Cellular Monolayer Research Articles

Activation of soluble AC10 induces cytoskeletal reorganisation

The cellular monolayer of the pulmonary endothelium forms a semi-selective barrier intersecting the vascular space, the underlying interstitium, and adjacent airspace. This endothelial barrier maintains tissue homeostasis critically regulating vessel permeability. Endothelial cAMP signals regulate endothelial permeability; however, cAMP signals are highly compartmentalized, such that cAMP signals generated by transmembrane adenylyl cyclase protect the barrier, while cAMP signals generated by soluble AC disrupt the barrier. Indeed, bicarbonate stimulation of soluble AC10 disrupts the endothelial barrier increasing permeability and is required for LPS-induced lung edema. Yet, how AC10 cAMP signals coordinate endothelial barrier regulation is unclear. The endothelial cytoskeleton alters cell shape which in turn regulates endothelial permeability. Thus, we hypothesized that bicarbonate stimulation of AC10 induces cytoskeletal remodeling. PMVECs were engineered to overexpress AC10 fused to a C-terminus hemagglutinin tag (HA). Gene and protein expression were controlled using a two-tier gene expression approach in which the doxycycline inducible promoter couples with a protein destabilizing variant of FKBP12 fused to the AC10-HA construct rendering the protein unstable in the absence of Shield1. In the presence of doxycycline and Shield1, AC10-HA protein began to appear 4-hours after induction and was absent in control cells. In this system, following addition of doxycycline and Shield1 and in the presence of elevated bicarbonate, whole cell cAMP increased over the same time course as protein expression while control cells showed no additional increase above normal bicarbonate stimulation. As cAMP increases above controls, endothelial cells begin to retract inducing inter-endothelial gaps between adjacent cells. This increase in inter-endothelial gaps was consistent with decreased resistance across the endothelial monolayer. To further address cytoskeletal mediated changes during AC10-induced endothelial barrier disruption, we examined actin cytoskeleton dynamics. Significant loss of the cortical actin rim and formation of actin stress fibers progress to complete actin cytoskeletal disruption by 24 hours. Interestingly, over the same course as the restructuring of the actin cytoskeleton, we detected phosphorylation of actin binding proteins, filamin and VASP, at PKA mediated sites. Thus, it appears that AC10 mediated pulmonary endothelial barrier disruption is coupled to actin remodeling.

Basement membrane proteins modulate cell migration on bovine pericardium extracellular matrix scaffold

Native bovine pericardium (BP) exhibits anisotropy of its surface ECM niches, with the serous surface (i.e., parietal pericardium) containing basement membrane components (e.g., Laminin, Col IV) and the fibrous surface (i.e., mediastinal side) being composed primarily of type I collagen (Col I). Native BP surface ECM niche anisotropy is preserved in antigen removed BP (AR-BP) extracellular matrix (ECM) scaffolds. By exploiting sideness (serous or fibrous surface) of AR-BP scaffolds, this study aims to determine the mechanism by which ECM niche influences human mesenchymal stem cells (hMSCs) migration. Human mesenchymal stem cells (hMSC) seeding on serous surface promoted more rapid cell migration than fibrous surface seeding. Gene analysis revealed that expression of integrin α3 and α11 were increased in cells cultured on serous surface compared to those on the fibrous side. Monoclonal antibody blockade of α3β1 (i.e., laminin binding) inhibited early (i.e. ≤ 6 h) hMSC migration following serous seeding, while having no effect on migration of cells on the fibrous side. Blockade of α3β1 resulted in decreased expression of integrin α3 by cells on serous surface. Monoclonal antibody blockade of α11β1 (i.e., Col IV binding) inhibited serous side migration at later time points (i.e., 6–24 h). These results confirmed the role of integrin α3β1 binding to laminin in mediating early rapid hMSCs migration and α11β1 binding to Col IV in mediating later hMSCs migration on the serous side of AR-BP, which has critical implications for rate of cellular monolayer formation and use of AR-BP as blood contacting material for clinical applications.

Organoid microphysiological system preserves pancreatic islet function within 3D matrix.

