are able to perform spatiotemporally resolved 3D TFM around migrating single cells. Overall, this dissertation highlights the critical role of mechanical cues in regulating cell migration and how bioengineering approaches can be leveraged to tease apart such contributions in the metastatic cascade