Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood-brain barrier model exemplifies tight-junction integrity.

The blood-brain barrier (BBB) consists of endothelial cells, astrocytes, and pericytes embedded in basal lamina (BL). Most in vitro models use nonhuman, monolayer cultures for therapeutic-delivery studies, relying on transendothelial electrical resistance (TEER) measurements without other tight-junction (TJ) formation parameters. We aimed to develop reliable, reproducible, in vitro 3-dimensional (3D) models incorporating relevant human, in vivo cell types and BL proteins. The 3D BBB models were constructed with human brain endothelial cells, human astrocytes, and human brain pericytes in mono-, co-, and tricultures. TEER was measured in 3D models using a volt/ohmmeter and cellZscope. Influence of BL proteins-laminin, fibronectin, collagen type IV, agrin, and perlecan-on adhesion and TEER was assessed using an electric cell-substrate impedance-sensing system. TJ protein expression was assessed by Western blotting (WB) and immunocytochemistry (ICC). Perlecan (10 µg/ml) evoked unreportedly high, in vitro TEER values (1200 Ω) and the strongest adhesion. Coculturing endothelial cells with astrocytes yielded the greatest resistance over time. ICC and WB results correlated with resistance levels, with evidence of prominent occludin expression in cocultures. BL proteins exerted differential effects on TEER, whereas astrocytes in contact yielded higher TEER values and TJ expression.-Maherally, Z., Fillmore, H. L., Tan, S. L., Tan, S. F., Jassam, S. A., Quack, F. I., Hatherell, K. E., Pilkington, G. J. Real-time acquisition of transendothelial electrical resistance in an all-human, in vitro, 3-dimensional, blood-brain barrier model exemplifies tight-junction integrity.

Fucosyltransferase 4 and 7 mediates adhesion of non-small cell lung cancer cells to brain-derived endothelial cells and results in modification of the blood-brain-barrier: in vitro investigation of CD15 and CD15s in lung-to-brain metastasis.

PURPOSE: Metastatic non-small cell lung (NSCLC) cancer represents one of the most common types of brain metastasis. The mechanisms involved in how circulating cancer cells transmigrate into brain parenchyma are not fully understood. The aim of this work was to investigate the role of fucosylated carbohydrate epitopes CD15 and sialyated CD15s in cancer adhesion to brain-derived endothelial cells and determine their influence in blood-brain barrier (BBB) disruption METHODS: Three distinct, independent methods were used to measure brain endothelial integrity and include voltohmmeter (EVOM™), impedance spectroscopy (CellZscope®) and electric cell-substrate impedance sensing system (ECIS™). Two fucosyltransferases (FUT4 and 7) responsible for CD15 and CD15s synthesis were modulated in four human cancer cell lines (three lung cancer and one glioma). RESULTS: Overexpression of CD15 or CD15s epitopes led to increase in adhesion of cancer cells to cerebral endothelial cells compared with wild-type and cells with silenced CD15 or CD15s (p < 0.01). This overexpression led to the disruption of cerebral endothelial cell monolayers (p < 0.01). Knockdown of FUT4 and FUT7 in metastatic cancer cells prevented disruption of an in vitro BBB model. Surprisingly, although the cells characterised as 'non-metastatic', they became 'metastatic' -like when cells were forced to over-express either FUT4 or FUT7. CONCLUSIONS: Results from these studies suggest that overexpression of CD15 and CD15s could potentiate the transmigration of circulating NSCLC cells into the brain. The clinical significance of these studies includes the possible use of these epitopes as biomarkers for metastasis.

CD15s/CD62E Interaction Mediates the Adhesion of Non-Small Cell Lung Cancer Cells on Brain Endothelial Cells: Implications for Cerebral Metastasis.

Expression of the cell adhesion molecule (CAM), Sialyl Lewis X (CD15s) correlates with cancer metastasis, while expression of E-selectin (CD62E) is stimulated by TNF-α. CD15s/CD62E interaction plays a key role in the homing process of circulating leukocytes. We investigated the heterophilic interaction of CD15s and CD62E in brain metastasis-related cancer cell adhesion. CD15s and CD62E were characterised in human brain endothelium (hCMEC/D3), primary non-small cell lung cancer (NSCLC) (COR-L105 and A549) and metastatic NSCLC (SEBTA-001 and NCI-H1299) using immunocytochemistry, Western blotting, flow cytometry and immunohistochemistry in human brain tissue sections. TNF-α (25 pg/mL) stimulated extracellular expression of CD62E while adhesion assays, under both static and physiological flow live-cell conditions, explored the effect of CD15s-mAb immunoblocking on adhesion of cancer cell-brain endothelium. CD15s was faintly expressed on hCMEC/D3, while high levels were observed on primary NSCLC cells with expression highest on metastatic NSCLC cells (p < 0.001). CD62E was highly expressed on hCMEC/D3 cells activated with TNF-α, with lower levels on primary and metastatic NSCLC cells. CD15s and CD62E were expressed on lung metastatic brain biopsies. CD15s/CD62E interaction was localised at adhesion sites of cancer cell-brain endothelium. CD15s immunoblocking significantly decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (p < 0.001), highlighting the role of CD15s-CD62E interaction in brain metastasis.

Improved Drug Delivery to Brain Metastases by Peptide-Mediated Permeabilization of the Blood-Brain Barrier.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood-brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

TNF-α enhancement of CD62E mediates adhesion of non-small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis.

BACKGROUND: CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non-small cell lung cancer (NSCLC). METHODS: CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. RESULTS: CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. CONCLUSION: This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E.

Receptors for hyaluronic acid and poliovirus: a combinatorial role in glioma invasion?

BACKGROUND: CD44 has long been associated with glioma invasion while, more recently, CD155 has been implicated in playing a similar role. Notably, these two receptors have been shown closely positioned on monocytes. METHODS AND FINDINGS: In this study, an up-regulation of CD44 and CD155 was demonstrated in established and early-passage cultures of glioblastoma. Total internal reflected fluorescence (TIRF) microscopy revealed close proximity of CD44 and CD155. CD44 antibody blocking and gene silencing (via siRNA) resulted in greater inhibition of invasion than that for CD155. Combined interference resulted in 86% inhibition of invasion, although in these investigations no obvious evidence of synergy between CD44 and CD155 in curbing invasion was shown. Both siRNA-CD44 and siRNA-CD155 treated cells lacked processes and were rounder, while live cell imaging showed reduced motility rate compared to wild type cells. Adhesion assay demonstrated that wild type cells adhered most efficiently to laminin, whereas siRNA-treated cells (p<0.0001 for both CD44 and CD155 expression) showed decreased adhesion on several ECMs investigated. BrdU assay showed a higher proliferation of siRNA-CD44 and siRNA-CD155 cells, inversely correlated with reduced invasion. Confocal microscopy revealed overlapping of CD155 and integrins (ß(1), α(v)ß(1) and α(v)ß(3)) on glioblastoma cell processes whereas siRNA-transfected cells showed consequent reduction in integrin expression with no specific staining patterns. Reduced expression of Rho GTPases, Cdc42, Rac1/2/3, RhoA and RhoB, was seen in siRNA-CD44 and siRNA-CD155 cells. In contrast to CD44-knockdown and 'double'-knockdown cells, no obvious decrease in RhoC expression was observed in CD155-knockdown cells. CONCLUSIONS: This investigation has enhanced our understanding of cell invasion and confirmed CD44 to play a more significant role in this biological process than CD155. Joint CD44/CD155 approaches may, however, merit further study in therapeutic targeting of infiltrating glioma cells.