Characterization Of a Novel In Vitro Circulation System Designed To Model The Migration Of Primary CLL Cells Across The Vascular Endothelium
Authors: Walsby Elisabeth J , Buggins Andrea GS , Devereux Stephen , Brennan Paul , Fegan Chris , Pepper Chris 
Published: October 2019

Abstract
There is growing evidence that lymphocyte trafficking contributes to the clinical course of chronic lymphocytic leukemia (CLL). However, no in vitro models exist to quantify migration from the peripheral vasculature or establish the key molecular events that drive this process. We have therefore established and characterized a novel dynamic in vitro model in which CLL cells are placed under shear forces equivalent to those experienced in the capillary beds and are made to flow through capillary-like hollow fibers lined with endothelial cells (HUVECs). Electronic microscopy demonstrated that HUVEC cells subjected to a shear force of 10 dynes/cm2 flattened against the wall of the hollow fibers giving confluent coverage and allowing formation of tight junctions in our system. Shear force was subsequently reduced to levels found in capillary beds (5 dynes/cm2) and CLL cells were introduced into the hollow fibers (106 CLL cells/ml). CLL cells circulated under these conditions increased their expression of selectin CD62L (associated with lymphocyte homing), CXCR4 (SDF-1 receptor) (both P<0.0001), lymphocyte adhesion molecule CD49d and the regulator of B cell receptor signaling, CD5 (both P=0.003) when compared to static cultures. We then demonstrated that CLL cells could migrate though the HUVEC-lined hollow fibers into the ‘extravascular’ space (EVS) and that this was an active process as 2mm fluorescent polystyrene beads were unable to move from the circulating compartment of the system to the EVS. CLL migration out of circulation into the EVS increased over time with a mean percentage migration of 1.37% ± 2.32% after 48h (n=22). Furthermore, CLL cells that migrated had significantly higher expression of CD49d (P=0.02), MMP-9 (breaks down extracellular matrix, P=0.004), the signaling molecule and activation marker CD38 (P=0.009), the co-stimulatory molecule CD80 (P=0.04) and the early activation marker CD69 (P=0.04) when compared with CLL cells that remained in the circulation. Similar results were also seen using HBMEC-lined hollow fibers suggesting that these effects were not HUVEC specific. Addition of an SDF-1 chemokine gradient from the EVS towards the circulating compartment resulted in significantly increased migration of CLL cells into the EVS after just 1h (P=0.04, n=8). Importantly, the degree of migration observed was strongly correlated with CD49d expression (r2=0.47, P=0.01) and in keeping with previously published data we showed a strong correlation between CD49, CD38 and MMP-9. Given these findings, we investigated the effects on migration of the CD49d blocking antibody, Natalizumab. This resulted in a significantly decreased migration (P=0.01) confirming that CD49d plays a critical role in CLL cell migration. Taken together, we have developed a dynamic in vitro model system of the peripheral vasculature. Using this unique model, we have identified some of the key pathophysiological events that drive CLL cell migration in contrast to normal cell migration and have shown the utility of our dynamic model to further understand the biology of CLL and as a novel potential drug testing platform.

Disclosures:
No relevant conflicts of interest to declare.