Measuring Changes in Brain Endothelial Barrier Integrity with Two Impedance-based Biosensors in Response to Cancer Cells and Cytokines.

The blood-brain barrier (BBB) protects the brain parenchyma against harmful pathogens in the blood. The BBB consists of the neurovascular unit, comprising pericytes, astrocytic foot processes, and tightly adhered endothelial cells. Here, the brain endothelial cells form the first line of barrier against blood-borne pathogens. In conditions like cancer and neuroinflammation, circulating factors in the blood can disrupt this barrier. Disease progression significantly worsens post barrier disruption, which permits access to or impairment of regions of the brain. This significantly worsens the prognoses, particularly due to limited treatment options available at the level of the brain. Hence, emerging studies aim to investigate potential therapeutics that can prevent these detrimental factors in the blood from interacting with the brain endothelial cells. The commercially available Electric Cell-Substrate Impedance Sensing (ECIS) and cellZscope instruments measure the impedance across cellular monolayers, such as the BBB endothelium, to determine their barrier strength. Here we detail the use of both biosensors in assessing brain endothelial barrier integrity upon the addition of various stimuli. Crucially, we highlight the importance of their high-throughput capability for concurrent investigation of multiple variables and biological treatments.

Melanoma Cells Produce Large Vesicular-Bodies That Cause Rapid Disruption of Brain Endothelial Barrier-Integrity and Disassembly of Junctional Proteins.

It is known that many cells produce extracellular vesicles, and this includes a range of different cancer cell types. Here we demonstrate the profound effects of large vesicular-like bodies produced by melanoma cells on the barrier integrity of human brain endothelial cells. These vesicular-bodies have not been fully characterised but range in size from ~500 nm to >10 µm, are surrounded by membrane and are enzymatically active based on cell-tracker incorporation. Their size is consistent with previously reported large oncosomes and apoptotic bodies. We demonstrate that these melanoma-derived vesicular-bodies rapidly affect brain endothelial barrier integrity, measured using ECIS biosensor technology, where the disruption is evident within ~60 min. This disruption involves acquisition of the vesicles through transcellular uptake into the endothelial cells. We also observed extensive actin-rearrangement, actin removal from the paracellular boundary of the endothelial cells and envelopment of the vesicular-bodies by actin. This was concordant with widespread changes in CD144 localisation, which was consistent with the loss of junctional strength. High-resolution confocal imaging revealed proximity of the melanoma vesicular-bodies juxtaposed to the endothelial nucleus, often containing fragmented DNA themselves, raising speculation over this association and potential delivery of nuclear material into the brain endothelial cells. The disruption of the endothelial cells occurs in a manner that is faster and completely distinct to that of invasion by intact melanoma cells. Given the clinical observation of large vesicles in the circulation of melanoma patients by others, we hypothesize their involvement in weakening or priming the brain vasculature for melanoma invasion.

Melanoma Mediated Disruption of Brain Endothelial Barrier Integrity Is Not Prevented by the Inhibition of Matrix Metalloproteinases and Proteases.

We have previously shown that human melanoma cells rapidly decrease human brain endothelial barrier strength. Our findings showed a fast mechanism of melanoma mediated barrier disruption, which was localised to the paracellular junctions of the brain endothelial cells. Melanoma cells are known to release molecules which cleave the surrounding matrix and allow traversal within and out of their metastatic niche. Enzymatic families, such as matrix metalloproteinases (MMPs) and proteases are heavily implicated in this process and their complex nature in vivo makes them an intriguing family to assess in melanoma metastasis. Herein, we assessed the expression of MMPs and other proteases in melanoma conditioned media. Our results showed evidence of a high expression of MMP-2, but not MMP-1, -3 or -9. Other proteases including Cathepsins D and B were also detected. Recombinant MMP-2 was added to the apical face of brain endothelial cells (hCMVECs), to measure the change in barrier integrity using biosensor technology. Surprisingly, this showed no decrease in barrier strength. The addition of potent MMP inhibitors (batimastat, marimastat, ONO4817) and other protease inhibitors (such as aprotinin, Pefabloc SC and bestatin) to the brain endothelial cells, in the presence of various melanoma lines, showed no reduction in the melanoma mediated barrier disruption. The inhibitors batimastat, Pefabloc SC, antipain and bestatin alone decreased the barrier strength. These results suggest that although some MMPs and proteases are released by melanoma cells, there is no direct evidence that they are substantially involved in the initial melanoma-mediated disruption of the brain endothelium.

Vmeasur: A software package for experimental and clinical measurement of mesenteric lymphatic contractile function over an extended vessel length.