Three-dimensional (3D) multicellular organoids recapitulate the native complexities of human tissue better than traditional cellular monolayers. As organoids are insufficiently supported using standard static culture, microphysiological systems (MPSs) provide a key enabling technology to maintain organoid physiology in vitro. Here, a polydimethylsiloxane-free MPS that enables continuous dynamic culture and serial in situ multiparametric assessments was leveraged to culture organoids, specifically human and rodent pancreatic islets, within a 3D alginate hydrogel. Computational modeling predicted reduced hypoxic stress and improved insulin secretion compared to static culture. Experimental validation via serial, high-content, and noninvasive assessments quantitatively confirmed that the MPS platform retained organoid viability and functionality for at least 10 days, in stark contrast to the acute decline observed overnight under static conditions. Our findings demonstrate the importance of a dynamic in vitro microenvironment for the preservation of primary organoid function and the utility of this MPS for in situ multiparametric assessment.

Methods in Screening Antiviral Drugs Against Enterovirus 71.

Enteroviruses 71 (EV71) is a single-stranded, neurotrophic RNA virus responsible for the numerous outbreaks of hand, foot, and mouth disease (HFMD) in the Asia-Pacific regions. HFMD primarily affects children to cause range of infection, from mild symptoms to acute flaccid paralysis, and hemorrhage. Despite increased incidence of EV71 epidemics globally and research against EV71 becoming prioritized, no antiviral agent against EV71 has yet been licensed and approved worldwide. In this chapter, detailed EV71 antiviral screening techniques are described, including plaque assay which determines viral titers through the use of a semisolid overlay, carboxymethyl cellulose to allow even viral spread and infection across the host cellular monolayers as well as a crystal violet, a distinct counterstain to visualize circular regions of infectious zones-plaques. qRT-PCR is used to quantify the viral genomic RNA in the infected samples and MTS cell viability assay to quantify the cell viability after infection or toxicity of the compound on the cells. Furthermore, various antiviral inhibition assays including prophylactic, post infection, and virucidal assays are demonstrated for estimation of the antiviral activity of potential antiviral drugs against EV71. These methods can be effectively utilized in virology laboratories for effective high-throughput screening of antiviral molecules against EV71 that can assist in the future development of antiviral drugs.

Effects of blood exposure, increased catecholamine levels and inflammatory mediators on brain microvascular endothelial cell integrity in vitro

Introduction: Recent studies have suggested the impact of increased catecholamine levels CAT and inflammatory mediators INF on blood on the integrity of the blood brain barrier (BBB), especially when occurring in combination with hemorrhagic stroke, a situation frequently observed in Taktosubo syndrome TTS. We investigated the possible effects of these stressors on the brain microvascular endothelium in vitro. Methods: As an in vitro model of the BBB we used an immortalized murine microvascular endothelial cell line from the cerebral cortex (cEND). The cEND were treated with medium or patient blood containing supraphysiological concentrations of stress factors such as catecholamines, TNF-α and Interleukin-6 as characteristically elevated in patient blood suffering from acute takotsubo syndrome TTS. We investigated the impact on monolayer integrity and cellular morphology by light microscopy and immunofluorescence staining. Furthermore, alterations of representative tight and adherence junction proteins as well as integrins were determined by RT-PCR and/or Western Blot. Results: After stress factor/ patient blood treatment, reduced viability of brain capillary endothelial cells in vitro was observed. This was concomitant with a disruption of cellular monolayer integrity and changed localisation of BBB-forming tight junction proteins as determined by real-time RT-PCR (qPCR), western blot and immunofluorescence labeling. Monolayer integrity was moreover determined by measurement of transendothelial electrical resistance. Conclusion: Cellular monolayer integrity and the expression of proteins forming the BBB are clearly affected by exposure to patient blood, catecholamines CAT and inflammatory mediators INF, respectively. Most of barrier proteins are downregulated, so a negative impact on brain endothelial cells forming the blood-brain barrier can be assumed.

