Electric Cell Impedance Sensing Research Articles

Abstract Tu029: Effects of the phosphodiesterase inhibitors, cilostazol and ensifentrine, on alveolar-capillary dysfunction caused by MRSA

Background: Acute lung injury (ALI) is characterized by excessive inflammation and dysfunction of the alveolar-capillary barrier, which consists of lung vascular endothelial and alveolar epithelial cells. Therapeutic interventions that protect this barrier are promising strategies for ALI treatment. Phosphodiesterase (PDE) inhibitors are potent modulators of inflammation. These include cilostazol, a PDE3 inhibitor, and ensifentrine, a dual PED3/4 inhibitor, which are both clinically available compounds that exhibit cytoprotective and anti-inflammatory properties. However, their roles in regulating alveolar-capillary function in ALI are unknown. The aim of this study is to investigate the effects of cilostazol and ensifentrine on lung endothelial and epithelial dysfunction caused by methicillin-resistant Staphylococcus aureus (MRSA), a common bacterial pathogen involved in ALI pathogenesis. Methods: Human primary lung endothelial cells (EC) and A549 (alveolar epithelial cell line) were pre-treated with cilostazol (CZ; 15 μM) or ensifentrine (Ens; 10 μΜ) for 1 hour and then challenged with heat-killed MRSA (2.5 x10 8 /ml). The Electrical Cell Impedance Sensing (ECIS) assay was used to assess EC barrier integrity. Pro-inflammatory signaling markers were measured by western blotting (WB) and qPCR. Cell conditioned media was analyzed for extracellular vesicle release by flow cytometry and WB. Results: MRSA-induced EC barrier disruption was attenuated by both CZ and Ens, although Ens exhibited superior barrier protective properties. CZ and Ens suppressed MRSA-induced upregulation of the inflammatory adhesion molecules, VCAM-1 and ICAM-1, in EC. Ens, but not CZ, reduced MRSA-induced ICAM-1 upregulation in A549. MRSA caused a significant increase in the release of EC-derived extracellular vesicles, which was partially inhibited by CZ or Ens pretreatment. Conclusions: Both cilostazol and ensifentrine exert anti-inflammatory properties in MRSA-treated lung EC. Compared to cilostazol, ensifentrine exhibits enhanced endothelial barrier protection and inhibits inflammation in both lung endothelial and epithelial cells. Future in vivo studies will evaluate the therapeutic efficacy of these inhibitors in bacterial-induced ALI.

Src kinase partially mediates cytokine-induced endothelial dysfunction

ObjectivesEndothelial dysfunction is known to be a key characteristic of preeclampsia (PE) and can contribute to progression of symptoms and injury to multiple organ systems. Delivery is the only treatment for progression of PE, but development of an endothelial-based therapy for PE presents a promising strategy. Growth factors and cytokines are dysregulated in PE and can impact endothelial function, manifesting changes in Ca2+ signaling and interruptions in monolayer barrier function that contribute to symptoms of hypertension, proteinuria, and edema. In this study, we highlight Src kinase as a partial mediator of growth factor and cytokine mediated endothelial dysfunction. Study designFura-2 Ca2+ imaging and Electrical Cell Impedance Sensing (ECIS) assays are performed on growth factor or cytokine exposed human umbilical vein endothelial cells (HUVECs). Inhibitors to MEK/ERK (U0126) or Src (PP2) are used to determine the contribution of kinase signaling pathways. Main outcome measuresDecreases in HUVEC Ca2+ signaling or monolayer resistance measure endothelial dysfunction. Reversal of endothelial dysfunction by kinase inhibitors reveals the respective contibutions of MEK/ERK and Src kinase. ResultsWe show that Src inhibition protects Ca2+ signaling responses against insults induced by VEGF165, bFGF, PlGF, TNFα, and IL-1β. Additionally, we show that Src inhibition protects the endothelial monolayer from the full impact of TNFα insult. Further, we find that MEK/ERK inhibition does not offer protection from growth factor-mediated endothelial dysfunction. ConclusionsThe results of this study suggest cytokine and growth factor-stimulated Src kinase plays a partial role on promoting endothelial dysfunction in HUVECs.

