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

Biosensors ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 56 ◽  
Author(s):  
Akshata Anchan ◽  
Panagiota Kalogirou-Baldwin ◽  
Rebecca Johnson ◽  
Dan T Kho ◽  
Wayne Joseph ◽  
...  
Keyword(s):  
Human Brain ◽  
Real Time ◽  
Melanoma Cell ◽  
Time Lapse ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Brain Endothelium ◽  
Impedance Sensing ◽  
Sensing Technology ◽  
Cell Substrate

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.

scholarly journals Comparison of Leading Biosensor Technologies to Detect Changes in Human Endothelial Barrier Properties in Response to Pro-Inflammatory TNFα and IL1β in Real-Time

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 159
Author(s):  
James J. W. Hucklesby ◽  
Akshata Anchan ◽  
Simon J. O'Carroll ◽  
Charles P. Unsworth ◽  
E. Scott Graham ◽  
...  
Keyword(s):  
Human Brain ◽  
Real Time ◽  
Barrier Properties ◽  
Endothelial Barrier ◽  
Endothelial Monolayer ◽  
Impedance Sensing ◽  
Endothelial Monolayers ◽  
Impedance Data ◽  
Cell Substrate ◽  
Limited Frequency

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 IL1β 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.

Recombinant arginase as a novel anti-melanoma agent

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 12032-12032 ◽  
Author(s):  
E. C. Hsueh ◽  
S. Knebel ◽  
I. Collier ◽  
M. Kadze ◽  
C. Hsueh ◽  
...  
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Large Scale ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Subtilis Strain ◽  
Argininosuccinate Synthetase ◽  
Melanoma Cell Lines

12032 Background: BCT-100 is a recombinant arginase comprised of 329 amino acid residues. Arginase converts arginine to urea and ornithine. Previous studies suggested that melanoma cells were auxotrophic for arginine due to absence of argininosuccinate synthetase (ASS) expression. Thus, we hypothesized that recombinant arginase, BCT-100, is cytotoxic to human melanoma cells and its cytotoxicity correlates with absence of ASS expression. Methods: BCT-100 pegylated recombinant human arginase was manufactured by large scale fermentation of a recombinant B. subtilis strain LLC101 encoded with a human arginase gene. Following fermentation, the recombinant protein was extracted, purified, pegylated, and ultra-dialyzed. Ten established human melanoma cell lines were used. Cells were grown to 90% confluence, harvested, and plated at 104 cells per well in a 96-well plate and co-cultured with increasing concentrations of pegylated BCT-100 for 72 hours. CellTiter 96 Aqueous Non-radioactive Cell Proliferation Assay (Promega, Madison, WI) was used to measure percent viability, with absorbances measured at 490 nm. Total cellular RNA was isolated from established melanoma cell lines converted to cDNA at a concentration of 5 ng/ul. Quantitative real time polymerase chain reaction was performed on a 7300 Real Time PCR System, using Gene Expression Assays for ASS and GAPDH (Applied Biosystems). 10,000 fold standard curves were generated for all samples using GAPDH expression. Results: All ten cell lines demonstrated decreased viability as concentrations of BCT-100 increased. Average IC50 value was 0.11 IU/ml. Eight of the 10 cells lines have IC50 values < 0.1 IU/ml. Of the 8 cell lines with IC50< 0.1 IU/ml, all of them have low or undetectable ASS expression using quantitative RT-PCR. Of the 2cell lines with IC50 > 0.1 IU/ml, ASS expression was detected in 1 of 2. Conclusions: Arginine depletion with recombinant arginase, BCT-100, was cytotoxic to melanoma cells in vitro. The cytotoxic effect of BCT-100 on melanoma cells correlated with expression of argininosuccinate synthetase. BCT-100 is a promising novel agent for treatment of melanoma. Further in vivo experiment with BCT-100 is ongoing. [Table: see text]

scholarly journals Synergy, Additivity, and Antagonism between Cisplatin and Selected Coumarins in Human Melanoma Cells

2021 ◽  
Vol 22 (2) ◽  
pp. 537
Author(s):  
Paula Wróblewska-Łuczka ◽  
Aneta Grabarska ◽  
Magdalena Florek-Łuszczki ◽  
Zbigniew Plewa ◽  
Jarogniew J. Łuszczki
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Type I ◽  
Antiproliferative Effects ◽  
Isobolographic Analysis ◽  
Natural Substances ◽  
Additive Interactions ◽  
Melanoma Cell Lines