Quercetin affects membrane lipids and apoptosis in three-dimensional fibroblast cultures

The polyphenol quercetin is associated with numerous beneficial health effects in various pathologies such as oxidative stress, neurodegenerative disorders, inflammation, neoplastic processes, age-related diseases, and so on. However, the molecular mechanisms underlying the effects of quercetin and its implication in the pharmaceutical practice require additional clarification. In this study we analyzed the biochemical mechanisms of quercetin effect on membrane lipids of fibroblasts cultured as three-dimensional (3D) cultures, which resemble living tissues more adequately compared to cellular monolayers. Quercetin treatment of 3D fibroblasts induced alterations of the plasma membrane phospholipid and fatty acid composition as well as in the phospholipid metabolizing enzymes sphingomyelinase and phospholipase A2. The level of sphingomyelin (SM), which acts as endogenous membrane antioxidant, was elevated due to quercetin action. Incubation of 3D fibroblasts with quercetin induced augmentation in the saturated fatty acids and reduction of all polyunsaturated fatty acids in the membrane phospholipid molecules of the plasma membranes, indicating an additional increase of the resistance to oxidative damage. Quercetin treatment reduced cholesterol susceptibility to oxidation, a phenomenon which is probably related to the observed increase in the level of SM. In addition, Western blot analysis showed augmented phosphorylation of Akt and expression of Bcl-2 in quercetin-treated cell, indicating that apoptosis was suppressed by quercetin. In conclusion, the reported results contribute to a better understanding of the beneficial effects of quercetin on the structure and functions of plasma membranes of 3D cell cultures, which are a more adequate model of living tissues.

Tracking collective cell motion by topological data analysis.

By modifying and calibrating an active vertex model to experiments, we have simulated numerically a confluent cellular monolayer spreading on an empty space and the collision of two monolayers of different cells in an antagonistic migration assay. Cells are subject to inertial forces and to active forces that try to align their velocities with those of neighboring ones. In agreement with experiments in the literature, the spreading test exhibits formation of fingers in the moving interfaces, there appear swirls in the velocity field, and the polar order parameter and the correlation and swirl lengths increase with time. Numerical simulations show that cells inside the tissue have smaller area than those at the interface, which has been observed in recent experiments. In the antagonistic migration assay, a population of fluidlike Ras cells invades a population of wild type solidlike cells having shape parameters above and below the geometric critical value, respectively. Cell mixing or segregation depends on the junction tensions between different cells. We reproduce the experimentally observed antagonistic migration assays by assuming that a fraction of cells favor mixing, the others segregation, and that these cells are randomly distributed in space. To characterize and compare the structure of interfaces between cell types or of interfaces of spreading cellular monolayers in an automatic manner, we apply topological data analysis to experimental data and to results of our numerical simulations. We use time series of data generated by numerical simulations to automatically group, track and classify the advancing interfaces of cellular aggregates by means of bottleneck or Wasserstein distances of persistent homologies. These techniques of topological data analysis are scalable and could be used in studies involving large amounts of data. Besides applications to wound healing and metastatic cancer, these studies are relevant for tissue engineering, biological effects of materials, tissue and organ regeneration.

Realtime Transepithelial/Endothelial Electrical Resistance Measurements in Multiple Transwell Culture Inserts

Epithelial and endothelial cells line the surface of the body. Adjacent cells of both epithelial and endothelial cells are connected to each other via intercellular junction called tight junction that regulates the diffusion of molecules and ions between the apical and basolateral side of the body. Quantification of this barrier is important to evaluate the suitability of in vitro cellular barrier models for drug toxicity or transport studies. Transepithelial/endothelial electrical resistance (TEER), a quantitative technique to access electrical resistance of the cellular monolayer during culture has become one of the most popular quantitative techniques to evaluate the integrity of tight junction. The major advantage of the TEER technique over traditional permeability measurements is that it is quick, label-free, and non-invasive. As such, the TEER technique has been widely used to evaluate in vitro models such as the blood-brain barrier, the intestine epithelial barrier, the alveolar epithelial barrier, and the blood-retina barrier.However, the reported normalized TEER values of a specific cell type in transwell inserts are inconsistent between different research groups. A major cause of data variation is the manual technique that is used for measuring TEER. Typically, a “chopstick” like electrode pair is immersed inside a cell cultured semi-porous transwell insert membrane to measure TEER. Due to the variation in the alignment of the electrodes every time a TEER value is measured, the actual values vary significantly between measurements. A second problem with the conventional manual measurement process is time gap between each measurement (typically 12h or 24h) since a more frequent measurement requires frequent opening and closing of cell culture incubator door that can disturb the cells and introduce potential contamination.Thus, to enable consistency in TEER measurement, and allow automated real-time data acquisition in multiple samples, we built an add-on device (used atop EVOM®, a commercially available TEER instrument) consisting of a 3D printed electrode holder, a temperature sensor, and a custom designed data logger. The 3D printed electrode holder allowed data variation to be minimized by allowing perfect alignment of electrodes during measurement. The real-time data acquisition and in-situ temperature measurement allowed detection and correction of data offset during medium replacement process. The concept and device demonstration presented here should enable research labs to minimize data variations allowing them to produce consistent TEER results without having to spend extra time manually measuring multiple samples.

