Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases.

BACKGROUND AND AIMS: Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. METHODS: We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib. RESULTS: siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection. CONCLUSIONS: The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases.

Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens.

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.

Distinct outcomes of CRL-Nedd8 pathway inhibition reveal cancer cell plasticity.

Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/ß-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR-ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR-ERG interaction.

Label-Free Electric Monitoring of Human Cancer Cells as a Potential Diagnostic Tool.

Dielectrophoresis is widely used for cell characterization, and the exerted force on cells depends on the difference of polarizability between the latter and the surrounding medium. This physical phenomenon is translated by the real part of the Clausius-Mossotti factor. It is mostly modeled from the imaginary part, measured by electrorotation. The method described here measures experimentally the real part of the Clausius-Mossotti factor. It relies on the cell velocity when submitted to pure dielectrophoresis, and it was conducted on several human cell lines, at different times. A variety of cell lines was evaluated, from different organs or representative of different stages of cancer, with promising findings for early cancer detection.

Involvement of membrane proteins and ion channels on the self-rotation of human cells in a non-rotating AC electric field.

Dielectrophoresis is a force that has been exploited in microsystems for label-free characterization and separation of cells, when their electrical signature is known. However, the polarization effect of cells at the transmembrane protein level is not well established. In this work, we have use the self-rotation effect of cells in a non-rotating field, known as the "Quincke effect," in order to measure the maximum rotation frequency (frotmax ) of different cell populations when modifying the composition of their membrane. We investigated the influence of active ionic transportation of membrane protein concentration on frotmax of HEK cells. Our results show that ionic transportation is responsible for the reduction of conductivity within the cytoplasm, which results in higher frotmax . However, the influence of the concentration of proteins in the membrane, achieved by silencing gene expression in cancer cells, changes significantly frotmax , which is not explained by the changes of ionic conductivity within the cell.

Facile bench-top fabrication of enclosed circular microchannels provides 3D confined structure for growth of prostate epithelial cells.

We present a simple bench-top method to fabricate enclosed circular channels for biological experiments. Fabricating the channels takes less than 2 hours by using glass capillaries of various diameters (from 100 µm up to 400 µm) as a mould in PDMS. The inner surface of microchannels prepared in this way was coated with a thin membrane of either Matrigel or a layer-by-layer polyelectrolyte to control cellular adhesion. The microchannels were then used as scaffolds for 3D-confined epithelial cell culture. To show that our device can be used with several epithelial cell types from exocrine glandular tissues, we performed our biological studies on adherent epithelial prostate cells (non-malignant RWPE-1 and invasive PC3) and also on breast (non-malignant MCF10A) cells We observed that in static conditions cells adhere and proliferate to form a confluent layer in channels of 150 µm in diameter and larger, whereas cellular viability decreases with decreasing diameter of the channel. Matrigel and PSS (poly (sodium 4-styrenesulphonate)) promote cell adhesion, whereas the cell proliferation rate was reduced on the PAH (poly (allylamine hydrochloride))-terminated surface. Moreover infusing channels with a continuous flow did not induce any cellular detachment. Our system is designed to simply grow cells in a microchannel structure and could be easily fabricated in any biological laboratory. It offers opportunities to grow epithelial cells that support the formation of a light. This system could be eventually used, for example, to collect cellular secretions, or study cell responses to graduated hypoxia conditions, to chemicals (drugs, siRNA, …) and/or physiological shear stress.

Comprehensive analysis of human cells motion under an irrotational AC electric field in an electro-microfluidic chip.

AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines.

The modulation of attachment, growth and morphology of cancerous prostate cells by polyelectrolyte nanofilms.

The behaviour of cancerous epithelial prostatic cells (PC3) growing on polyelectrolytes (PE) coatings was compared to the behaviour of immortalized normal prostatic cells (PNT-2). The cell behaviour was evaluated and quantified in terms of initial cell attachment, growth, metabolic activity, morphometry, adhesion, apoptosis and stress related gene expression. Both the anionic PSS (poly(sodium 4-styrenesulphonate))-terminated surface and cationic PAH (poly(allylamine hydrochloride))-terminated surfaces were not cytotoxic. The initial attachment of cells was better on the PAH-terminated surface compared to fibronectin. However, the proliferation rate of PC3 cells was reduced on the PAH-terminated surface and slightly increased on the PSS coatings. Only PAH prevented the clustering phenotype of PC3 and reduced the number of focal adhesion points as compared to fibronectin or PSS coatings. In contrast, none of the PE surfaces significantly affected the biological responses of PNT-2 cells. PAH-terminating films provide a tool to preferentially modulate the growth of some cancerous phenotypes, in this case as a micro-environment that reduces the growth of metastatic PC3 cells.

