Parallelizable Microfluidic Platform to Model and Assess In Vitro Cellular Barriers: Technology and Application to Study the Interaction of 3D Tumor Spheroids with Cellular Barriers.

Endothelial and epithelial cellular barriers play a vital role in the selective transport of solutes and other molecules. The properties and function of these barriers are often affected in case of inflammation and disease. Modelling cellular barriers in vitro can greatly facilitate studies of inflammation, disease mechanisms and progression, and in addition, can be exploited for drug screening and discovery. Here, we report on a parallelizable microfluidic platform in a multiwell plate format with ten independent cell culture chambers to support the modelling of cellular barriers co-cultured with 3D tumor spheroids. The microfluidic platform was fabricated by microinjection molding. Electrodes integrated into the chip in combination with a FT-impedance measurement system enabled transepithelial/transendothelial electrical resistance (TEER) measurements to rapidly assess real-time barrier tightness. The fluidic layout supports the tubeless and parallelized operation of up to ten distinct cultures under continuous unidirectional flow/perfusion. The capabilities of the system were demonstrated with a co-culture of 3D tumor spheroids and cellular barriers showing the growth and interaction of HT29 spheroids with a cellular barrier of MDCK cells.

A novel microfluidic 3D platform for culturing pancreatic ductal adenocarcinoma cells: comparison with in vitro cultures and in vivo xenografts.

The integration of microfluidics and cell biology has reached a significant milestone with the development of "organ-on-chips", smart technological platforms that, once applied to the study of human diseases, such as cancer, might ultimately contribute to design personalised treatments and hence improve health outcomes. This paper reports that the combination of microfluidics and dielectrophoresis (DEP) allows to culture different pancreatic ductal adenocarcinoma (PDAC) human cell lines into a cyclic olefin polymer (COP) chamber (HepaChip®), enriched by the extracellular matrix (ECM) protein collagen. We show that PDAC cells cultured into the HepaChip® (1) are vital and grow, provided they properly attach to collagen; (2) show morphological appearance and growth characteristics closer to those of cells grown as spheroids than as classical 2 dimensional (2D) in vitro cultures. Finally, preliminary experiments show that PDAC cells respond to high doses of Cisplatin perfused through the chip. Overall, the present microfluidic platform could be exploited in the future for a personalised approach to PDAC.

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation.

A non-canonical function of eukaryotic elongation factor 1A1: regulation of interleukin-6 expression.

Interleukin-6 is one of the most prominent triggers of inflammatory processes. We have shown recently that heteroarylketones (HAKs) interfere with stimulated interleukin-6 expression in astrocytes by suppression of STAT3 phosphorylation at serine 727. Surprisingly, this effect is not based on the inhibition of STAT3-relevant kinases. Therefore, we here used the structurally modified HAK compound biotin-HAK-3 in a reverse chemical approach to identify the relevant molecular target in UV-mediated cross-linking experiments. Employing streptavidin-specific 2D-immunoblotting followed by mass spectrometry we identified nine proteins putatively interacting with biotin-HAK-3. After co-immunoprecipitation, co-immunofluorescence, surface plasmon resonance analyses and RNAi-mediated knock-down, the eukaryotic elongation factor 1A1 (eEF1A1) was verified as the relevant target of HAK bioactivity. eEF1A1 forms complexes with STAT3 and PKCδ, which are crucial for STAT3(S727) phosphorylation and for NF-κB/STAT3-enhanced interleukin-6 expression. Furthermore, the intracellular HAK accumulation is strongly dependent on eEF1A1 expression. Taken together, the results reveal a novel molecular mechanism for a non-canonical role of eEF1A1 in signal transduction via direct modulation of kinase-dependent phosphorylation events.

Heteroarylketones inhibit astroglial interleukin-6 expression via a STAT3/NF-κB signaling pathway.