Transport Properties of Statins by Organic Anion Transporting Polypeptide 1A2 and Regulation by Transforming Growth Factor-β Signaling in Human Endothelial Cells.

Our in vivo rodent studies have shown that organic anion transporting polypeptide (Oatp) 1a4 is critical for blood-to-brain transport of statins, drugs that are effective neuroprotectants. Additionally, transforming growth factor-β (TGF-β) signaling via the activin receptor-like kinase 1 (ALK1) receptor regulates Oatp1a4 functional expression. The human ortholog of Oatp1a4 is OATP1A2. Therefore, the translational significance of our work requires demonstration that OATP1A2 can transport statins and is regulated by TGF-β/ALK1 signaling. Cellular uptake and monolayer permeability of atorvastatin, pravastatin, and rosuvastatin were investigated in vitro using human umbilical vein endothelial cells (HUVECs). Regulation of OATP1A2 by the TGF-β/ALK1 pathway was evaluated using bone morphogenetic protein 9 (BMP-9), a selective ALK1 agonist, and LDN193189, an ALK1 antagonist. We showed that statin accumulation in HUVECs requires OATP1A2-mediated uptake but is also affected by efflux transporters (i.e., P-glycoprotein, breast cancer resistance protein). Absorptive flux (i.e., apical-to-basolateral) for all statins was higher than secretory flux (i.e., basolateral-to-apical) and was decreased by an OATP inhibitor (i.e., estrone-3-sulfate). OATP1A2 protein expression, statin uptake, and cellular monolayer permeability were increased by BMP-9 treatment. This effect was attenuated in the presence of LDN193189. Apical-to-basolateral statin transport across human endothelial cellular monolayers requires functional expression of OATP1A2, which can be controlled by therapeutically targeting TGF-β/ALK1 signaling. Taken together with our previous work, the present data show that OATP-mediated drug transport is a critical mechanism in facilitating neuroprotective drug disposition across endothelial barriers of the blood-brain barrier. SIGNIFICANCE STATEMENT: Transporter data derived from rodent models requires validation in human models. Using human umbilical vein endothelial cells, this study has shown that statin transport is mediated by OATP1A2. Additionally, we demonstrated that OATP1A2 is regulated by transforming growth factor-β/activin receptor-like kinase 1 signaling. This work emphasizes the need to consider endothelial transporter kinetics and regulation during preclinical drug development. Furthermore, our forward-thinking approach can identify effective therapeutics for diseases for which drug development has been challenging (i.e., neurological diseases).

Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy.

Under physiological conditions, retinal pigment epithelium (RPE) is a cellular monolayer composed of mitotically quiescent cells. Tight junctions and adherens junctions maintain the polarity of RPE cells, and are required for cellular functions. In proliferative vitreoretinopathy (PVR), upon retinal tear, RPE cells lose cell-cell contact, undergo epithelial-mesenchymal transition (EMT), and ultimately transform into myofibroblasts, leading to the formation of fibrocellular membranes on both surfaces of the detached retina and on the posterior hyaloids, which causes tractional retinal detachment. In PVR, RPE cells are crucial contributors, and multiple signaling pathways, including the SMAD-dependent pathway, Rho pathway, MAPK pathways, Jagged/Notch pathway, and the Wnt/β-catenin pathway are activated. These pathways mediate the EMT of RPE cells, which play a key role in the pathogenesis of PVR. This review summarizes the current body of knowledge on the polarized phenotype of RPE, the role of cell-cell contact, and the molecular mechanisms underlying the RPE EMT in PVR, emphasizing key insights into potential approaches to prevent PVR.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin.

SummaryEstrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ERα) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosin-dependent and reversible by estrogens. Detailed investigations showed that the ERα target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

Co-culture model of Caco-2/HT29-MTX cells: A promising tool for investigation of phycotoxins toxicity on the intestinal barrier

Most lipophilic phycotoxins have been involved in human intoxications but some of these toxins have never been proven to induce human gastro-intestinal symptoms, although intestinal damage in rodents has been documented. For investigating the in vitro toxicological profile of lipophilic phycotoxins on intestine, the epithelial Caco-2 cell line has been the most commonly used model. Nevertheless, considering the complexity of the intestinal epithelium, in vitro co-cultures integrating enterocyte-like and mucus-secreting cell types are expected to provide more relevant data. In this study, the toxic effects (viability, inflammation, cellular monolayer integrity, modulation of cell type proportion and production of mucus) of four lipophilic phycotoxins (PTX2, YTX, AZA1 and OA) were evaluated in Caco-2/HT29-MTX co-cultured cells. The four toxins induced a reduction of viability from 20% to 50% and affected the monolayer integrity. Our results showed that the HT29-MTX cells population were more sensitive to OA and PTX2 than Caco-2 cells. Among the four phycotoxins, OA induced inflammation (28-fold increase of IL-8 release) and also a slight increase of both mucus production (up-regulation of mucins mRNA expression) and mucus secretion (mucus area and density). For PTX2 we observed an increase of IL-8 release but weaker than OA. Intestinal cell models integrating several cell types can contribute to improve hazard characterization and to describe more accurately the modes of action of phycotoxins.

Inhibitory effect of resveratrol on the growth and angiogenesis of human endometrial tissue in an In Vitro three-dimensional model of endometriosis

Endometriosis is a chronic estrogen-dependent disorder and one of the most common causes of infertility in women. Resveratrol (RES) is a polyphenolic and phytoestrogenic compound with anti-oxidative, anti-inflammatory, anti-angiogenic, and anti-estrogenic properties. The aim of the present study was to investigate the effect of different concentrations of RES on human endometrial growth and angiogenesis in an in vitro three-dimensional (3D) model of endometriosis.Human endometrial tissues of endometriosis (endometriotic) and normal (endometrial) subjects (n = 9/groups) were biopsied in sterile conditions and cut into 1 × 2 mm pieces. Tissue fragments of each biopsy were given concentrations of 0 (control), 10, 50, 100 and 200 μM RES for 21 days in 3D culture condition using fibrin as an extracellular matrix. Scoring methods were used for tissue changes, including; cellular invasion, monolayer formation and angiogenesis. Nitric oxide (NO) was measured using Griess's reaction, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the apoptotic gene expression.The mean of growth scores of endometriotic and endometrial tissue showed a significant dose dependent inhibition (P < 0.05). The levels of NO also significantly decreased in different groups. Apoptotic genes (P53, Bax, Bcl2 and caspase 3) and Sirt1 showed a significant increase in various concentrations of RES in both tissues (P < 0.05).RES exert dose- and time-dependent inhibitory effects on human endometrial tissue, and its higher doses suggested it as a natural supplement to inhibit the growth and treatment of endometriosis.

Cs-131 as an experimental tool for the investigation and quantification of the radiotoxicity of intracellular Auger decays in vitro