(1) Cisplatin (CDDP) is used in melanoma chemotherapy, but it has many side effects. Hence, the search for natural substances that can reduce the dose of CDDP, and CDDP-related toxicity, is highly desired. Coumarins have many biological properties, including anticancer and antiproliferative effects. (2) An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on two human melanoma cell lines (FM55P and FM55M2) examined the antitumor properties of CDDP and five naturally occurring coumarins (osthole, xanthotoxin, xanthotoxol, isopimpinellin, and imperatorin). The antiproliferative effects produced by combinations of CDDP with the coumarins were assessed using type I isobolographic analysis. (3) The most potent anticancer properties of coumarins were presented by osthole and xanthotoxol. These compounds were characterized by the lowest median inhibitory concentration (IC50) values relative to the FM55P and FM55M2 melanoma cells. Isobolographic analysis showed that for both melanoma cell lines, the combination of CDDP and osthole exerted synergistic and additive interactions, while the combination of CDDP and xanthotoxol exerted additive interactions. Combinations of CDDP with xanthotoxin, isopimpinellin, and imperatorin showed antagonistic and additive interactions in two melanoma cell lines. (4) The combination of CDDP and osthole was characterized by the most desirable synergistic interaction. Isobolographic analysis allows the selection of potential candidates for cancer drugs among natural substances.

scholarly journals Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Matthew R. Pennington ◽  
Gerlinde R. Van de Walle
Keyword(s):  
Real Time ◽  
Cellular Response ◽  
Cellular Responses ◽  
Cellular Damage ◽  
Impedance Sensing ◽  
Novel Technologies ◽  
Antiviral Therapies ◽  
Mucosal Surfaces ◽  
Cell Substrate ◽  
Hsv 1

ABSTRACT Alphaherpesviruses, including those that commonly infect humans, such as HSV-1 and HSV-2, typically infect and cause cellular damage to epithelial cells at mucosal surfaces, leading to disease. The development of novel technologies to study the cellular responses to infection may allow a more complete understanding of virus replication and the creation of novel antiviral therapies. This study demonstrates the use of ECIS to study various aspects of herpesvirus biology, with a specific focus on changes in cellular morphology as a result of infection. We conclude that ECIS represents a valuable new tool with which to study alphaherpesvirus infections in real time and in an objective and reproducible manner. Electric cell-substrate impedance sensing (ECIS) measures changes in an electrical circuit formed in a culture dish. As cells grow over a gold electrode, they block the flow of electricity and this is read as an increase in electrical impedance in the circuit. ECIS has previously been used in a variety of applications to study cell growth, migration, and behavior in response to stimuli in real time and without the need for cellular labels. Here, we demonstrate that ECIS is also a valuable tool with which to study infection by alphaherpesviruses. To this end, we used ECIS to study the kinetics of cells infected with felid herpesvirus type 1 (FHV-1), a close relative of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, and compared the results to those obtained with conventional infectivity assays. First, we demonstrated that ECIS can easily distinguish between wells of cells infected with different amounts of FHV-1 and provides information about the cellular response to infection. Second, we found ECIS useful in identifying differences between the replication kinetics of recombinant DsRed Express2-labeled FHV-1, created via CRISPR/Cas9 genome engineering, and wild-type FHV-1. Finally, we demonstrated that ECIS can accurately determine the half-maximal effective concentration of antivirals. Collectively, our data show that ECIS, in conjunction with current methodologies, is a powerful tool that can be used to monitor viral growth and study the cellular response to alphaherpesvirus infection. IMPORTANCE Alphaherpesviruses, including those that commonly infect humans, such as HSV-1 and HSV-2, typically infect and cause cellular damage to epithelial cells at mucosal surfaces, leading to disease. The development of novel technologies to study the cellular responses to infection may allow a more complete understanding of virus replication and the creation of novel antiviral therapies. This study demonstrates the use of ECIS to study various aspects of herpesvirus biology, with a specific focus on changes in cellular morphology as a result of infection. We conclude that ECIS represents a valuable new tool with which to study alphaherpesvirus infections in real time and in an objective and reproducible manner.

scholarly journals Identification and characterization of a low-affinity granulocyte- macrophage colony-stimulating factor receptor on primary and cultured human melanoma cells

Blood ◽  
1991 ◽  
Vol 78 (3) ◽  
pp. 609-615 ◽  
Author(s):  
GC Baldwin ◽  
DW Golde ◽  
GF Widhopf ◽  
J Economou ◽  
JC Gasson
Keyword(s):  
Cell Lines ◽  
Melanoma Cell ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Melanoma Cell Lines