Abstract B001: Heterogeneity of cancer network biophysical phenotypes is required for tumor muscle invasion in vivo

Abstract Bladder, colon, gastric, prostate, and uterine cancers originate from the organ’s epithelium, and each organ is surrounded by smooth muscle. In prostate cancer, organ-confined tumors, without extracapsular extension (ECE) through muscle, have a 5-year survival rate of 99% compared to 31% for metastatic disease. Previously, we modeled tumor cluster ECE and reported that a CRISPR-Cas9 integrin mutation (α6AA) blocked ECE by switching from a cell-ECM to a cell-cell biophysical phenotype. Since tumors are mosaics and migrate as heterogeneous drug-resistant populations, we tested the functional heterogeneity of the two biophysical cell phenotypes in ECE. Experimental procedures included the in vivo mouse xenograft model of ECE as a functional endpoint of muscle invasion, the MTT assay for cell survival, and using electric cell impedance sensing (ECIS) measurements to directly compare the biophysical properties of tumor cells expressing the invasion permissive α6WT integrin, cells expressing only the α6AA mutation, or a mixed population expressing both integrin types. The α6AA integrin mutation or α6KO knockout produced cells unable to invade into and through the smooth muscle of the mouse as compared to the α6WT integrin cells, as previously reported by us. The new unpublished results are that cell adhesion mediated drug resistance was detected in the α6AA population since their LD50 to Bortezomib, Gemcitabine, and Taxotere was shifted to 33.71nM, 64.66nM, and 5.77nM respectively from 16.29nM, 31.74nM, and 3.52nM in the α6WT cells as measured by MTT assay at 72 hours of incubation. In contrast, the α6AA cells were sensitized to the NAMPT inhibitor, FK866, with LD50 shifting from 27.46nM in α6WT to 7.354nM in the α6AA cells. A mixture of α6AA and α6WT cells allowed invasion into and through the muscle in the mouse of the mutant α6AA integrin cells that could not invade on their own. Examining the live cellular biophysical parameters revealed that the integrin mutation produced a 4-fold increase in cell-cell resistance (400Hz measurement) and a 12-fold recovery time delay in re-establishing a cell-cell resistance monolayer after wounding, with response complexity decreased two-fold. Cell-ECM resistance (40,000Hz measurement) in α6AA versus α6WT population was 1.5-fold decreased with a 6-fold recovery time delay in the cell-ECM resistance monolayer and response complexity decreased 2-fold. A heterogenous population containing up to a 4:1 proportion of α6AA to α6WT cells synergistically delayed cell-cell recovery time of the wounded monolayer up to 6-fold compared to the α6WT cells. Conversely, recovery of the wounded cell-ECM resistance monolayer with 1:1 or 4:1 mixture of α6AA cell only delayed recovery time by 1.5–1.7-fold. Taken together, these data suggest that a heterogenous tumor population provides functional advantages for drug-resistant tumor cells to invade and traverse the muscle barrier. Current work is ongoing to optimize the drug combinations to eliminate both phenotypes in the invasive tumor network and prevent ECE. Citation Format: Kendra D. Marr, Jaime M.C. Gard, William L. Harryman, Elijah J. Keeswood, Allan I. Paxson, Charles Wolgemuth, Lori A. Hazlehurst, Raymond B. Nagle, Anne E. Cress. Heterogeneity of cancer network biophysical phenotypes is required for tumor muscle invasion in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B001.

O006 Hepatitis A virus cellular receptor 1 (HAVCR1) influences the integrity of blood brain barrier (BBB) of cerebral endothelial cells

Abstract Introduction Hepatitis A virus cellular receptor 1 (HAVcR1) has been established to play an important role in tight junction (TJ) in cancer cells and in peripheral endothelial cells. Its role in blood brain barrier (BBB) of cerebral endothelium remains unknown. This study aimed to investigate the association and functionality of HAVcR1 in BBB. Methods HAVcR1 was knocked down by siRNA in human cerebral microvessel endothelial cell/D3 (hCMEC/D3) cell lines. Changes in tight junction (TJ) behaviour were assessed using electric cell impedance sensing (ECIS), transendothelial resistance (TER), and paracellular permeability (PCP). The expression of HAVcR1 and the binding partners of HAVcR1 protein was assessed using quantitative polymerase chain reaction (qPCR). Results The HAVcR1 gene transcript, highly expressed in hCMEC/D3 cells, was successfully knocked down from the cell by anti-HAVcR1 siRNA. HAVcR1 knockdown resulted in an increase in four TJ molecules and a decrease in eight TJ molecules as demonstrated by transcript analysis. HAVcR1 knockdown cells displayed lower electric resistance compared with wild type cells during initial attachment and spreading in hCMEC/D3 cells shown by ECIS. Similarly, TER in HAVcR1 knockdown cells were also reduced. Higher PCP fluorescence signals were detected in HAVcR1 knockdown cell lines compared with wild type. Conclusion HAVcR1 regulates paracellular leakage of cerebral endothelial cells and loss of HAVcR1 renders the cells with weaker tight junction by influencing TJ protein expression. HAVcR1 have a function as part of the regulatory apparatus for TJ in BBB.