Purpose In this work, we set out to provide an experimental setup, using Cs-131, with associated dosimetry for studying relative biological effectiveness (RBE) of Auger emitters. Material and methods Cs-131 decays by 100% electron capture producing K- (9%) and L- (80%) Auger electrons with mean energies of 26 keV and 3.5 keV, respectively, plus ≈ 9.4 very low energy electrons (<0.5 keV) per decay. Cs-131 accumulates in the cells through the Na+/K+-ATPase. By this uptake mechanism and the alkali chemistry of Cs+, we argue for its intracellular homogeneous distribution. Cs-131 was added to the cell culture medium of HeLa and V79 Cells. The bio-kinetics of Cs-131 (uptake, release, intracellular distribution) was examined by measuring its intracellular activity concentration over time. Taking advantage of the 100% confluent cellular monolayer, we developed a new and robust dosimetry that is entrusted to a quantity called S C-value. Results The S C-values evaluated in the cell nucleus are almost independent of the nuclear size and geometry. We obtained dose-rate controlled RBE-values for intracellular Cs-131 decay. Using the γH2AX assay, the RBE was 1 for HeLa cells. Using the clonogenic cell survival, it was 3.9 for HeLa cells and 3.2 for V79 cells. Conclusion This experimental setup and dosimetry provides reliable RBE-values for Auger emitters in various cell lines.

The role of single cell mechanical behavior and polarity in driving collective cell migration.

The directed migration of cell collectives is essential in various physiological processes, such as epiboly, intestinal epithelial turnover, and convergent extension during morphogenesis as well as during pathological events like wound healing and cancer metastasis. Collective cell migration leads to the emergence of coordinated movements over multiple cells. Our current understanding emphasizes that these movements are mainly driven by large-scale transmission of signals through adherens junctions. In this study, we show that collective movements of epithelial cells can be triggered by polarity signals at the single cell level through the establishment of coordinated lamellipodial protrusions. We designed a minimalistic model system to generate one-dimensional epithelial trains confined in ring shaped patterns that recapitulate rotational movements observed in vitro in cellular monolayers and in vivo in genitalia or follicular cell rotation. Using our system, we demonstrated that cells follow coordinated rotational movements after the establishment of directed Rac1-dependent polarity over the entire monolayer. Our experimental and numerical approaches show that the maintenance of coordinated migration requires the acquisition of a front-back polarity within each single cell but does not require the maintenance of cell-cell junctions. Taken together, these unexpected findings demonstrate that collective cell dynamics in closed environments as observed in multiple in vitro and in vivo situations can arise from single cell behavior through a sustained memory of cell polarity.

P168 Modelling macrophage-fibroblast interaction in scleroderma to evaluate new treatments

Abstract Background Alternatively-activated M2-like macrophages are known to express CD206 and are thought to have an important role in pathological fibrosis in scleroderma through stimulation of fibroblasts. RP peptides are 10-12 mer synthetic peptides which have been developed to engage with human macrophages via CD206, but the selectivity of this receptor for macrophages requires clarification. In this study, we investigate the relative expression levels of CD206 in SSc macrophages and fibroblasts, and explore two approaches in modelling macrophage-fibroblast interactions to evaluate the efficacy of the RP peptides. Methods Macrophages were derived from peripheral blood mononuclear cells by culture of buffy layer cells in RPMI supplemented by M-CSF (4ng/ml) for 7 days. Skin fibroblasts were obtained by explant culture of 4 mm punch biospies from the involved forearm skin of SSc patients and healthy controls, maintained in DMEM with 10-% FCS and studied at passage 3-5. Stimulation with TGFβ (4ng/ml) was used to further enhance the activation state of fibroblasts. Macrophages and skin fibroblast lysates were evaluated using qPCR to determine the level of expression of CD206. Macrophages were co-cultured with fibroblasts within respective monolayers on 50kPa gels (models stiff scleroderma skin) for four days with or without the peptide inhibitors. Collagen mRNA expression from the cellular monolayers were quantified by qPCR and Western blot. In the second approach, macrophages and fibroblasts were co-cultured together with or without the peptide inhibitors in a collagen gel contraction assay. The gel was weighed and imaged after 48 hours of incubation and the collagen gel contraction was then quantified as a measure of fibrotic activity. Results Expression of CD206 was specific to macrophages and not seen in fibroblast cultures (relative expression level in macrophages 26.63 versus 0.004 in unstimulated fibroblasts versus 0.04 in TGFβ-stimulated fibroblasts. Fibroblasts co-cultured with macrophages in monolayers show increased CTGF and collagen mRNA by qPCR. There was a dose response reduction in CTGF and collagen mRNA in assays cultured with the RP peptide inhibitor. CTGF mRNA was positively correlated with CD206 expression (r2=0.81). Co-culture of SSc macrophages led to enhanced contraction of collagen gels (0.1g in macrophages, 0.08g in fibroblasts, 0.062 for fibroblasts co-cultured with macrophages) which was fully reversed by the RP peptide treatment (0.1g). Conclusion SSc macrophages express CD206 which has potential as a biomarker of ongoing fibrotic activity whereas fibroblasts express very minimal levels of CD206 even when stimulated by the pro-fibrotic TGFβ. RP peptides that are specific for CD206 on macrophages led to a suppression of the macrophage signature and inhibition of the pro-fibrotic cross talk in macrophages and fibroblasts in both monolayer and 3D collagen gels. Both approaches of evaluating macrophage-fibroblast interactions in scleroderma are viable approaches to evaluate new treatments. Disclosures L. Lei None. B. Abdi None. A. Mujkavnovic None. X. Shi Wen None. H. Lopez Shareholder/stock ownership; Scientific director at Riptide Biosciences. R. Stratton None.