Label-free analysis of prostate acini-like 3D structures by lensfree imaging.

We present a lensfree imaging method to analyze polarity in RWPE1 prostate epithelial cells that form polarized acini with lumen under standard tridimensional (3D) culture conditions. The first event in epithelial carcinogenesis is loss of polarity, followed by uncontrolled proliferation leading to metastasis. We demonstrate that it is possible to use optical signatures to discriminate 3D objects with distinct polarities in a large field of view. The three metrics we present here are designed as image processing tools to discriminate acini from spheroids without any 3D reconstruction. To demonstrate that our lensfree imaging platform may be used to study the 3D organization of epithelial cells, we analyzed and quantified the modulation of dynamic processes, e.g., the polarity of acini and the merging of polarized structures, upon transforming growth factor beta-1 (TGF beta-1) addition to the culture media. Hence, coupling lensfree microscopy with 3D cell culture provides an innovative tool to study epithelial tissue morphogenesis in a large field of view and to elucidate the regulation of growth, morphogenesis and differentiation in normal and cancerous human prostate cells. Moreover, such biosensor would be a powerful tool to follow cancer progression and to evaluate anti-cancer drugs.

The miR-17 family links p63 protein to MAPK signaling to promote the onset of human keratinocyte differentiation.

The p63 protein plays a key role in regulating human keratinocyte proliferation and differentiation. Although some p63-regulating microRNAs (miRNAs) have been identified in the control of epidermal homeostasis, little is known about miRNAs acting downstream of p63. In this paper, we characterized multiple p63-regulated miRNAs (miR-17, miR-20b, miR-30a, miR-106a, miR-143 and miR-455-3p) and elucidated their roles in the onset of keratinocyte differentiation. We identified RB, p21 and multiple MAPKs as targets of these p63-controlled miRNAs. Upon inhibition of most of these miRNAs, we observed defects in commitment to differentiation that could be reversed by siRNA-mediated silencing of their targets. Furthermore, knockdown of MAPK8 and MAPK9 efficiently restored expression of the early differentiation markers keratin 1 and keratin 10 in p63-silenced primary human keratinocytes. These results highlight new mechanistic roles of multiple miRNAs, particularly the miR-17 family (miR-17, miR-20b and miR-106a), as regulatory intermediates for coordinating p63 with MAPK signaling in the commitment of human mature keratinocytes to early differentiation.

A miniaturized planar patch-clamp system for transportable use.

In the last decade, planar patch-clamp (PPC) has emerged as an innovative technology allowing parallel recordings of cellular electrophysiological activity on planar substrates. If PPC is widely adopted by the pharmaceutical sector, it remains poorly extended to other areas (i.e. environment and safety organizations) probably because of the large, expensive and non-easily transportable format of those commercial equipments. The present work describes for the first time a new compact and transportable planar patch-clamp system (named Toxint'patch or TIP, for Toxin detection with integrated patch-clamp) focusing on environmental matters and meant to be used in coastal laboratories, for direct on-site monitoring of the seawater and shellfish quality. The TIP system incorporates silicon chips tailored to monitor cellular ionic currents from cultured cells stably expressing a phycotoxin molecular target. The functionality of this novel briefcase-sized PPC system is described in terms of fluidic control, electronic performances with amplifying and filtering boards and of user interface for data acquisition and control implemented on a computer.

The development of high quality seals for silicon patch-clamp chips.

Planar patch-clamp is a two-dimensional variation of traditional patch-clamp. By contrast to classical glass micropipette, the seal quality of silicon patch-clamp chips (i.e. seal resistance and seal success rate) have remained poor due to the planar geometry and the nature of the substrate and thus partially obliterate the advantages related to planar patch-clamp. The characterization of physical parameters involved in seal formation is thus of major interest. In this paper, we demonstrate that the physical characterization of surfaces by a set of techniques (Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), surface energy (polar and dispersive contributions), drop angles, impedance spectroscopy, combined with a statistical design of experiments (DOE)) allowed us discriminating chips that provide relevant performances for planar patch-clamp analysis. Analyses of seal quality demonstrate that dispersive interactions and micropore size are the most crucial physical parameters of chip surfaces, by contrast to surface roughness and dielectric membrane thickness. This multi-scale study combined with electrophysiological validation of chips on a diverse set of cell-types expressing various ion channels (IRK1, hERG and hNa(v)1.5 channels) unveiled a suitable patch-clamp chip candidate. This original approach may inspire novel strategies for selecting appropriate surface parameters dedicated to biochips.

A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles.

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.