Poster S9 - Effects of Heparan Sulfate Modulation on Breast Cancer Cell MDA-MB-231 Adhesion and Transmigration across an in vitro Blood-Brain Barrier

Introduction: On the surface of every mammalian cell, there is a matrix-like glycocalyx layer of a mucopolysaccharide structure consisting of glycoproteins, proteoglycans and glycosaminoglycans (GAGs) including heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). Tumor cell adhesion to and transmigration across the endothelial barrier forming the microvessel wall are two critical steps in its metastasis. Disruption of endothelial heparan sulfate (HS) is an early event in tumor cell metastasis across vascular barriers. Our recent study showed that while vascular endothelial growth factor (VEGF-A165), a tumor secretion, greatly reduces HS at an in vitro blood-brain barrier (BBB) formed by human cerebral microvascular endothelial cells (hCMECs), it significantly enhances HS on a breast cancer cell, MDA-MB-231 (MB231). Here we tested the hypothesis that this differential effect of VEGF on the HS favors MB231 adhesion and transmigration. We also tested if agents such as a plasma protein, orosomucoid, and a plasma sphingolipid, sphingosine-1-phosphate (S1P) that enhance endothelial HS may disrupt HS of MB231 and reduce its adhesion and transmigration.

Materials and

Methods: Human cerebral microvascular endothelial cells (hCMECs) from Millipore Sigma and human breast carcinoma cells MDA-MB-231 from ATCC were used in the study. In vitro BBBs were generated by culturing hCMEC monolayers either on a glass bottom dish (No. 1.5 from MetTek) or on a Transwell filter (8 μm pore, 0.33 cm2 bottom area) as described in our previous studies. The Trans-endothelial electrical resistance (TEER) and permeability to Dex-70k were first measured for the in vitro BBB. The HS at the BBB and that at MB231 were quantified by immunostaining and confocal microscopy as well as the adherent and transmigrated MB231 cells.

Results, Conclusions, and Discussions:

Results: We found that 1) VEGF decreases the HS of the BBB to 29% of the control while increases that of MB231 to 1.5 folds of the control. Although orosomucoid and S1P enhance the HS of the BBB to 3.4 and 1.9 folds of the control, respectively, surprisingly, they reduce the HS of MB231 to 64% and 73% of the control, correspondingly; 2) While VEGF significantly increases the BBB permeability to Dex-70k to 1.5 folds of the control, it slightly decreases the TEER of the BBB by 9%. Instead, orosomucoid and S1P reduce the BBB permeability to Dex-70k to 71% and 81% of the control, and moderately increase the TEER by 11% and 13% that of the control, correspondingly; 3) Treatments with VEGF enhance MB231 adhesion by 2.5 folds, and transmigration by 1.9 folds, respectively, compared to that with no treatments. While treatments with orosomucoid and S1P reduce MB231 adhesion to 62% and 66% and transmigration to 55% and 64%, correspondingly, compared to those with no treatments.

Conclusion: Our results demonstrated that modulation of HS at MB231 and at the BBB significantly affects MB231 adhesion to and transmigration across the in vitro BBB. Specifically, the reduced BBB HS and enhanced MB231 HS by VEGF increase, while enhanced BBB HS and reduced MB231 HS by orosomucoid and S1P decrease MB 231 adhesion to and transmigration across the BBB, respectively. Our findings suggest a therapeutic intervention by targeting the HS-mediated breast cancer brain metastasis.

Discussion: It is understandable that the degradation of HS on the BBB by VEGF enables more VEGF receptors and ligands of cell adhesion molecules (CAMs) at MB231 to interact with VEGF and CAMs at MB231 to enhance their adhesion. It is quite puzzling why the enhanced HS of MB231 by VEGF increases MB231 adhesion and transmigration. One possibility is that the HS-associated heparan sulfate proteoglycans (HSPGs) of MB231 can bind to CAMs of endothelial cells (the BBB) and extracellular matrix (ECM) proteins for adhesion and transmigration. Further investigations will be conducted to clarify the mechanisms.

Acknowledgements (Optional): The study was partially supported by NIH RO1NS101362, 1UG3UH3TR002151 and ARO W911NF2310189