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International audience; A recent decline in the discovery of novel medications challenges the widespread use of 2D monolayer cell assays in the drug discovery process. As a result, the need for more appropriate cellular models of human physiology and disease has renewed the interest in spheroid 3D culture as a pertinent model for drug screening. However, despite technological progress that has significantly simplified spheroid production and analysis, the seeming complexity of the 3D approach has delayed its adoption in many laboratories. The present report demonstrates that the use of a spheroid model may be straightforward and can provide information that is not directly available with a standard 2D approach. We describe a cost-efficient method that allows for the production of an array of uniform spheroids, their staining with vital dyes, real-time monitoring of drug effects, and an ATP-endpoint assay, all in the same 96-well U-bottom plate. To demonstrate the method performance, we analyzed the effect of the preclinical anticancer drug MLN4924 on spheroids formed by VCaP and LNCaP prostate cancer cells. The drug has different outcomes in these cell lines, varying from cell cycle arrest and protective dormancy to senescence and apoptosis. We demonstrate that by using high-content analysis of spheroid arrays, the effect of the drug can be described as a series of EC50 values that clearly dissect the cytostatic and cytotoxic drug actions. The method was further evaluated using four standard cancer chemotherapeutics with different mechanisms of action, and the effect of each drug is described as a unique multi-EC50 diagram. Once fully validated in a wider range of conditions, this method could be particularly valuable for phenotype-based drug discovery.

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/β-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.

Background Photosensitizers are used in photodynamic therapy (PDT) to destruct tumor cells, however, their limited solubility and specificity hampers routine use, which may be overcome by encapsulation. Several promising novel nanoparticulate drug carriers including liposomes, polymeric nanoparticles, metallic nanoparticles and lipid nanocomposites have been developed. However, many of them contain components that would not meet safety standards of regulatory bodies and due to difficulties of the manufacturing processes, reproducibility and scale up procedures these drugs may eventually not reach the clinics. Recently, we have designed a novel lipid nanostructured carrier, namely Lipidots, consisting of nontoxic and FDA approved ingredients as promising vehicle for the approved photosensitizer m-tetrahydroxyphenylchlorin (mTHPC). Results In this study we tested Lipidots of two different sizes (50 and 120 nm) and assessed their photodynamic potential in 3-dimensional multicellular cancer spheroids. Microscopically, the intracellular accumulation kinetics of mTHPC were retarded after encapsulation. However, after activation mTHPC entrapped into 50 nm particles destroyed cancer spheroids as efficiently as the free drug. Cell death and gene expression studies provide evidence that encapsulation may lead to different cell killing modes in PDT. Conclusions Since ATP viability assays showed that the carriers were nontoxic and that encapsulation reduced dark toxicity of mTHPC we conclude that our 50 nm photosensitizer carriers may be beneficial for clinical PDT applications. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0221-x) contains supplementary material, which is available to authorized users.

Considering toxicity requirements for clinical translation of fluorescence imaging applications, the use of biocompatible carriers for designing near infrared emitting contrast agents appears as an attractive alternative to semiconductor nanocrystals. Lipid nanoparticles (LNP) have been designed to serve as carriers for indocyanine green (ICG), the presently only human-use approved near infrared dye. The cytotoxicity and hemocompatibility of these nanoparticle-based probes are determined in vitro, respectively in mouse 3T3 fibroblasts and human blood samples. Comparative biodistribution of free ICG and ICG-LNP in mice is monitored, and an ex vivo fluorescence organ quantification is performed considering large animal cohorts. Good tolerability and very low hemolytic activity are demonstrated for naked and ICG-loaded LNP. Interestingly, ICG-LNP lead to long-term plasma fluorescence (> 24 hours) but also a partial intestinal reabsorption of ICG between 5 and 24 hours after injection. This novel ICG nanoformulation is foreseen to expand rapidly the field of clinical fluorescence imaging applications.