OBJECTIVE: Conventionally, in vivo mesenteric lymphatic contractile function is measured using a high magnification transmission microscope (field of view 0.3-1.5 mm), which precludes visualization of extended lengths of vessels embedded in mesenteric fat. Existing software is not optimized for imaging at a low magnification using a contrast agent. We aimed to develop a simple and clinically transferable method for in situ visualization, image analysis, and quantitative assessment of mesenteric lymphatic contractile function over an extended area. METHODS: Subserosal injection of various blue dyes was taken up by mesenteric lymphatics and visualized under a stereomicroscope (25×), allowing for video recording of 1.4 × 1.1 cm of intact mesentery. A new R package ("vmeasur") that combines multiple high-performance image analyses into a single workflow was developed. The edges of each vessel were determined by applying an automated threshold to each frame (with real-time manual verification). The vessel width at every point in each frame was plotted to provide contractile parameters over time and along the lymphatic vessel length. RESULTS: Contractile parameters and their differences along the length of the vessel were accurately calculated in a rodent model. In a human mesenteric lymphatic, the algorithm was also able to measure changes in diameter over length. CONCLUSION: This software offers a low cost, rapid, and accessible method to measure lymphatic contractile function over a wide area, showing differences in contractility along the length of a vessel. Because the presence of mesenteric fat has less of an impact on imaging, due to the use of an exogenous contrast agent, there is potential for clinical application.

Comparison of Leading Biosensor Technologies to Detect Changes in Human Endothelial Barrier Properties in Response to Pro-Inflammatory TNFα and IL1ß in Real-Time.

Electric Cell-Substrate Impedance Sensing (ECIS), xCELLigence and cellZscope are commercially available instruments that measure the impedance of cellular monolayers. Despite widespread use of these systems individually, direct comparisons between these platforms have not been published. To compare these instruments, the responses of human brain endothelial monolayers to TNFα and IL1ß were measured on all three platforms simultaneously. All instruments detected transient changes in impedance in response to the cytokines, although the response magnitude varied, with ECIS being the most sensitive. ECIS and cellZscope were also able to attribute responses to particular endothelial barrier components by modelling the multifrequency impedance data acquired by these instruments; in contrast the limited frequency xCELLigence data cannot be modelled. Consistent with its superior impedance sensing, ECIS exhibited a greater capacity than cellZscope to distinguish between subtle changes in modelled endothelial monolayer properties. The reduced resolving ability of the cellZscope platform may be due to its electrode configuration, which is necessary to allow access to the basolateral compartment, an important advantage of this instrument. Collectively, this work demonstrates that instruments must be carefully selected to ensure they are appropriate for the experimental questions being asked when assessing endothelial barrier properties.

Analysis of Melanoma Secretome for Factors That Directly Disrupt the Barrier Integrity of Brain Endothelial Cells.

We have recently demonstrated that invasive melanoma cells are capable of disrupting the brain endothelial barrier integrity. This was shown using ECIS biosensor technology, which revealed rapid disruption via the paracellular junctions. In this paper, we demonstrate that melanoma cells secrete factors (e.g., cytokines) that weaken the endothelial barrier integrity. Through proteome profiling, we attempt to identify the barrier-disrupting cytokines. Melanoma conditioned media were collected from three New Zealand melanoma lines. ECIS technology was used to assess if the conditioned media disrupted the endothelial barrier independent of the melanoma cells. The melanoma cell secretome was assessed using cytometric bead array (CBA), Luminex immunoassay and multiplex Proteome Profilers, to detect the expression of secretory proteins, which may facilitate metastasis. Finally, ECIS technology was used to assess the direct effects of secreted proteins identified as candidates from the proteome screens. We show that melanoma-conditioned media significantly disrupted the brain endothelial barrier, however, to a much lesser extent than the cells from which they were collected. Cytokine and proteome profiling of the conditioned media showed evidence of high concentrations of approximately 15 secreted proteins (including osteopontin, IL-8, GDF-15, MIF and VEGF). These 15 secreted proteins were expressed variably across the melanoma lines. Surprisingly, the addition of these individually to the brain endothelial cells did not substantially affect the barrier integrity. ANGPTL-4 and TGFß were also produced by the melanoma cells. Whilst TGFß-1 had a pronounced effect on the barrier integrity, surprisingly ANGPTL-4 did not. However, its C-terminal fragment did and within a very similar period to the conditioned media, albeit not to the same extent. Herein we show that melanoma cells produce a wide-range of soluble factors at high concentrations, which most likely favour support or survival of the cancer cells. Most of these, except for TGFß-1 and the C-terminal fragment of ANGPTL-4, did not have an impact on the integrity of the brain endothelial cells.

Real-Time Measurement of Melanoma Cell-Mediated Human Brain Endothelial Barrier Disruption Using Electric Cell-Substrate Impedance Sensing Technology.

Electric cell-substrate impedance sensing (ECIS) is an impedance-based method for monitoring changes in cell behaviour in real-time. In this paper, we highlight the importance of ECIS in measuring the kinetics of human melanoma cell invasion across human brain endothelium. ECIS data can be mathematically modelled to assess which component of the endothelial paracellular and basolateral barriers is being affected and when. Our results reveal that a range of human melanoma cells can mediate disruption of human brain endothelium, primarily involving the paracellular route, as demonstrated by ECIS. The sensitivity of ECIS also reveals that the paracellular barrier weakens within 30-60 min of the melanoma cells being added to the apical face of the endothelial cells. Imaging reveals pronounced localisation of the melanoma cells at the paracellular junctions consistent with paracellular migration. Time-lapse imaging further reveals junctional opening and disruption of the endothelial monolayer by the invasive melanoma cells all within several hours. We suggest that the ability of ECIS to resolve changes to barrier integrity in real time, and to determine the route of migration, provides a powerful tool for future studies investigating the key molecules involved in the invasive process of cancer cells.