Development and Optimization of a Modular Microfluidic Device to Study the Effects of Deformation on Metastatic Breast Cancer

Human cells experience a variety of biophysical forces in vivo during metastasis that can result in changes in both biological processes and cellular phenotype, driving pro-survival behavior. Prior studies have shown how matrix stiffness, fluid viscosity, and fluid shear stress enhance the proliferation of metastatic breast cancer. One additional biophysical force cells experience is deformation, which can occur during transendothelial migration during extravasation metastatic events and during transit through narrow capillaries while circulating throughout the body. While the biophysical changes in cells due to deformation have been well studied (e.g., changes in size, shape, recovery time), the phenotypic changes induced by deformation driving pro-survival behavior have not been fully elucidated. The goal of this work was to provide new insight into the biochemical changes, such as altered proliferation or protein phosphorylation during biophysical interrogation in single breast cancer cells. This was accomplished by developing a modular microfluidic device consisting of two unique devices to first mimic the biophysical forces that are endured by the cell during metastasis and to second interrogate single cells using immunostaining for changes in proliferation and protein phosphorylation. Single breast cancer cells were deformed through a venturi-style constriction channel device and collected in a microwell array device for single-cell immunostaining utilizing fluorescence microscopy. Two different breast cancer subtypes, estrogen receptor positive (ER+) and triple negative breast cancer (TNBC), were studied due to prior work that has shown how these different subtypes deform differently, which suggests that the biochemical pathways altered as a result of these biophysical forces could also be variable. Device optimization steps were performed to confirm that 2,051 MDA-MB-231 cells/min were deformed resulting in >98.6% of intact, viable cells after deformation. Single MCF-7 (a model ER+ cell line) deformed cells exhibited decreased levels of the proliferative marker Ki67 and no change in phosphorylated AKT (p-AKT) compared to the non-deformed control cells. Single MDA-MB-231 (a model TNBC cell line) deformed cells exhibited increased levels of p-AKT and decreased levels of Ki67. These preliminary findings suggest that proliferation is diminished immediately after deformation in both subtypes; however, the increase in p-AKT in the TNBC subtype suggests another biochemical pathway is activated due to deformation. These initial findings support that the biochemical changes under deformation appear subtype specific.