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Nano and Micro Technologies

Nano and Micro Technologies - Poster Session A

Poster J12 - A microscale approach to study how exposure to fluid shear stress alters cancer cell extravasation

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Breast cancer is one of the most common diagnoses for American women with one in eight women developing this terrible disease and >90% of cancer-related deaths due to the metastatic burden. Cancer metastasis occurs when circulating tumor cells (CTCs) intravasate from the primary tumor into the vasculature, traverse the bloodstream, and then extravasate at a distal location to form secondary tumors which has been shown to be more aggressive and resistant to therapeutics. The precise mechanism(s) driving this altered phenotype are currently unknown; however, some hypothesize that the biophysical forces that cells are exposed to in the vasculature can drive this pro-survival phenotype. One force CTCs experience is fluid shear stress (FSS), the unit area of force acting on the cell parallel to a small element of the surface (the endothelium), which has been shown to alter their phenotype. Previous studies have suggested that exposure to FSS induces the proliferation and protein phosphorylation of estrogen receptor positive (ER+) breast cancer cells driving this pro-survival phenotype at the metastatic site. Based on prior work by us and others, we hypothesize that exposure to FSS can result in other altered behaviors in metastatic cancer including enhanced cellular migration. The change in migratory behavior is pivotal to how extravasation occurs and could lead to modifications in secondary tumor formation.

Materials and

Methods:  The design of the modular microfluidic system consists of two devices to expose the cells to well-defined magnitudes of FSS and study their one-dimensional (1D) migration. The first device, referred to as the shearing module (Fig. 1A), consisted of a 1 m long, 70 μm wide, and 100 μm tall serpentine microfluidic channel that exposed cells to physiologically relevant magnitudes and durations of FSS. The second device, referred to as the migration device (Fig. 1C-D), contained two parallel channels connected by an array of 7 µm wide and 200 µm tall channels to facilitate 1D migration for a minimum of 50 cells for the sheared and nonsheared cohort. 1D migration was chosen as it has been shown to more accurately mimic 3D migration. A model triple negative breast cancer cell line, MDA-MB-231, was sheared and seeded into the bottom channel inlet of the migratory device, incubated for 6 h, and allowed to migrate within the device for 12 hours (Fig. 1E). Data was collected using a 37°C temperature-controlled light microscope that collected time-lapsed images every 3 minutes. Each cell with observable movement was manually tracked with the ImageJ plugin, MTrackJ (Fig. 1F). The migratory behavior of the sheared cells was compared to the nonsheared group using established metrics including displacement, distance, and speed by analyzing the tracking data. The full dataset of the two cohorts was then binned into three subpopulations based on quantitative increments for displacement [0-49 (low), 50-99 (intermediate), 100< pixels (high)] and compared using the above metrics.

Results, Conclusions, and Discussions: A direct comparison of the entire cohort of sheared vs. non-sheared cells showed no significant statistical differences in their migratory behavior including displacement, distance travelled, or average speed. However, visual examination of their corresponding migration tracks during the 12 h migration suggested distinct subpopulations of cells with substantially different migratory behavior. This led towards clustering the cells into three different subpopulations (low, intermediate, and high) based on the total displacement (Fig. 2). The sheared cells were found to exhibit a statistically significant increase in both distance travelled and average speed in the high and intermediate subpopulations when compared to the non-sheared cells. No difference between sheared and non-sheared cells was observed in the low cohort most likely due to the majority of cells travelling a very short distance, oftentimes not even into the channel. While the distance and speed metrics are compelling, it is also necessary to evaluate the role of FSS exposure on the directionality of the migrating cells. Current work is underway to evaluate this by calculating the migrating angles to evaluate the directionality of the cells in addition to determining how the cells migrate within and outside the 1D channel across all three subpopulations in both the sheared and non-sheared cohorts. The modular microfluidic approach has provided new insight on the role of FSS on the 1D migration of TNBC and suggests that exposure to shear enhances the migratory potential of subpopulations of metastasizing cancer cells. Future studies are planned to expand upon this by incorporating a chemotactic component would allow observation of how both a physical cue (FSS) and a chemical cue (chemotaxis) could influence extravasation. It is imperative to consider new factors to the study that will enable scientists to further refine research of targeted therapies for patients with TNBC, and primarily improve lives.

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