Abstract Hematopoietic growth factor receptors are present on cells of normal nonhematopoietic tissues such as endothelium and placenta. We previously demonstrated functional human granulocyte-macrophage colony- stimulating factor (GM-CSF) receptors on small cell carcinoma of the lung cell lines, and others have reported that certain solid tumor cell lines respond to GM-CSF in clonogenic assays. In the current study, we examine human melanoma cell lines and fresh specimens of melanoma to determine whether they have functional GM-CSF receptors. Scatchard analyses of 125I-GM-CSF equilibrium binding to melanoma cell lines showed a mean of 542 +/- 67 sites per cell with a kd of 0.72 +/- 0.14 nmol/L. Cross-linking studies in the melanoma cell line, M14, showed a major GM-CSF receptor species of 84,000 daltons. Under the conditions tested, the M14 cells did not have a proliferative response to GM-CSF in vitro, nor was any induction of primary response genes detected by Northern analysis in response to GM-CSF. Studies to determine internal translocation of the receptor-ligand complex indicated less than 10% of the 125I-GM-CSF internalized was specifically bound to receptors. Primary melanoma cells from five surgical specimens had GM-CSF receptors; Scatchard analysis was performed on one sample, showing 555 sites/cell with a kd of 0.23 nmol/L. These results indicate that human tumor cells may express a low-affinity GM-CSF receptor protein that localizes to the cell surface and binds ligand, but lacks functional components or accessory factors needed to transduce a signal.

A monoclonal antibody (LYP18) directed against the blood platelet glycoprotein IIb/IIIa complex inhibits human melanoma growth in vivo

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 909-912
Author(s):  
H Boukerche ◽  
O Berthier-Vergnes ◽  
M Bailly ◽  
JF Dore ◽  
LL Leung ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Melanoma Cell ◽  
Blood Platelet ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Platelet Glycoprotein ◽  
Novel Approach ◽  
Growth Of Tumor

A monoclonal antibody (MoAb) (LYP18), generated against human platelet glycoprotein IIb/IIIa (GPIIb/IIIa), immuno-precipitated a IIb/IIIa-like GP complex from a highly tumorigenic human melanoma cell line (M3Dau). The M3Dau melanoma cells specifically bound 125I-labeled LYP18. To study the biologic role of these IIb/IIIa-like glycoproteins, M3Dau melanoma cells were incubated with LYP18 or a control MoAb directed against another melanoma cell-surface antigen and implanted subcutaneously (SC) in nude mice. LYP18 dramatically inhibited the growth of tumor in vivo. LYP18 was not directly cytotoxic to the melanoma cells. These results demonstrate that the IIb/IIIa-like GPs are present on melanoma cells and play a crucial role in tumor cell growth. MoAbs directed against tumor cytoadhesive receptors may represent a novel approach in tumor treatment.

Effect of small inhibitors of nuclear export (SINE) on growth inhibition and apoptosis of human melanoma cells.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13549-e13549
Author(s):  
Gregory B. Lesinski ◽  
Jennifer Yang ◽  
Matthew A Bill ◽  
Yosef Landesman ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Nuclear Export ◽  
Melanoma Cells ◽  
Human Melanoma ◽  
Human Melanoma Cell ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Melanoma Cell Lines

e13549 Background: Inhibition of nuclear export can promote re-activation of tumor suppressor pathways. CRM1 (chromosomal regional maintenance 1) or XPO1 (exportin 1) is the major protein that mediates nuclear export. We hypothesized that CRM1 mediated nuclear export represents a novel therapeutic target that can be manipulated to inhibit melanoma cell survival. Methods: The growth inhibitory and pro-apoptotic effects of KPT-185, KPT-276 and KPT-330, small molecules selective inhibitor of nuclear export (SINE) were evaluated in human melanoma cell lines using an MTT assay and Annexin V/PI staining, respectively. Fluorescence microscopy and immunoblots were used to assess nuclear accumulation of tumor suppressor proteins. The trans-isomer of KPT-185 and DMSO (vehicle) were used as a negative controls in all assays. The pharmacokinetic (PK) profile of all compounds was evaluated in mice. Results: CRM1 protein was highly expressed in human melanoma cell lines with diverse molecular profiles (i.e., B-Raf, NRAS, p53). KPT-SINE inhibited melanoma cell growth in a concentration-dependent manner and induced apoptosis at nanomolar concentrations. Importantly, there was no evidence that B-Raf V600 mutational status influenced melanoma cell response to these agents. Nuclear accumulation and/or induction of p53, p21, FOXO3a, STAT1 and BAD, and reduction of MCL-1 occurred in melanoma cells at time points prior to apoptosis as shown by increase in cleaved PARP and caspase 3 levels. PK studies were conducted in mice following oral administration of 10 mg/kg, to guide drug selection for our ongoing efficacy studies in murine melanoma models. KPT-185 showed limited bioavailability and systemic exposure, while KPT-276 and KPT-330 showed >50% bioavailability reaching Cmax >5µM. Conclusions: This study represents the first report of CRM1 inhibition in melanoma. These data indicate that the novel SINE compounds can effectively inhibit CRM1-mediated nuclear export and induce apoptosis in melanoma cells. KPT-330 is currently under development as orally bioavailable, small molecule inhibitors for a human clinical trial.

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