YAP/TEAD involvement in resistance to paclitaxel chemotherapy in lung cancer.

The Yes-associated protein (YAP) oncoprotein has been linked to both metastases and resistance to targeted therapy of lung cancer cells. We aimed to investigate the effect of YAP pharmacological inhibition, using YAP/TEA domain (TEAD) transcription factor interaction inhibitors in chemo-resistant lung cancer cells. YAP subcellular localization, as a readout for YAP activation, cell migration, and TEAD transcription factor functional transcriptional activity were investigated in cancer cell lines with up-regulated YAP, with and without YAP/TEAD interaction inhibitors. Parental (A549) and paclitaxel-resistant (A549R) cell transcriptomes were analyzed. The half-maximal inhibitory concentration (IC50) of paclitaxel or trametinib, which are Mitogen-Activated protein kinase and Erk Kinase (MEK) inhibitors, combined with a YAP/TEAD inhibitor (IV#6), was determined. A three-dimensional (3D) microfluidic culture device enabled us to study the effect of IV#6/paclitaxel combination on cancer cells isolated from fresh resected lung cancer samples. YAP activity was significantly higher in paclitaxel-resistant cell lines. The YAP/TEAD inhibitor induced a decreased YAP activity in A549, PC9, and H2052 cells, with reduced YAP nuclear staining. Wound healing assays upon YAP inhibition revealed impaired cell motility of lung cancer A549 and mesothelioma H2052 cells. Combining YAP pharmacological inhibition with trametinib in K-Ras mutated A549 cells recapitulated synthetic lethality, thereby sensitizing these cells to MEK inhibition. The YAP/TEAD inhibitor lowered the IC50 of paclitaxel in A549R cells. Differential transcriptomic analysis of parental and A549R cells revealed an increased YAP/TEAD transcriptomic signature in resistant cells, downregulated upon YAP inhibition. The YAP/TEAD inhibitor restored paclitaxel sensitivity of A549R cells cultured in a 3D microfluidic system, with lung cancer cells from a fresh tumor efficiently killed by YAP/TEAD inhibitor/paclitaxel doublet. Evidence of the YAP/TEAD transcriptional program's role in chemotherapy resistance paves the way for YAP therapeutic targeting.

Flowmetering for microfluidics.

Originally designed for chromatography, electrophoresis, and printing technologies, microfluidics has since found applications in a variety of domains such as engineering, chemistry, environmental, and life sciences. The fundamental reason for this expansion has been the development of miniature components, allowing the handling of liquids at the microscale. For the maturation of microfluidic technologies, the need for affordable, reliable, and quantitative techniques to measure flow rates from 1 nL min-1 to 1 mL min-1 appears as a strong challenge. We review herein the different technologies available and those under development, and discuss their sensing principles and industrial maturity. Given the need of traceability of these measurements, we then focus on the developments of primary standards to measure microfluidic flow rates by metrological institutes. We conclude this review with some perspectives and pending challenges for microfluidic flowmeters.

Controlling the distance of highly confined droplets in a capillary by interfacial tension for merging on-demand.

Droplet microfluidics is a powerful technology that finds many applications in chemistry and biomedicine. Among different configurations, droplets confined in a capillary (or plugs) present a number of advantages: they allow positional identification and simplify the integration of complex multi-steps protocols. However, these protocols rely on the control of droplet speed, which is affected by a complex and still debated interplay of various physico-chemical parameters like droplet length, viscosity ratio between droplets and carrier fluid, flow rate and interfacial tension. We present here a systematic investigation of the droplet speed as a function of their length and interfacial tension, and propose a novel, simple and robust methodology to control the relative distance between consecutive droplets flowing in microfluidic channels through the addition of surfactants either into the dispersed and/or into the continuous phases. As a proof of concept application, we present the possibility to accurately trigger in space and time the merging of two confined droplets flowing in a uniform cross-section circular capillary. This approach is further validated by monitoring a conventional enzymatic reaction used to quantify the concentration of H2O2 in a biological sample, showing its potentialities in both continuous and stopped assay methods.

On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation.

Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.

The power of solid supports in multiphase and droplet-based microfluidics: towards clinical applications.

Multiphase and droplet microfluidic systems are growing in relevance in bioanalytical-related fields, especially due to the increased sensitivity, faster reaction times and lower sample/reagent consumption of many of its derived bioassays. Often applied to homogeneous (liquid/liquid) reactions, innovative strategies for the implementation of heterogeneous (typically solid/liquid) processes have recently been proposed. These involve, for example, the extraction and purification of target analytes from complex matrices or the implementation of multi-step protocols requiring efficient washing steps. To achieve this, solid supports such as functionalized particles (micro or nanometric) presenting different physical properties (e.g. magnetic, optical or others) are used for the binding of specific entities. The manipulation of such supports with different microfluidic principles has both led to the miniaturization of existing biomedical protocols and the development of completely new strategies for diagnostics and research. In this review, multiphase and droplet-based microfluidic systems using solid suspensions are presented and discussed with a particular focus on: i) working principles and technological developments of the manipulation strategies and ii) applications, critically discussing the level of maturity of these systems, which can range from initial proofs of concept to real clinical validations.

A simple and low-cost chip bonding solution for high pressure, high temperature and biological applications.

The sealing of microfluidic devices remains a complex and time-consuming process requiring specific equipment and protocols: a universal method is thus highly desirable. We propose here the use of a commercially available sealing tape as a robust, versatile, reversible solution, compatible with cell and molecular biology protocols, and requiring only the application of manually achievable pressures. The performance of the seal was tested with regards to the most commonly used chip materials. For most materials, the bonding resisted 5 bars at room temperature and 1 bar at 95 °C. This method should find numerous uses, ranging from fast prototyping in the laboratory to implementation in low technology environments or industrial production.

Continuous chemical operations and modifications on magnetic γ-Fe2O3 nanoparticles confined in nanoliter droplets for the assembly of fluorescent and magnetic SiO2@γ-Fe2O3.

We present a microfluidic platform that allows undergoing different chemical operations in a nanoliter droplet starting from the colloidal suspension of magnetic iron oxide (γ-Fe2O3) nanoparticles "NPs" (ferrofluid). These operations include: mixing, flocculation, magnetic decantation, colloidal redispersion, washing, surface functionalization, heating and colloidal assembly. To prove the platform capabilities, we produced fluorescent and magnetic nanoassemblies composed of fluorescent silica and magnetic NPs.