The Capacity of High-Grade Serous Ovarian Cancer Cells to Form Multicellular Structures Spontaneously along Disease Progression Correlates with Their Orthotopic Tumorigenicity in Immunosuppressed Mice.

Many studies have examined the biology, genetics, and chemotherapeutic response of ovarian cancer’s solid component; its liquid facet, however, remains critically underinvestigated. Floating within peritoneal effusions known as ascites, ovarian cancer cells form multicellular structures, creating a cancer niche in suspension. This study explores the pathobiology of spontaneously formed, multicellular, ovarian cancer structures derived from serous ovarian cancer cells isolated along disease evolution. It also tests their capacity to cause peritoneal disease in immunosuppressed mice. Results stem from an analysis of cell lines representing the most frequently diagnosed ovarian cancer histotype (high-grade serous ovarian cancer), derived from ascites of the same patient at distinct stages of disease progression. When cultured under adherent conditions, in addition to forming cellular monolayers, the cultures developed areas in which the cells grew upwards, forming densely packed multilayers that ultimately detached from the bottom of the plates and lived as free-floating, multicellular structures. The capacity to form foci and to develop multicellular structures was proportional to disease progression at the time of ascites extraction. Self-assembled in culture, these structures varied in size, were either compact or hollow, irregular, or spheroidal, and exhibited replicative capacity and an epithelial nature. Furthermore, they fully recreated ovarian cancer disease in immunosuppressed mice: accumulation of malignant ascites and pleural effusions; formation of discrete, solid, macroscopic, peritoneal tumors; and microscopic growths in abdominal organs. They also reproduced the histopathological features characteristic of high-grade serous ovarian cancer when diagnosed in patients. The following results encourage the development of therapeutic interventions to interrupt the formation and/or survival of multicellular structures that constitute a floating niche in the peritoneal fluid, which in turn halts disease progression and prevents recurrence.

