(72c) Disturbed Flow Induced Endothelial Glycocalyx Degradation Promotes Cancer Cell Attachment to the Endothelium

Authors: 
Mensah, S., Northeastern University
Nersesyan, A., Northeastern University
Harding, I., Northeastern University
Niedre, M., Northeastern University
Torchilin, V., Northeastern University
Ebong, E. E., Northeastern University
INTRODUCTION

Cancer metastasis has been identified as one of the major causes of cancer-related deaths [1, 2]. During metastasis, small amounts of primary tumor cells migrate from the parent tumor to other tissues, where they form secondary tumors [3]. The mechanism for secondary tumor homing to new tissues locations remains under investigation, but it is well established that cancer cell migration is enabled largely by circulating cancer cells crossing the vascular endothelial barrier and traveling via the blood vessels [3, 4]. It has been suggested that the migration of cancer cells across the endothelium, in search of locations to colonize for secondary tumor formation, could be due to glycocalyx (GCX) dysfunction, which allows for cancer cell attachment to endothelial cells [4].

Endothelial glycocalyx dysfunction could be a result of multiple factors, and the most common way of GCX dysfunction is through inflammatory stress and enzyme activities which is mostly characterized by the specific loss of GCX components [5]. Currently, evidence is also emerging to suggest that disturbed flow patterns within the vascular system could also result in GCX dysfunction. Harding et al recently showed that disturbed flow patterns resulted in the decrease in the expression of GCX in rat cells [6].

We hypothesize that disturbed flow patterns in the vascular system will enhance the attachment of cancer cells to the endothelium due to disturbed flow induced dysfunctions in the GCX. Our results provide evidence to support the fact that hemodynamics and the endothelial GCX, together, play an important role in mediating cancer-endothelial cell interactions relevant to secondary tumor formation.

METHODS

A parallel plate flow chamber [6], was used to create both disturbed and uniform laminar flow patterns similar to what is observed in vivo. After introducing disturbed and uniform flow patterns to human umbilical vein endothelial cells (HUVEC) for 4 hours at 15 dynes/cm2, we co-incubated HUVEC monolayers with circulating Red Cell Tracker labeled stage IV breast cancer cell (4T1) or human breast adenocarcinoma cell (MCF7) , for an hour at 1 dynes/cm2.

We investigated endothelial cell expression of GCX and the adhesion molecule E-selectin and made correlations to the level of attachment of 4T1 or MCF7 breast cancer cells.

In addition, we confirmed our results in vivo, 14 hours after no treatment or intravenous treatment of Balb/c mice with 5 units (U) of neuraminidase (an enzyme that degrades one component of the GCX: sialic acid). Red Cell Tracker labeled 4T1 breast cancer cells were injected into these mice intravenously and allowed to circulate for an hour. Mice were then sacrificed. Endothelial integrity was confirmed in mice aortas. GCX integrity or degradation was confirmed in mice aortas. 4T1 breast cancer cell homing to the lungs of Balb/c mice was quantified.

RESULTS

Disturbed flow enhances the attachment of 4T1 and MCF7 breast cancer cells to cultured HUVEC.

Labeled 4T1 or MCF7 breast cancer cells were co-incubated with flow conditioned HUVEC. Compared to uniform flow conditions, 4T1 breast cancer cell attachment to the endothelium in disturbed flow conditions was increased by 2.35±0.29 fold. For MCF7 cells, exposure to disturbed flow resulted in a 2.34±0.42-fold increase in attachment to HUVEC.

Differences in breast cancer cell attachment to HUVEC do not correlate to expression of E-selectin adhesion molecules.

In fact, there is non-differential expression of E-selectin adhesion molecules between disturbed and uniform flow regions. We found that no statistically significant difference between the HUVEC expression of E-selectin adhesion molecules in disturbed versus laminar flow regions.

Elevated breast cancer cell attachment to HUVEC coincides with HUVEC GCX degradation in disturbed flow regions, and is a result of GCX degradation.

Expression of GCX was assessed by wheat germ agglutinin (WGA) staining and showed an intact GCX on the surface of HUVEC monolayers in uniform flow conditions. After introducing HUVEC monolayers to disturbed flow patterns, the GCX coverage of and thickness on HUVEC were both reduced by about 60%. We attributed the increased breast cancer cell attachment to this reduction in GCX coverage. To confirm the role of the GCX in cancer-endothelial cell adhesion, we degraded the GCX in uniform flow conditions and successfully induced cancer cell attachment in the same region.

In vivo, neuraminidase-induced GCX degradation leads to increased homing of 4T1 breast cancer cells to the lung, presumably attached to the pulmonary blood vessel wall endothelium. When in vivo experiments were performed to confirm in vitro findings, first we investigated the expression of GCX before and after treating Balb/c mice with 5 U of neuraminidase. We found that there was a statistically significant 20% decrease in the thickness of GCX in neuraminidase treated mice, in comparison with non-treated mice. After treating Balb/c mice with 5 U of Neur, in comparison with non-treated mice conditions there was a statistically significant increase in the homing of 4T1 breast cancer cells to the lungs, likely at the blood vessel walls. Specifically, we observed a 2.2±0.11-fold increase in the homing of 4T1 breast cancer cells to the lungs of neuraminidase treated mice, compared to untreated mice.

DISCUSSION

In this study, we showed evidence to support the importance of flow-regulated endothelial cell GCX in mediating cancer cell attachment to the endothelium. In summary, we found that conditioning endothelial cells in disturbed flow conditions results in an increase in cancer cell attachment to the endothelium, which is independent of E-selectin expression and in correlation with reduced GCX expression. We also confirmed in vitro and in vivo that the increase in cancer cell attachment occurs as a direct result of decreased GCX expression. Our results provide new insight into the possible pathway leading to secondary tumor formation during the progression of cancer.

ACKNOWLEDGEMENTS

We appreciate funding from National Institutes of Health (K01 HL125499), the National Science Foundation (DGE-096843), and the Northeastern University Provost’s Tier 1 Grant.

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