Abstract 3835: Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia

Abstract The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) migration, invasion, metastasis, and decreased cancer-specific survival. Kindlin-2 (FERMT2) is a β1 integrin adaptor and mechanosensory focal adhesion (FA) protein that activates and clusters integrins in response to structural ECM alterations in the tumor microenvironment. Our goal was to determine if integrin-kindlin-2 adhesion complexes (kindlin-2:α6β1) were responsive to hypoxia, a physiologically relevant and altered microenvironment in PCa progression. Five different endpoints were tested including the biochemical analysis of kindlin-2 complexes, qRT-PCR, immunoblotting, immunocytochemistry, and electric cell impedance sensing (ECIS). Using DU145 prostate cancer cells grown under hypoxia (1% O2) for up to 16 hours, the results showed a reversible increase in kindlin-2:α6β1 complexes with maximal assembly within 4 hours and disassembly starting by 8 hours. Notably, kindlin-2:α6β1 complexes were found exclusively within membrane projections and were not observed within hypoxia-inducible paxillin (PXN)-containing FAs. The hypoxia induced kindlin-2:α6β1 complexes and classical FAs were dependent on kindlin-2 as determined by CRISPR-Cas9 heterozygous deletion of FERMT2. Protein co-localization of α6 integrin and PXN with kindlin-2 within membrane projections and FAs, respectively, was also induced under hypoxia. Further, non-invasive ECIS measurements in live cells confirmed functional cell-cell and cell-ECM dynamics driven by hypoxia and requiring kindlin-2. Our results indicate that the kindlin-2:α6β1 complexes are uniquely associated with FA-independent membrane projections induced by hypoxia, a tumor microenvironment associated with aggressive prostate cancer. The novel kindlin-2:α6β1 complexes may represent an actionable pharmacological target for blocking escape of organ confined disease and metastasis promoting steps of human prostate cancer. (Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226). Citation Format: Daniel Hernandez-Cortes, Jaime M.C. Gard, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Kindlin-2 complexes containing α6β1 integrin are responsive to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3835.

Expression and activation of the ketone body receptor HCAR2/GPR109A promotes preservation of retinal endothelial cell barrier function

Preservation of retinal barrier function is critical to maintenance of retinal health. Therefore, it is not surprising that loss of barrier integrity is a pathologic feature common to degenerative retinal diseases such as diabetic retinopathy. Our prior studies demonstrate the importance of hydroxycarboxylic acid receptor 2/GPR109A (HCAR2/GPR109A) expression in the retinal pigment epithelium (RPE) to outer retinal barrier integrity. However, whether HCAR2/GPR109A is expressed in retinal endothelial cells and has a similar relationship to inner blood retinal barrier regulation is not known. In the current study, we examined relevance of receptor expression to endothelial cell dependent-blood retinal barrier integrity. siRNA technology was used to modulate HCAR2/GPR109A expression in human retinal endothelial cells (HRECs). Cells were cultured in the presence or absence of VEGF, a pro-inflammatory stimulus, and/or various concentrations of the HCAR2/GPR109A-specific agonist beta-hydyroxybutyrate (BHB). HCAR2/GPR109A expression was monitored by qPCR and electrical cell impedance sensing (ECIS) was used to evaluate barrier function. Complementary in vivo studies were conducted in wildtype and HCAR2/GPR109A knockout mice treated intraperitoneally with lipopolysaccharide and/or BHB. Vascular leakage was monitored using fluorescein angiography and Western blot analyses of albumin extravasation. Additionally, retinal function was evaluated by OptoMotry. Decreased (siRNA knockdown) or absent (gene knockout) HCAR2/GPR109A expression was associated with impaired barrier function both in vitro and in vivo. BHB treatment provided some protection, limiting disruptions in retinal barrier integrity and function; an effect that was found to be receptor (HCAR2/GPR109A)-dependent. Collectively, the present studies support a key role for HCAR2/GPR109A in regulating blood-retinal barrier integrity and highlight the therapeutic potential of the receptor toward preventing and treating retinal diseases such as diabetic retinopathy in which compromised barrier function is paramount.

Role of the Epithelial Glycocalyx in Maintaining the Alveolar‐Capillary Barrier During Acute Lung Injury

