(20f) A Modular Microfluidic Platform to Study How Fluid Shear Stress Alters Estrogen Receptor Phenotype in Single Breast Cancer Cells

Approximately 70% of breast cancer patients present with an estrogen receptor-positive (ER+) subtype. Many of these patients respond to endocrine therapy which is designed to specifically target the estrogen receptor; however, following metastatic spread many of these patients develop a resistance to endocrine therapy resulting in a poorer prognosis. Cancer cells are exposed to a range of chemical and physical cues during metastasis that can result in both phenotypic and genotypic changes; however, the underlying cause of endocrine resistance is currently unknown. During metastasis, cancer cells are exposed to fluid shear stress (FSS) while traversing the circulatory system which can result in both deformation and DNA damage. FSS can be understood as the internal frictional force between moving layers in laminar flow. The goal of this work is to further study the effects of fluid shear stress on its role in the developed resistance to endocrine therapies. The most common approach to study FSS is to flow cells through tubing using either a peristaltic pump or syringe pump. This approach has been effective; however, the individual cells can experience a range of FSS magnitudes due to the larger diameter of the tubing and parabolic velocity profile associated with laminar flow. To overcome this limitation, a micron-scale fluidic channel was developed to expose single ER+ breast cancer cells (MCF-7) to similar FSS magnitudes and durations. ‘This shearing module’ was then paired with a single cell ‘trapping module’ containing an array of 3000 traps to allow for on-chip immunostaining of MCF-7 cells for biomarkers associated with endocrine resistance. A strength of the modular approach is that the shearing module could be used by itself to shear a large number of cells for off-chip analysis using standard techniques including PCR and Western blot for both population analysis and single-cell resolution. In this way, for bulk analysis, the cells can be collected and frozen after shearing in a microtube for subsequent analysis. For single-cell resolution, they are trapped in the wells by pairing the shearing module. MCF-7 cells were found to exhibit both enhanced proliferation and increased protein phosphorylation following exposure to 10 dyn/cm² FSS. Interestingly, the bulk analysis showed no change in the phosphorylation of Akt; however, the single-cell resolution showed a significant change in pAkt levels. Analyzing this data showed that only a small subpopulation, which could correspond to an endocrine resistance population, exhibited enhanced pAkt levels validating the need for single-cell analysis. Phosphorylated Akt plays an important role in colonization and tumorigenesis since it promotes cell survival by inhibiting apoptosis and mediating cells proliferation. Finally, immunostaining for phosphorylation of the estrogen receptor showed diminished levels in cells exposed to shear when compared to the non-sheared control. This finding supports a potential mechanism for endocrine resistance by FSS-induced loss of the estrogen receptor at the metastatic site.

Figure 1. Experimental set-up diagram, COMSOL modeling, and devices pictures. A) Top view of the shearing device showing some dimensions, the inlet, and the outlet. B) Top view of the trapping array showing the usual distribution of cells once they are trapped. C) Cross-section of the shearing device showing the dimensions and the velocity profile inside. D) Side view of the trapping array. E) Brightfield image of MCF-7 cells after being sheared and trapped. F) Picture of the shearing device with red dye inside. G) Picture of the trapping array with red dye inside.