Abstract 4269: Interstitial fluid pressure alters cell motility and collective invasion via EMT marker expression in an engineered model of a human breast tumor

Abstract

Abstract We developed a three-dimensional (3D) engineered model of a solid human breast tumor to study the effects of interstitial fluid pressure (IFP) on collective invasion and the expression levels of epithelial-mesenchymal transition (EMT) markers. Many solid tumors exhibit elevated IFP; as these tumors grow, intra-tumoral vascular and lymphatic vessels collapse. The non-functioning lymphatic system impairs drainage, and immature hyperpermeable blood vessels cause fluid to accumulate within the interstitial space. As a result, IFP rises steeply beyond the tumor periphery and plateaus at pressures as high as 50 mm Hg above normal at the tumor core. This pressure profile, in turn, leads to outward fluid flow from the core of the tumor. IFP has been shown to affect the migratory behavior of individual cells in 3D cell culture models, though its role in collective cancer invasion remains unknown. Moreover, the underlying molecular mechanisms linking IFP to changes in cell motility remain unclear. We sought to address these questions using our engineered model. Our 3D culture model consists of an aggregate of MDA-MB-231 breast cancer cells (mimicking a solid tumor) embedded within a 3D collagen gel that is flanked by two media reservoirs. The IFP profile experienced by the cancer cells is established by altering the heights of the media reservoirs on either side of the collagen, creating a hydrostatic pressure gradient. Transcript levels of EMT markers in the aggregates subjected to a variety of pressure profiles were determined using quantitative real-time PCR. Expression of these markers was also manipulated ectopically through the creation of stable cell lines. Time-lapse imaging and cell tracking were used to determine the persistence and motility of individual cells within the aggregates. We found that the direction of IFP-induced flow determines the invasive phenotype of tumor cells. Additionally, high expression levels of both mesenchymal (Snail1, vimentin) and epithelial (E-cadherin, keratin-8) markers were characteristic of collectively invading aggregates, suggesting that partial EMT is important for collective invasion. Ectopic expression and knockdown of EMT markers revealed that they are necessary and sufficient for collective invasion in response to IFP. Time-lapse imaging analysis demonstrated that IFP and EMT marker expression also affect the motility and persistence of individual cells within the aggregates, further confirming that IFP is an important regulator of collective invasion. In conclusion, we used a robust culture model of a human breast tumor to gain insight into the mechanisms guiding collective invasion from primary tumors in response to IFP; IFP alters expression levels of EMT markers, thereby regulating collective invasion. Citation Format: Alexandra S. Piotrowski-Daspit, Joe Tien, Celeste M. Nelson. Interstitial fluid pressure alters cell motility and collective invasion via EMT marker expression in an engineered model of a human breast tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4269.