RationaleDisruption of the alveolar‐capillary barrier is a signature feature of acute lung injury. Previous studies have shown the importance of the glycocalyx in maintaining the integrity and homeostasis of the lung endothelium during acute lung injury. However, the role of the glycocalyx on lung epithelial barriers during acute lung injury has not been fully explored. We hypothesize that the lung epithelial glycocalyx is important in maintaining lung epithelial tight junctions in the setting of acute lung injury.MethodsCultured mouse lung epithelial cells (MLE12) were treated with functional or heat‐inactivated heparinase I/III. Additionally, some MLE12 cells were treated with LPS after pretreatment with functional heparinase I/III. Glycocalyx shedding was measured by immunohistochemistry for heparan sulfate (HS). To assess paracellular permeability, electrical cell‐impedance sensing (ECIS) was used to measure the resistance across the epithelial monolayer. To determine whether loss of the glycocalyx affects epithelial tight junctions we measured gene and protein expression of ZO‐1, by PCR, western blot, and immunofluorescence.ResultsMLE12 cells in culture express a robust glycocalyx that is completely shed following treatment with active heparinase I/III at 24 hours. Active heparinase I/III did not cause significant change in monolayer resistance from baseline compared to inactive heparinase I/III by ECIS (Active −142±39.2 v Inactive −118.9±57.9 ohms, p=0.75). Active heparinase I/III did not decrease protein expression of ZO‐1 compared to inactive heparinase I/III (Active 1.55±0.33 v Inactive 1.8±0.25 fold change ZO‐1/Actin, p=0.61). MLE12 cells treated with activated heparinase I/III and LPS had a significantly greater decrease in resistance from baseline compared to inactive heparinase and LPS (Figure , Active + LPS −145±8.8 vs Inactive + LPS − 7.7±10.8 ohms, p=0.0022)ConclusionTreating MLE12 cells with functional heparinase I/III degrades the epithelial glycocalyx. Loss of the glycocalyx alone was not sufficient to cause permeability defects nor alter the expression of tight junction proteins. However, glycocalyx shedding did increase the susceptibility of MLE12 cells to LPS‐induced injury. In clinical situations, the shedding of the glycocalyx in ALI is often accompanied by compounding factors such as infection, inflammation, or mechanical stress. The current findings suggest that damage to the epithelial glycocalyx may play a key role in maintaining the alveolar epithelial barrier in acute lung injury.Support or Funding InformationSupported by a research grant from Department of DefenseMLE12 cells treated with active heparinase + LPS had a significantly greater decrease in resistance compared to baseline than cells treated with inactive heparinase + LPS p=0.0022.Figure 1

Placental growth factor regulates the pentose phosphate pathway and antioxidant defense systems in human retinal endothelial cells

The molecular mechanisms whereby placental growth factor (PlGF) mediates its effects in nonproliferative diabetic retinopathy (DR) are unknown. To better understand the role of PlGF in DR, we used tandem mass tags (TMT)-labeled quantitative proteomics to human retinal endothelial cells (HRECs), treated anti-PlGF antibody, and PBS as a control. Functional annotation and pathway enrichments were performed, which suggested that the differentially expressed proteins (DEPs) were involved in key metabolic processes, protein binding, and membrane, pentose phosphate pathway (PPP) and adherens junction. We conducted integrated gene profiles of our previously published transcriptomic data to the TMT-labeled proteomics data. The results showed the sixty genes were found to be changed at the proteome level. The functional annotation conducted for the sixty proteins suggested that 58.3% of proteins were involved in PPP, 25% of proteins were in interleukin-12 singling and 16.7% of proteins were involved in glycolysis and gluconeogenesis pathway. Mass spectrometry results were validated by transendothelial electrical resistance measurement by an electrical cell-impedance sensing (ECIS) and western blot analysis of VE-cadherin, G6PD. These findings suggest that the PPP proteins and antioxidants may act as a downstream target of PlGF and may play a decisive role in HREC biological functions in DR. SignificancePlGF (Placental growth factor) is known to play a pivotal role in pathological angiogenesis and inflammation by stimulating endothelial cell migration and by recruiting pericytes and inflammatory cells such as microglia and macrophages. Despite the well-defined pathophysiological roles of PlGF, the underlying molecular and cellular mechanisms are not completely understood, especially the exact relationships between biochemical events and molecular pathways regulated by PlGF, whose inhibition exhibits a protective role in DR. This study provides new insights into protein expression patterns and enables the identification of many attractive candidates for investigation of PPP pathway role in the activation of the antioxidant defense system in DR. Our findings suggest that the PPP proteins and antioxidants (PRDX6, HMOX1, NQO1 and YES1) may act as downstream targets of PlGF and may play a decisive role in HREC biological functions in DR.

