E-Selectin-Mediated Rolling and Firm Adhesion of Pancreatic Cancer Cells in Shear Flow

E-Selectin-mediated Rolling and Firm Adhesion of Pancreatic Cancer Cells in Shear Flow

Daniel J Shea¹, Yi Wai Li¹ and  Konstantinos Konstantopoulos¹

¹Department of Chemical and Biomolecular Engineering, Johns Hopkins University

Introduction: Metastasis is a highly-regulated, multistep process, in which tumor cells migrate from the primary tumor and enter the circulatory system where they interact extensively with host cells before extravasating and colonizing a distal organ. Extravasation of a tumor cell from a blood vessel first requires the rolling and arrest of the cell to the vessel wall through the formation of specific bonds [1,2].  Our lab recently identified the sialofucosylated surface protein Podocalyxin (PODXL) to be overexpressed in metastatic pancreatic cancer cells and to bind to E/L-selectin [3]. PODXL-E-selectin mediated rolling velocities were found to depend on single bond kinetics and could be predicted by the single receptor-ligand off-rate and the number of bonds [4].  We looked to build on this and develop a quantitative understanding of how selectin-ligand tethering facilitates firm adhesion at elevated levels of shear stress.  We also determined the rate-limiting parameters in this cascade of events, such as the lengths of coated regions with selectins and hyaluronic acid, necessary to support downstream firm adhesion.  The quantitative analysis of the selectin-ligand binding kinetics of known pancreatic cancer surface proteins in the presence of shear flow will enable us to further our understanding of the metastatic process.

Materials and Methods: Using standard microfabrication principles we patterned a glass slide with E-selectin and hyaluronic acid (HA) patches with lengths varying from 10-160μm in geometrically defined regions with a gap distance between the two regions of between 30-120μm (Fig. 1A). Using a microfluidic chamber, PODXL expressing metastatic pancreatic cancer cells (Pa03c) were then perfused over the patches and all cells were tracked using a modified code package produced by Eric M. Furst (University of Delaware).  Using a custom Matlab program, we identified both the rolling and firm adhesion regions and separate these regions into ten binned sections of increasing patch length. The program then analyzes each cell track with respect to these regions and indicates the position and velocity of the cell, while also determining if the cell binds in either the rolling or firm adhesion regions.

Results and Discussion: Experiments were performed with Pa03c pancreatic cancer cells using devices coated with E-selectin (rolling) and HA (firm adhesion).  Cells were tracked as they pass over the rolling, gap and firm adhesion regions, respectively (Fig. 1A). Cells interacting with E-selectin showed a clear rolling behavior. The extent of interacting cells decreased with shear stress.  However, it increased with longer E-selectin patch lengths due to increased ligand-selectin contact times (Fig. 1B). We determined that cells bind infrequently to hylaronic acid-coated regions, however the binding frequency increases when a cell first rolls on E-selectin (Fig. 1C).  Interestingly, for rolling cells, firm adhesion is most frequent when cells roll on shorter E-selectin patch lengths (<40μm), likely because these cells are bound immediately before entering the gap thus having a slower velocity. Together these data indicate that rolling on E-selectin can facilitate firm adhesion with HA and can do so even at small E-selectin patch lengths.

Figure 1. (A) Device schematic with rolling region (red) and firm adhesion region (green). (B) Frequency of rolling events at increasing patch lengths. (C) Frequency of firm adhesion events for cells that roll (blue) or that do not roll (red) before firmly adhering. 30μm gap.

Conclusions: This work impacts a broad range of areas including cell-biophysics, engineering and cancer research and is critically important as it advances the knowledge of receptor-ligand processes as they pertain to cancer metastasis.  Knowledge of the properties of receptor-ligand-mediated tumor cell adhesion, in physiologically relevant environments is critical to the design parameters necessary to engineer sensors, to target biological entities based on recognition, and to design molecules to interrupt adverse or pathological adhesion events.

References: (1) Cheung, L. S. L. et al., Phys Biol, 2011, 8(1), 015013. (2) Stroka, K. M., and Konstantopoulos, K. Am J Physiol Cell Physiol, 2014, 306(2), 98-109. (3) Dallas, M. R. et al., Am J Physiol Cell Physiol, 2012, 303, C616–624. (4) Shea, D. J. et al., Oncotarget, 2015, 6(28), 24842.