(E-178) Mesenchymal Phenotype Confers Mechanical Resistance to Fluid Shear Stress in Migratory Breast Cancer Subpopulations

Introduction:: Metastasis is a complex process during which cancer cells navigate through the tumor microenvironment and withstand the mechanical stresses of the circulatory system. Recent studies suggest that the application of fluid shear stress (FSS) to cancer cells may induce an epithelial-mesenchymal transition (EMT) and increase cell migration. However, we propose here an alternate explanation of these results: E/M state affects shear stress survival, such that shear stress may be selecting for more mesenchymal cells rather than (or in addition to) inducing a mesenchymal phenotype. Understanding these two possibilities (fluid shear as an inducer of EMT versus fluid shear stress as a selector for a mesenchymal phenotype) is critical for identifying which cell characteristics are essential for escaping the primary tumor and surviving as circulating tumor cells (CTCs). While most studies focus on a cancer cell's ability to complete a single step of the metastatic cascade, our work aims to investigate the process as a multi-step journey and uncover the characteristics that enable cells to transition from one step to the next. To achieve this, we employ migration-based sorted cells to examine how different migratory and mesenchymal phenotypes influence FSS resistance.


Materials and Methods:: Parental MDA-MB-231 cells were sorted using consecutive transwell assays, resulting in highly and weakly migratory cells (Fig. 1A). RNA sequencing (RNAseq) was conducted at the Vanderbilt Technologies for Advanced Genomics Core. Shear stress experiments were done utilizing a Harvard Apparatus PHD 2000 Infusion system (10 pulses, 4000 dyn/cm²). Cells were counted before and after pulses were administered, and cell survivability was calculated as being: [Post Shear (cells/mL)] / [Pre Shear (cells/mL)]. Vimentin integrated density measurements obtained utilizing mouse monoclonal anti-vimentin antibody (Sigma #V6630) followed by Alexa Fluor 488 donkey-anti-mouse IgG. Cells fixed in 3.2% paraformaldehyde and stained directly following shear stress exposure. Imaged on Agilent Cytation5 plate imager. Cells treated for 24 hours with TGFβ utilizing a final concentration of 5 ng/mL. GraphPad Prism 9.3.1 was used for statistical analysis.

Results, Conclusions, and Discussions:: We used RNAseq to quantify genotypic differences in our sorted subpopulations. Specifically, we analyzed the transcriptomic abundance of genes related to the "Hallmarks of EMT" gene set to investigate the EMT-related differences between the highly and weakly migratory cells. Our results indicate that highly migratory cells have a greater expression of EMT-related genes (Fig. 1B) and that is phenotypically corroborated by a higher aspect ratio than their weakly migratory counterparts (Fig. 1C). Furthermore, when exposed to high pulsatile fluid shear stress, weakly migratory cells showed a 22% decrease in survivability when compared to the static condition (Fig. 1D), suggesting that migratory and mesenchymal cells are better equipped to survive in CTC-like conditions. To further explore this phenomenon, we stained static and surviving sheared cells for vimentin, a common EMT marker. Our analysis revealed that the sheared weakly migratory subpopulation lacked a population of epithelial cells (low vimentin expression) present in the static group (Fig. 1F). This led us to hypothesize that the cells getting shredded under high pulsatile fluid shear stress are epithelial cells and that promoting EMT in the weakly migratory subpopulation would counteract this effect. To test this hypothesis, we induced EMT in the weakly migratory MDAs using TGFβ and observed an increase in cell survival when sheared 24 hours later (Fig. 1G). Our findings suggest that a cell's ability to survive high pulsatile fluid shear stress is associated with its EMT state. By promoting EMT, we recovered the survival of 22% of cells in the weakly migratory group, indicating that fluid shear stress selects for cells with a higher migratory potential and mesenchymal phenotype. This study highlights the importance of migratory and mesenchymal cells in both escaping the primary tumor and surviving as CTCs. Our findings could aid in developing more accurate and specific cancer treatments that target cells expressing a mesenchymal phenotype.

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