PCB-3D-Printed, Reliable and Reusable Wells for Impedance Spectroscopy of Aqueous Solutions

Impedance Spectroscopy (IS) has been shown to be a non-invasive and reliable technique for the electrical characterization of biological materials. This paper presents the design and implementation of reliable, reusable wells that are used to perform IS measurements of aqueous solutions. These reusable wells are detachable, easy to clean and low-cost and they are made up of a platen on a Printed Circuit Board (PCB) and the chambers are manufactured using 3D-printing technology. In this case, in order to verify its functionality, IS measurements of electrolytic and non-electrolytic aqueous solutions were carried out. Initially, as a reference, the impedance spectrum of a Hanks’ solution was obtained following a proposed measurement protocol. Then, we analyse this spectrum and we propose an Equivalent Electrical Model (EEM) for validating the reusable wells. Finally, IS measurements are carried out on aqueous solutions of molecular D-glucose and sodium chloride prepared in Hanks’ solution and deionized water, by considering physiological concentrations. The parameter values of the EEMs of each solution tested were obtained using genetic algorithms and Matlab and, from these values, it is possible to conclude that the measurements performed are unable to differentiate the physiological concentration of glucose in the aqueous solution used. Also, from these results, it can be concluded that the designed wells are suitable for IS measurements of aqueous solutions and that they can be used in Electrical Cell Impedance Sensing (ECIS) or applications that require electrical characterization of solutions.

Real time in vitro monitoring and impedance modeling of mammalian cell activities on planar ECIS and micro/nano patterned cytosensors

Real time monitoring of cellular activities like cell adhesion, spreading, proliferation, apoptosis and wound healing is of significant importance for understanding the mechanisms involved in cell–cell and cell–matrix communication, which eventually is expected to unfold some of the mysteries related to the functioning of diseased and normal cells. Towards this direction, surface topography plays a crucial role to provide a distinct in vitro environment for better manipulation and understanding of in vivo cell behavior. In this review, we focus on the real time monitoring of mammalian cell activities along with distributed electrical models using planar electric cell impedance sensing (ECIS) substrates and micro/nano patterned topographic cytosensors.

Electronic platform for real-time multi-parametric analysis of cellular behavior post-exposure to single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWCNTs) implementation in a variety of biomedical applications from bioimaging, to controlled drug delivery and cellular-directed alignment for muscle myofiber fabrication, has raised awareness of their potential toxicity. Nanotubes structural aspects which resemble asbestos, as well as their ability to induce cyto and genotoxicity upon interaction with biological systems by generating reactive oxygen species or inducing membrane damage, just to name a few, have led to focused efforts aimed to assess associated risks prior their user implementation. In this study, we employed a non-invasive and real-time electric cell impedance sensing (ECIS) platform to monitor behavior of lung epithelial cells upon exposure to a library of SWCNTs with user-defined physico-chemical properties. Using the natural sensitivity of the cells, we evaluated SWCNT-induced cellular changes in relation to cell attachment, cell-cell interactions and cell viability respectively. Our methods have the potential to lead to the development of standardized assays for risk assessment of other nanomaterials as well as risk differentiation based on the nanomaterials surface chemistry, purity and agglomeration state.

In vitro and in vivo effects of suppressor of cytokine signalling 7 knockdown in breast cancer: the influence on cellular response to hepatocyte growth factor.

Purpose. Suppressor of cytokine signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with phospholipase Cγ-1 (PLCγ-1), a key downstream mediator of the hepatocyte growth factor (HGF)/C-MET axis. Here, we report our observations of the effect of knocking down SOCS7 gene on the behaviour of breast cancer cells both in vitro and in vivo and to elucidate whether this involves HGF/C-MET pathway using the PLCγ-1 blocker U73122. Methods. MCF7 and MDA-MB-231 breast cancer cells were transfected with anti-SOCS7 ribozymal transgene, to create sublines with SOCS7 knockdown. The in vitro growth and migration of the cells were evaluated in basic conditions and with HGF and U73122 treatment using growth assays, scratch-wound, and electrical cell impedance sensing (ECIS) migration assays. MCF7 and MDA-MB-231 in vivo tumour xenograft growth were also studied. Results. Basal in vitro growth and migration of both cellular lines and the in vivo MCF7 xenograft growth were significantly enhanced with SOCS7 knockdown. In vitro HGF treatment has further influenced the growth and migration when SOCS7 gene was knocked-down in both cellular lines (P < 0.05). PLCγ-1 pharmacological inhibition of the HGF/C-MET cascade during their in vitro growth and migration seemed to only occur when SOCS7 gene was knocked down. Conclusions. We report a unique regulatory role for SOCS7 in controlling the malignant behaviour of breast cancer lines MCF7 and MDA-MB-231 in vitro and the MCF7 tumour xenografts in vivo. We also report a regulatory role for SOCS7 during the in vitro HGF-induced growth and migration in these cells as HGF treatment and SOCS7 loss have synergistically enhanced these functions. This SOCS7 knockdown-attributed effect could be due to a precise anti-PLCγ-1 role.

