470027 The Role of Glycocalyx and Shear Stress on Endothelial-Cancer Cell Attachment

Monday, November 14, 2016: 12:48 PM
Continental 7 (Hilton San Francisco Union Square)
Solomon Mensah1, Ian Harding1, Mark Niedre1,2, Vladimir Torchilin3 and Eno Essien Ebong1,4, (1)Department of Bioengineering, Northeastern University, Boston, MA, (2)Department of Electrical and Computer Engineering, Northeastern University, Boston, MA, (3)Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, (4)Department of Chemical Engineering, Northeastern University, Boston, MA

Introduction: Cancer and endothelial cell (EC) interactions [Giavazzi, R., et al., Journal of Clinical Investigation, 1993] are likely mediated by glycocalyx (GCX), a complex of sugar molecules that envelop both cell types and can regulate the interactions of their intercellular adhesion molecules. It was recently shown that metastatic cancer cells up-regulate the expression of their surface sugar molecules, which mechanically regulate integrin-mediated intercellular interactions and amplify cancer cell growth and survival [Paszek, M.J., et al., Nature, 2014]. Compared to cancer GCX, endothelial GCX composition, structure and function during intercellular interactions are understudied but of great clinical importance. EC GCX consists of membrane bound glycoproteins, proteoglycans, and glycosaminoglycans side chains [Pries, A.R., et al., Pflugers Arch, 2000; Boddohi, S., et al., Adv Mater, 2010]. EC GCX structure is dependent on active EC shedding and synthesis of GCX, in response to the microenvironment [Arisaka, T., et al., Ann N Y Acad Sci, 1995; Elhadj, S., et al., J Cell Biochem, 2002; Koo, A., et al., Am J Physiol Cell Physiol, 2013].The glycocalyx structure is particularly responsive to shear stress and has been shown to remodel when exposed to flow [Tarbell, J., et al., PloS one, 2014]. Typically, degraded GCX coincides with diseased states, whereas intact EC GCX coincides with normal or healthy conditions. For example, endothelial cell glycocalyx is often degraded in areas prone to atherosclerosis, coinciding with abnormal levels of shear stress in these regions. It is therefore believed that EC GCX is substantially degraded in settings of secondary tumor formation due to irregularities in the tumor vessels; which are mostly disorderly organized [Jain, R., J Clin Oncol, 2013]. The resultant structure and composition of the GCX is presumed to pave the way for cancer cells to invade the endothelium for transendothelial migration. The goal of this present study is to test the hypothesis that GCX controls the attachment of cancer cells onto the endothelium. Materials and Methods: To test this hypothesis, we conducted both static enzyme degradation experiments and shear stress induced flow experiments using rat fat pad endothelial cells (RFPECs), which naturally express a robust glycocalyx. We verified the condition of the RFPECs’ GCX by immunostaining the sialic acid glycosaminoglycans of the GCX. Sialic acid was used to characterize GCX due to its implication in the attachment of cancer cells to the endothelium. For the static experiments, degradation of glycosaminoglycans in vivo was mimicked by using neuraminidase to degrade sialic acid, respectively. Immunofluorescence confocal interrogation was used to examine the effects of degrading enzymes on sialic acid thickness and coverage on RFPECs. RFPEC monolayers with intact or enzyme degraded glycocalyx were co-incubated with metastatic mouse breast cancer cells (4T1) labeled with CellTracker Red. The fluorescently-labeled 4T1 cells were easily identified during co-incubation with glycocalyx-rich or glycocalyx-deficient RFPEC monolayers, enabling quantification of the effect of glycosaminoglycan removal on cancer cell attachment to ECs. For the shear stress induced flow experiments, a custom parallel plate-step flow chamber was developed to expose RFPECs to shear stresses at 10 dynes/cm2. Hypothetically, areas of recirculation, characterized by low shear stresses, will produce RFPECs with degraded glycocalyx, representing degraded EC GCX in vivo. Attachment of cancer cells to RFPEC monolayers from shear stress induced flow experiments will be tested for cancer cell attachment using the same methods as previously described. Results and Discussion: In control conditions, the thickness of sialic acid was 1.6 ± 0.01 µm. Enzyme doses of 15, 135, 1215, and 3645 mU/ml reduced thickness to 1.41 ± 0.04 µm, 0.97 ± 0.03 µm, 0.88 ± 0.02 µm and 0.80 ± 0.01 µm respectively. These results indicate that an enzyme concentration of 3645 mU/ml was required to degrade ~50% of the thickness of sialic acid in RFPEC GCX. To see the impact of sialic acid thickness reduction on cancer cell attachment, we co-incubated CellTracker Red stained 4T1 breast cancer cells with untreated RFPEC versus neuraminidase-treated RFPEC. On control RFPEC the number of attached cancer cells was 310.77 ± 30.40 for every 1 cm2 (or for every 2000 adherent RFPEC at confluence). RFPEC treated with 15, 135, 1215 and 3645 mU/ml of enzyme showed cancer cell attachment levels of 305.43 ± 43.07, 244.92 ± 29.16, 432.71 ± 39.23 and 740.66 ± 92.20, respectively. In flow conditions, it is expected that areas close to the disturbed flow step will experiment irregular flow patterns which will result in the disorganization and subsequent degradation of GCX. This will then lead to the increased attachment of cancer cells in the disturbed flow areas in comparison with laminar and static conditions. The enzyme degradation results point to the fact that degrading GCX to ~50% of its thickness could be very instrumental in the attachment of circulating cancer cells at secondary tumor formation sites. We hope to see a disrupted GCX at disturbed flow areas which should result in an increase in cancer cell attachment. Conclusion: We have shown that GCX degradation may initiate secondary tumor formation. As a future direction we hope to dive deeper into the subject of whether other glycosaminoglycans are also partly responsible for enhancing circulating cancer cell attachment to the endothelium. We also look forward to examining the role of enzymatic GCX degradation in cancer metastasis in vivo. Acknowledgements: Northeastern University provided start-up and pilot funding to support this work.

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See more of this Session: Engineering in Cancer Biology and Therapy II
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