Owing to its simplicity, lensless imaging system is adept at continuous monitoring of adherent cells inside the incubator. The setup consists of a CMOS sensor with pixel pitch of 2.2 μm and field of view of 24 mm2, LED with a dominating wavelength of 525 nm, along with a pinhole of 150 μm as the source of illumination. The in-line hologram obtained from cells depends on the degree of cell-substrate adhesion. Drastic difference is observed between the holographic patterns of floating and adherent cells. In addition, the well-established fact of reduction of cell-substrate contact during cell division is observed with our system based on corresponding spontaneous transition in the holographic pattern. Here, we demonstrate that by recognizing this specific holographic pattern, number of cells undergoing mitosis in a cell culture with a population of approximately 5000 cells, can be estimated in real-time. The method is assessed on comparison with Edu-based proliferation assay. The approach is straightforward and it eliminates the use of markers to estimate the proliferation rate of a given cell culture. Unlike most proliferation assays, the cells are not harvested enabling continuous monitoring of cell culture.

International audience; The use of fluorescent nanostructures can bring several benefits on the signal to background ratio for in vitro microscopy, in vivo small animal imaging, and image-guided surgery. Fluorescent quantum dots (QDs) display outstanding optical properties, with high brightness and low photobleaching rate. However, because of their toxic element core composition and their potential long term retention in reticulo-endothelial organs such as liver, their in vivo human applications seem compromised. The development of new dye-loaded (DiO, DiI, DiD, DiR, and Indocyanine Green (ICG)) lipid nanoparticles for fluorescence imaging (lipidots) is described here. Lipidot optical properties quantitatively compete with those of commercial QDs (QTracker(®)705). Multichannel in vivo imaging of lymph nodes in mice is demonstrated for doses as low as 2 pmols of particles. Along with their optical properties, fluorescent lipidots display very low cytotoxicity (IC(50) > 75 nM), which make them suitable tools for in vitro, and especially in vivo, fluorescence imaging applications.

International audience; When surface-supported chemical reactions are performed in microsystems, high production rate cannot be obtained due to the intrinsically low surface area. Increasing the active surface area of silicon microsystems is a challenge that is addressed in this paper using two original approaches: (i) modifying the structure of silicon by creating nanostructures (black silicon) using conventional etching processes of silicon micromachining or (ii) depositing a layer of porous _-alumina by washcoating a colloidal suspension of boehmite onto the silicon surface. The catalytic oxidation of carbon monoxide on platinum was chosen as a first test reaction in the domain of heterogeneous catalysis. In order to perform this reaction, platinum was either deposited by sputtering on silicon devices with black silicon nanostructuration, or impregnated inside the porosity of an alumina layer previously deposited on a silicon device. For specific biological applications, such as proteins analysis, some biological reactions could be advantageously achieved in microsystems using surface-supported species. As an example, an enzymatic reaction was carried out usingsilicon devices modified with black silicon nanostructuration and further functionalized with trypsin as a model enzyme. The catalytic activity was compared between silicon devices with the same two types of catalysts, comprising or not an enhancement of the surface activity. A minimum 10- fold increase in catalytic activity was estimated from kinetic measurements and represents the augmentation of the active surface really available for reactions. It was also shown how these catalytic materials were integrated in a microreactor increasing its catalytic active surface without modifying its global size.

Enzymatic digestion and peptide separation are basic steps for preparation of protein samples prior to their analysis by mass spectrometry. Micro-pillar reactors for digestion and reverse phase liquid chromatography were designed and constructed using semiconductor technologies. The performances of the micro-machined reactors were evaluated: complete Cytochrome C digestion was achieved in 6 min for a concentration up to 25 pmol.μl -1 and the separation micro-column was seen to exhibit separation capabilities and capacity close to those obtained with a commercial column. Furthermore, a comparative study between hydrodynamic and electroosmotic driven flows was performed for each peptide of a Cytochrome C digest. It was demonstrated that parasitic electrophoretic phenomena disturbed peptide mobility but not protein identification.

Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D-a cell proliferation inhibitor.

International audience; A new contrast agent, LipImage™ 815, has been designed and compared to previously described indocyanine green (ICG)-loaded lipid nanoparticles (ICG-lipidots®). Both contrast agents display similar size (50-nm diameter), zeta potential, high IC50 in cellular studies, near-infrared absorption and emission wavelengths in the "imaging window," long-term shelf colloidal and optical stabilities with high brightness (>106 L mol-1 cm-1) in ready-to-use storage conditions in aqueous buffer (4°C in dark), therefore being promising fluorescence contrast agents for in vivo imaging. However, while ICG-lipidots® display a relatively short plasma lifetime, LipImage™ 815 circulates in blood for longer times, allowing the efficient uptake of fluorescence signal in human prostate cancer cells implanted in mice. Prolonged tumor labeling is observed for more than 21 days.