Microfluidic Chip with Integrated Electrical Cell-Impedance Sensing for Monitoring Single Cancer Cell Migration in Three-Dimensional Matrixes

Cell migration has been recognized as one hallmark of malignant tumor progression. By integrating the method of electrical cell-substrate impedance sensing (ECIS) with the Boyden chamber design, the state-of-the-art techniques provide kinetic information about cell migration and invasion processes in three-dimensional (3D) extracellular matrixes. However, the information related to the initial stage of cell migration with single-cell resolution, which plays a unique role in the metastasis-invasion cascade of cancer, is not yet available. In this paper, we present a microfluidic device integrated with ECIS for investigating single cancer cell migration in 3D matrixes. Using microfluidics techniques without the requirement of physical connections to off-chip pneumatics, the proposed sensor chip can efficiently capture single cells on microelectrode arrays for sequential on-chip 2D or 3D cell culture and impedance measurement. An on-chip single-cell migration assay was successfully demonstrated within several minutes. Migration of single metastatic MDA-MB-231 cells in their initial stage can be monitored in real time; it shows a rapid change in impedance magnitude of approximately 10 Ω/s, whereas no prominent impedance change is observed for less-metastasis MCF-7 cells. The proposed sensor chip, allowing for a rapid and selective detection of the migratory properties of cancer cells at the single-cell level, could be applied as a new tool for cancer research.

Observations on the effects of Suppressor of Cytokine Signaling 7 (SOCS7) knockdown in breast cancer cells: their in vitro response to Insulin Like Growth Factor I (IGF-I)

Suppressor of cytokine signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with phospholipase Cγ-1 (PLCγ-1), one of the insulin-like growth factor-I (IGF-I) receptor downstream molecules. In this study, we sought to observe the effect of knocking down SOCS7 gene on breast cancer cells in vitro growth and migration and to elucidate whether this involves IGF-I-PLCγ1 route using the PLCγ-1 blocker U73122. Suitable breast cancer cells (MCF7 and MDA-MB-231) were transfected with anti-SOCS7 ribozymal transgene, to create sub-lines with SOCS7 knockdown verified by RT-PCR. The growth and migration of the cells were evaluated in the presence or absence of IGF-I and PLCγ-1 inhibitor using growth assay, scratch-wound and electrical cell impedance sensing (ECIS) migration assays. IGF-I treatment produced more pronounced influence on MCF7 growth and migration and on MDA-MB-231 migration when SOCS7 gene was knocked down in both lines (p < 0.05). The absence of IGF-I-induced growth response in MDA-MB-231 could be due to the intrinsic characteristics of these cells. PLCγ-1 pharmacological inhibition during their in vitro migration seemed to only occur when SOCS7 gene was knocked down. To the best of our knowledge, this is the first report of the SOCS7 regulatory role in IGF-I induced in vitro functions in ER-positive and ER-negative breast cancer cells. IGF-I treatment and SOCS7 loss have synergistically resulted in increased growth and migration of MCF7 and in increased migration of MDA-MB-231 cells. The migratory effects could be due to a precise anti-PLCγ-1 role.

Ultrasound-mediated drug delivery with real-time cell permeability measurements

Ultrasound-mediated drug and gene delivery offers a variety of novel possibilities for improved localized treatment of vascular- and cancer-related diseases. This therapeutic application benefits from the use of acoustic radiation force, which facilitates the exposure for enhanced binding from ligand-receptor interactions. The unique merits of ultrasound are the transient increase of cell permeability without any detrimental and irreversible side-effects. The related underlying molecular and cellular pathways of ultrasound-induced permeability and the subsequent recovery of cells are topics of on-going research. Real-time studies of cell behavior during and past-ultrasound exposure have been limited by the lack of appropriate techniques. The electric-cell impedance sensing (ECIS) technique is a suitable way of studying cell permeability changes in real-time. Its nanoscale sensitivity and speedy acquisition of data allows for the accurate and timely monitoring of cell behavior. Our preliminary results suggest that cells recover within 24–36 h post-exposure. During this time window the cells undergo drastic changes exhibiting an increased permeability of 2.4 ± 0.6 Ω•cm2 compared to 3.8 ± 0.5 Ω•cm2 that normal untreated cells exhibit.

Abstract P1-04-03: Knocking down Suppressor of Cytokine Signaling 7 in breast cancer: The role in Insulin-like Growth Factor - I/Phospholipase Cγ-1 signaling