Abstract P2-20-04: Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease

Abstract Introduction: Brain metastatic disease (BMD) is attributed to significant mortality and morbidity in breast cancer patients. Strict policing of molecular traffic across the blood-brain barrier (BBB) precludes brain parenchymal penetration of most small molecule drugs, chemotherapy included. Consequently, the treatment of BMD remains difficult, particularly if the BBB remains intact during disease progression. Artemisinin has been efficacious in treating multi-drug resistant cerebral malaria likely indicative of an ability to traverse the BBB. Thus, Artemisinin may be able to modulate the restrictive nature of the BBB and provide a potential adjunctive therapy to established drug therapy in the treatment of BMD. Emerging evidence suggests that Artemisinin itself may harbour potent anti-cancer properties. Aim: To investigate in vitro the effect on barrier function in particular paracellular permeability of brain vascular endothelial cells compared to non-brain vascular endothelial cells when treated with Artemisinin. Materials and Methods: Inter-individual BBB differences were modelled using the human brain vascular endothelial cell lines hCMEC-D3, TY09 and TY10. In addition to the brain cell lines, a HUVEC cell line was treated with incremental concentrations of artemisinin. Adhesion, migration and electrical resistance across cellular monolayers were quantified using Electric Cell-Substrate Impedance Sensing (ECIS). Trans- Endo/Epithelial Resistance (TEER) and Paracellular Permeability (PCP) were used to assess barrier function. Immunofluorescence was used to qualitatively assess tight junction (TJ) integrity of all cell lines. RNAseq was used to determine changes in message after treatment with Artemisinin in all 4 cell lines. Results: A significant reduction in electrical resistance across all cell lines was observed when treated with 1mM Artemisinin and may be suggestive of a cytotoxic concentration. In comparison when treated with 0.01mM Artemisinin the electrical resistance decreased which could suggest restrictive barrier properties were enhanced, in addition evidencing Artemisinin may exhibit biphasic activity. The reduction in electrical resistance across the brain endothelial cell lines was significant (p&amp;lt;0.05) but deemed not significant across HUVEC, perhaps mirroring Artemisinin’s pharmacological action at differing vascular interfaces in vivo. Higher Artemisinin concentrations significantly reduced TEER across the brain cell lines and increased paracellular permeability, the same trends were observed in HUVEC. IF demonstrated that Artemisinin was able to effect changes in TJ protein location which supports the hypothesis that Artemisinin reduces barrier function across the brain endothelial lines, suggested by an increased permeability in the presence of Artemisinin. Electrical resistance across all cell lines significantly reduced when treated with 1mM Artemisinin. When compared to negative control, there was a significant decrease in electrical resistance across all cell lines treated with 1mM Artemisinin. RNAseq comparisons revealed that in TY10 cells, 1167 genes were upregulated and 94 downregulated in comparison to HUVEC where 185 and 0 genes were up or downregulated respectively and that in all 3 brain endothelial cell lines that the TJ protein CLDN5 exhibited a significant reduction in expression at gene level (p=0.03). Conclusion: The results demonstrate that Artemisinin may have the capacity to transiently increase or decrease the permeability of the BBB. Therefore, Artemisinin may have a role in facilitating drug delivery to the cerebral parenchyma providing potential therapeutic benefit for the treatment of BMD or other related neurological conditions difficult to treat due to the BBB. Citation Format: Emily Appadurai, Fiona Ruge, Andrew Sanders, Wen G Jiang, Tracey A. Martin. Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-04.

Morphogenesis is Stressful - Elastic Properties of Folding Cell Sheets

Living tissues are intelligent materials that can change their mechanical properties while they develop. In spite of extensive studies in multiple model organisms we are only just beginning to understand these dynamic properties and their role in tissue development. Although many tissues are known to exhibit visco-elastic properties, it is unclear which properties dominate three-dimensional shape changes of cellular monolayers, such as epithelia. The embryonic inversion process in the micro-algal family Volvocales is uniquely suited for comparative studies on epithelial morphogenesis. Volvocalean embryos consist of cup-shaped or spherical cellular monolayers which invert their curvature in order to expose their flagella. Volvox globator exhibits one of the most striking processes of cell sheet folding: Through inwards folding at the equator of the initially spherical cell sheet adopts a mushroom shape and eventually turns itself entirely inside-out through an anterior opening [1]. These global deformations are driven by several waves of active cell shape changes [2, 3]. A combination of advanced imaging and computational analyses is used to explore the role of tissue contractility in the occurring invagination and involution. The associated internal stresses as well as the elastic properties of the dynamic cell sheet are determined through laser ablation experiments [manuscript in preparation]. [1] Höhn S and Hallmann A. BMC Biology 9, 89 (2011). [2] Höhn S, Honerkamp-Smith AR, Haas PA, Khuc Trong P, and Goldstein RE. Physical Review Letters 114, 178101 (2015). [3] Haas PA, Höhn S, Honerkamp-Smith AR, Kirkegaard JB, and Goldstein RE. PLOS Biology 16, e2005536 (2018).

Developing a Microfluidic Device with an Integrated Electrode System for Measuring the Impedance Spectra of Cellular Models in Real Time

A mathematical model is presented that describes the distribution of the potential and electric field strength in cells of a microfluidic device for culturing a cellular monolayer with built-in electrodes of different geometries. The design of the measuring cell and the geometry of the electrodes are selected by analyzing the results from calculations.