Temple University, Department of Mechanical Engineering
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Culturing tumor endothelial cell (HBTAEC) in the vascular compartment for 48 h significantly decreased permeability compared to bMTM with no cells. Permeability of the fluorescent 40 kD dextran from vascular compartment to the tumor compartment in a cell-free bMTM were quantified by normalizing the fluorescent intensity in the tumor to the vascular compartment while fluorescent dextrans diffuse from the vascular compartment to the tumor compartment (panel A). Permeability increased when endothelial cells were treated with TNF-α but was still significantly lower than in cell-free bMTM (panel B). Data are presented as mean ± SEM (n = 3). *Significant difference by ANOVA.
Tumor conditioned media (TCM) (48 h treatment) from highly metastatic MDA-MB-231 tumor cells increased liposome extravasation into the tumor compartment as indicated by the ratio of fluorescent liposome intensity in the tumor compartment to the vascular compartment (panel A). Liposomes extravasated more after MDA-MB-231 conditioned media or TNF-α treatment, but were not affected by TCM obtained from nonmetastatic MCF-7 tumor cells (panels B–E). Data are presented as mean ± SEM (n = 3). *Significant difference by ANOVA.
Highly metastatic MDA-MB-231 tumor cells reduced endothelial cell tight junction (ZO-1) molecule expression. When cultured without tumor cells, tight junctions were fully established between adjacent cells. When co-cultured with MDA-MB-231 tumor cells, tight junctions were intermittently expressed along the edge of contacting endothelial cells, while no change was observed when HBTAEC were co-cultured with MCF-7 tumor cells. Cell tight junction molecule was stained with ZO-1 antibody (red); cell F-actin microfilament was stained with phalloidin (green); cell nucleus was counterstained with Hoechst 33342 (blue).
Permeability of HBTAEC increased significantly with increasing co-culture duration with highly metastatic MDA-MB-231 cells (panel A) and was accompanied by morphological changes in endothelial cells (panels B–D). HBTAEC cultured without tumor cells showed no change in morphology for 120 h (panel B) but endothelial cell morphology changed significantly when co-cultured with MDA-MB-231 cells for 120 h (panel C). HBTAEC co-cultured with MCF-7 cells for 120 h showed fewer morphological changes (panel D). Data are presented as mean ± SEM (n = 3). *Significant difference by two-way ANOVA.
Schematic of the bMTM (A) with magnified view of the vascular compartment, vascular-tumor compartment interface and the tumor compartment (B). Optical image of the bMTM (C) with HBTAEC cultured in the vascular compartment (D) and MDA-MB-231 cultured in the tumor compartment (E). HBTAEC cultured under flow in the vascular compartment of bMTM form a complete lumen as shown with 3D reconstruction of confocal images of HBTAEC cultured in bMTM stained with f-actin (green) and Draq5 (red) after 4 days in culture maintained under flow of 0.05 μl/min (F–I); images are shown with a Y-axis rotation of 0, 60, 180 and 240 degrees in (F,G,H and I) respectively.
Highly metastatic MDA-MB-231 tumor cells reduced endothelial cell adherens junction (VEcadherin) molecule expression. When cultured without the presence of tumor cells, adherens junctions were fully established between adjacent cells. When co-cultured with MDA-MB-231 tumor cells, VE-cadherin was partially expressed between adjacent cells, while no change in VE-cadherin expression was observed when HBTAEC were co-cultured with MCF-7 tumor cells. Cell adherens junction molecule was stained with VEcadherein antibody (red); cell F-actin microfilament was stained with phalloidin (green); cell nucleus was counterstained with Hoechst 33342 (blue).
HBTAEC lumen formation as indicated by VE-cadherin (red) is shear flow dependent. Cell nucleus were stained with Hoechst 33342 (blue). At low shear regions (white arrows), the red staining were intermittent indicating less VE-cadherin formation (panel A). When co-cultured with highly metastatic MDA-MB-231 tumor cells, VE-cadherin expression in HBTEAC were inhibted (panel B). The presence of less metastatic MCF-7 tumor cells did not change the formation of adherens junctions (panel C).