Abstract BACKGROUND: Suppressor of Cytokine Signaling 7 (SOCS7) is a member of the SOCS family and is known to interact with Phospholipase Cγ-1 (PLCγ-1), one of the Insulin like Growth Factor - I (IGF-I) receptor downstream molecules. In the present study, we sought to examine the effect of knocking down SOCS7 gene on breast cancer cells growth and migration and to elucidate whether this has involved IGF-I — PLCγ-1 signaling using the PLCγ-1 blocker U73122. METHODS: Suitable breast cancer cells (MCF7 and MDA-MB-231) were transfected with anti-SOCS7 ribozymal transgene, to create sublines with SOCS7 knockdown that was verified by RT-PCR. The growth and migration of the cells were evaluated in the presence or absence of IGF-I and PLCγ-1 inhibitor using in vitro growth assay and Electrical Cell Impedance Sensing (ECIS) migration assay. RESULTS: MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 (SOCS7 knockdown) were constructed. Both sublines showed a higher rate of growth compared to control cells with and without IGF-I stimulation. U73122 treatment did not appear to change this growth outcome. Using ECIS migration assay, it was shown that knocking down SOCS7 had a significant positive effect on the migration of MCF7 and MDA-MB-231 cells, and that both IGF-I treatment and SOCS7 knockdown had a synergistic positive influence on their migration (p &amp;lt; 0.05). We further demonstrated that the impact of U73122 on the IGF-I migratory effect was dependent upon SOCS7 knockdown as it has significantly blocked the stimulatory effect of IGF-I on MCF7ΔSOCS7 and MDA-MB-231ΔSOCS7 migration but not that of the control cells. While SOCS7 has acted to control the IGF-I effect, it appeared to cancel the inhibitory function of U73122, indicating a specific anti - PLCγ-1 role for SOCS7 in IGF-I induced breast cancer cellular migration. CONCLUSION: SOCS7 loss resulted in increased growth and migration of breast cancer cells and this had a synergistic effect on their response to IGF-I. This role could be related to its interaction with PLCγ-1 during the cellular migration but not the growth. It may be possible that SOCS7 acts through different mechanism to control the cellular growth. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-04-03.

Tumour suppressor function of MDA-7/IL-24 in human breast cancer

IntroductionMelanoma differentiation associated gene-7 (MDA-7), also known as interleukin (IL)-24, is a tumour suppressor gene associated with differentiation, growth and apoptosis. However, the mechanisms underlying its anti-neoplastic activity, tumour-specificity and efficacy across a spectrum of human cancers have yet to be fully elucidated. In this study, the biological impact of MDA-7 on the behavior of breast cancer (BC) cells is evaluated. Furthermore, mRNA expression of MDA-7 is assessed in a cohort of women with BC and correlated with established pathological parameters and clinical outcome.MethodsThe human BC cell line MDA MB-231 was used to evaluate the in-vitro impact of recombinant human (rh)-MDA-7 on cell growth and motility, using a growth assay, wounding assay and electric cell impedance sensing (ECIS). Localisation of MDA-7 in mammary tissues was assessed with standard immuno-histochemical methodology. BC tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription, MDA-7 transcript levels were determined using real-time quantitative PCR. Transcript levels were analyzed against tumour size, grade, oestrogen receptor (ER) status, nodal involvement, TNM stage, Nottingham Prognostic Index (NPI) and clinical outcome over a 10 year follow-up period.ResultsExposure to rh-MDA-7 significantly reduced wound closure rates for human BC cells in-vitro. The ECIS model demonstrated a significantly reduced motility and migration following rh-MDA-7 treatment (p = 0.024). Exposure to rh-MDA-7 was only found to exert a marginal effect on growth. Immuno-histochemical staining of human breast tissues revealed substantially greater MDA-7 positivity in normal compared to cancer cells. Significantly lower MDA-7 transcript levels were identified in those predicted to have a poorer prognosis by the NPI (p = 0.049) and those with node positive tumours. Significantly lower expression was also noted in tumours from patients who died of BC compared to those who remained disease free (p = 0.035). Low levels of MDA-7 were significantly correlated with a shorter disease free survival (mean = 121.7 vs. 140.4 months, p = 0.0287) on Kaplan-Meier survival analysis.ConclusionMDA-7 significantly inhibits the motility and migration of human BC cells in-vitro. MDA-7 expression is substantially reduced in malignant breast tissue and low transcript levels are significantly associated with unfavourable pathological parameters, including nodal positivity; and adverse clinical outcomes including poor prognosis and shorter disease free survival. MDA-7 offers utility as a prognostic marker and potential for future therapeutic strategies.

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.

The Role of Claudin- 5 and the Paracellular Barrier Function in Endothelial Cells during Invasion of Breast Cancer Cells.