BACKGROUND: Elevated brain levels of the pleiotropic cytokine interleukin-6, which is mainly secreted from activated local astrocytes, contribute to pathological events including neuroinflammation and neurodegeneration. Thus, inhibition of pathological IL-6 expression provides a rationale strategy for targeting the onset or further progression of neurological disorders including Alzheimer's disease, multiple sclerosis, Parkinson's disease and traumatic brain injury. The purpose of this study was to identify and to characterize new potent inhibitors of astrocytic IL-6 expression for further therapeutic development of novel anti-inflammatory and neuroprotective drugs. METHODS: Oncostatin M (OSM)-treated human glioma U343 cells were used as model for induction of astrocytic IL-6 expression. This model was characterized by immunoblotting, siRNA technique, ELISA and qRT-PCR and used to screen low molecular weight compound libraries for IL-6-lowering effects. To validate bioactive compounds identified from library screens, bacterial lipopolysaccharide was used to induce IL-6 expression in cultivated primary astrocytes and in mice in vivo. To dissect underlying molecular mechanisms, protein extracts from OSM-treated U343 cells were analyzed by phospho-specific immunoblotting and immunocytochemistry as well as by co-immunoprecipitation. RESULTS: OSM-treatment (100 ng/ml; 24 h) led to 30-fold increase of IL-6 secretion from U343 cells. The temporal profile of IL-6 mRNA induction displayed a biphasic induction pattern with peak synthesis at 1 h (6.5-fold) and 16 h (5.5-fold) post stimulation. IL-6 protein release did not show that biphasic pattern and was detected as early as 3 h post stimulation reaching a maximum at 24 h. The screen of compound libraries identified a set of heteroarylketones (HAKs) as potent inhibitors of IL-6 secretion. HAK compounds affected the second peak in IL-6 mRNA synthesis, whereas the first peak was insensitive to HAK treatment. HAK compounds also suppressed lipopolysaccharide-induced IL-6 expression in primary murine astrocytes as well as in brain and plasma samples from lipopolysaccharide-treated mice. Finally, HAK compounds were demonstrated to specifically suppress the OSM-induced phosphorylation of STAT3 at serine 727 and the physical interaction of pSTAT3S727 with p65. CONCLUSION: Heteroarylketone compounds are potent inhibitors of IL-6 expression in vitro and in vivo and may represent a new class of potent anti-inflammatory and neuroprotective drugs.

Brain prolyl endopeptidase expression in aging, APP transgenic mice and Alzheimer's disease.

Prolyl endopeptidase (PEP) is believed to inactivate neuropeptides that are present in the extracellular space. However, the intracellular localization of PEP suggests additional, yet unidentified physiological functions for this enzyme. Here we studied the expression, enzymatic activity and subcellular localization of PEP in adult and aged mouse brain as well as in brains of age-matched APP transgenic Tg2576 mice and in brains of Alzheimer's disease patients. In mouse brain PEP was exclusively expressed by neurons and displayed region- and age-specific differences in expression levels, with the highest PEP activity being present in cerebellum and a significant increase in hippocampal but not cortical or cerebellar PEP activity in aged mouse brain. In brains of young APP transgenic Tg2576 mice, hippocampal PEP activity was increased compared to wild-type littermates in the pre-plaque phase but not in aged mice with beta-amyloid plaque pathology. This "accelerated aging" with regard to hippocampal PEP expression in young APP transgenic mice might be one factor contributing to the observed cognitive deficits in these mice in the pre-plaque phase and could also explain in part the cognition-enhancing effects of PEP inhibitors in several experimental paradigms.

Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion.

For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double-labelling procedures and localization of PEP-enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two-hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U-343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing-associated neurodegenerative diseases, cell-permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.

Modulation of inositol 1,4,5-triphosphate concentration by prolyl endopeptidase inhibition.

Prolyl endopeptidase (PEP) is a proline-specific oligopeptidase with a reported effect on learning and memory in different rat model systems. Using the astroglioma cell line U343, PEP expression was reduced by an antisense technique. Measuring different second-messenger concentrations revealed an inverse correlation between inositol 1,4,5-triphosphate [Ins(1,4,5)P3] concentration and PEP expression in the generated antisense cell lines. However, no effect on cAMP generation was observed. In addition, complete suppression of PEP activity by the specific inhibitor, Fmoc-Ala-Pyrr-CN (5 micro m) induced in U343 and other cell lines an enhanced, but delayed, increase in Ins(1,4,5)P3 concentration. This indicates that the proteolytic activity of PEP is responsible for the observed effect. Furthermore, the reduced PEP activity was found to amplify Substance P-mediated stimulation of Ins(1,4,5)P3. The effect of reduced PEP activity on second-messenger concentration indicates a novel intracellular function of this peptidase, which may have an impact on the reported cognitive enhancements due to PEP inhibition.