Abstract Material and Methods: Human endothelial (HECV) and breast cancer cells (MDA-MB-231) were used to assess the effect of over-expression (HECVCL-5, MDACL-5) or knockdown (HECVDCL-5, MDADCL-5) of Claudin-5. Cell adhesion and growth were evaluated using in vitro assays. An in vivo tumour model using athymic nude mice was used to assess growth of tumours. Transendothelial and transepithelial resistance (TER) was employed to measure changes in TJ function. Cellular motility was analyzed using Cytodex-2 bead motility assay. ECIS (electrical cell impedance sensing) was used to assess the attachment and migration of the cells.Results: There were no significant differences in in vitro growth of cells that over-expressed or had knockdown of Claudin-5 (both HECV and MDA-MB-231 cells). Expression of CL5 in both HECV and MDA-MB-231 did not have a significant impact on in vivo growth. There were however, significant differences in adhesion between control cells (HEVCpEF6) and HECVCL-5, HECVDCL-5 (changes in adhesion: HEVC pEF6 vs. HECVCL-, P&amp;gt;0.05; HECVDCL-5, P=0.046). There was a distinct modulation of TJ function as assessed using TER. HECVDCL-5 cells exhibited increased resistance compared to HEVC pEF6 and HECVCL-5 (change in TER: HEVC pEF6: -151.33±6.11 vs. HECVCL-5 -187±1.73, HECVDCL-5-89.33±2.08, P &amp;lt;0.01). There was no difference in TER in MDA CL-5 and MDADCL-5cells. HGF, a powerful motogen and modulator of TJ function, caused a significant increase in TER in HECVCL-5 and HECVDCL-5, but resulted in a significant decrease in TER in MDA-MB-231 cells. Motility studies revealed that HECVCL-5 and MDA CL-5 after treatment with HGF cells were significantly more motile than cells without treatment (HECVCL-5 vs. HECVCL-5+HGF, P=0.015; MDACL-5 vs. MDACL-5+HGF, P=0.033). There was no difference in motility in HECVDCL-5 cells, however, MDADCL-5 cells were significantly less motile after treatment with HGF (P=0.017). Interestingly, ECIS results showed that the ROCK inhibitor Y27632 inhibited the attachment and migration of HECVCL-5 cells but was no longer able to inhibit attachment and migration of HECVDCL-5 cells.Discussion: These results demonstrate for the first time that Claudin-5 can be modulated to effect changes in cell motility, attachment and TJ function in human endothelial and breast cancer cells. Moreover, there appears to be a link between Claudin-5 expression and cell motility, particularly with respect to the ROCK signalling cascade. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6170.

NWASP, the Neural Wiskott-Aldrich Syndrome Protein, Plays a Pivotal Role in the Control of Endothelial Migration and Angiogenesis.

Abstract Introduction. nWASP, the neural Wiskott-Aldrich Syndrome Protein, is a member of the Wiskott-Aldrich syndrome (WAS) protein family which are known regulators of actin polymerization. Via interactions with actin, ERM (ezrin/moesin/radixin) family proteins, and other cytoskeletal associated proteins, nWASP is a key cell migration mediator. It has been shown that nWASP is aberrantly expressed in human breast cancer and that a reduction of nWASP is linked to a poor clinical outcome. Over-expression of nWASP in breast cancer cells also reduces their aggressiveness in vitro. In the present study, we investigated the potential influence of nWASP on the biological functions of endothelial cells and the angiogenic aspect of endothelial cells.Materials and methods. A panel of human cell lines of variety origins (n=43) were screened for the presence/absence of nWASP expression. A human vascular endothelial cell line, HECV, was found to be negative for nWASP expression and was chosen as a cell model in the present study. An expression construct for human nWASP was prepared using a mammalian expression vector, pcDNA3/V5/GFP/TOPO vector. HECV cells were transfected with the expression construct, or control vectors. The impact of nWASP over-expression in the endothelial cell line on the adhesiveness, migration and in vitro angiogenesis was assessed using a panel of in vitro models.Results. HECV cells, following transfection with the nWASP construct, successfully expressed nWASP, a subline subsequently established from which was so named as HECVnWASPexp7. Using an electric cell impedance sensing (ECIS) method, it was shown that HECVnWASPexp7 cells showed a significantly reduced adhesiveness to matrix surface (333.4±1.4 ohm), p&amp;lt;0.01 when compared with the wild type cells (HECVwt 419.7±1.6 ohm) and transfection control (HECVpControl 387.9±1.46 ohm). In a similar fashion, HECVnWASPexp7 cells also displayed a marked reduction in cell migration when compared with control cells. Using an in vitro microtubule formation assay, it was shown that both HECVwt and HECVpControl cells responded well in their microtubule formation to an angiogenic factor, hepatocyte growth factor (HGF). However, HECVnWASPexp7cells were less responsive to HGF when assessed for microtubules. We have further shown that in response to an nWASP inhibitor, Wiskostatin, HECVnWASPexp7 cells markedly increased their tubule formation, a clear contrast to the wild type and control cells.Conclusion. It is concluded that nWASP is a key regulator of the migratory property of vascular endothelial cells and that this effect on cell migration is reflected by changes in the angiogenic activities of the cells. Collectively, nWASP is a potential intrinsic angiogenic regulator in endothelial cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2156.