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Smart microgels (μGels) made of polymeric particles doped with inorganic nanoparticles have emerged recently as promising multifunctional materials for nanomedicine applications. However, the synthesis of these hybrid materials is still a challenging task with the necessity to control several features, such as particle sizes and doping levels, in order to tailor their final properties in relation to the targeted application. We report herein an innovative modular strategy to achieve the rational design of well-defined and densely filled hybrid particles. It is based on the assembly of the different building blocks, i.e., μGels, dyes, and small gold nanoparticles (4 nm), and the tuning of nanoparticle loading within the polymer matrix through successive incubation steps. The characterization of the final hybrid networks using UV-vis absorption, fluorescence, transmission electron microscopy, dynamic light scattering, and small-angle X-ray scattering revealed that they uniquely combine the properties of hydrogel particles, including high loading capacity and stimuli-responsive behavior, the photoluminescent properties of dyes (rhodamine 6G, methylene blue and cyanine 7.5), and the features of gold nanoparticle assembly. Interestingly, in response to pH and temperature stimuli, the smart hybrid μGels can shrink, leading to the aggregation of the gold nanoparticles trapped inside the polymer matrix. This stimuli-responsive behavior results in plasmon band broadening and red shift toward the near-infrared region (NIR), opening promising prospects in biomedical science. Particularly, the potential of these smart hybrid nanoplatforms for photoactivated hyperthermia, photoacoustic imaging, cellular internalization, intracellular imaging, and photothermal therapy was assessed, demonstrating well controlled multimodal opportunities for theranostics.

Supplemental Materials. Supplemental Table 1: Patient mutational status and agreement between qPCR, dPCR and sequencing results. Supplemental Table 2: Analysis of different tissue samples coming from the same patient. Supplemental Figure 1: Fraction of mutated alleles (including multiple subclones) in the patients detected by both conventional and droplet-digital PCR procedures. Supplemental Figure 2: Fraction of mutated DNA observed for the different tested mutations of KRAS and BRAF.

Purpose:KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis.Experimental Design: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction.Results: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors.Conclusions: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies. Clin Cancer Res; 21(5); 1087–97. ©2014 AACR.

The development of anti-counterfeiting inks based on surface-enhanced Raman scattering (SERS) labels have attracted great interest in recent years for their use as security labels in anti-counterfeiting applications. Indeed, they are promising alternatives to luminescent inks, which suffer from several limitations including emission peak overlap, toxicity and photobleaching. Most of the reported SERS security labels developed so far rely on the use of thiolate self-assembled monolayers (SAMs) for the immobilization of Raman reporters on metallic nanoparticle surface. However, SAMs are prone to spontaneous desorption and degradation under laser irradiation, thereby compromising the ink long-term stability. To overcome this issue, we develop herein a new generation of SERS security labels based on silver nanoparticles (Ag NPs) functionalized by aryl diazonium salts, carrying various substituents (-NO

Surface-enhanced Raman spectroscopy (SERS) is an emerging powerful vibrational technique offering unprecedented opportunities in biomedical science for the sensitive detection of biomarkers and the imaging and tracking of biological samples. Conventional SERS detection is based on the use of plasmonic substrates (e.g., Au and Ag nanostructures), which exhibit very high enhancement factors (EF = 10

International audience; The surface functionalization of silver nanoparticles (NPs) by Raman reporters has stimulated a wide interest in recent years for the design of Surface-Enhanced Raman Spectroscopy (SERS) labels. However, silver NPs are prone to oxidation and aggregation, which strongly limits their applications. The design of stable SERS tags based on Ag NPs still represents a major challenge for Raman bioimaging. We address this issue herein by taking advantage of aryl diazonium salt chemistry to obtain stable Ag NPs functionalized by multifunctional polyaryl layers bearing different Raman reporters (-NO2,-CN,-CCH). The resulting SERS-encoded Ag NPs were characterized by UV-vis absorption, transmission electron microscopy (TEM) and SERS. The formation of multilayers at the surface of Ag NPs gives access to new spectrally distinguishable SERS codes thus broadening the library of available Raman tags. Proof-of-concept Raman imaging experiments were performed on cancer cells (HeLa) after NP uptake, highlighting the large potentials of diazonium salt chemistry to design Ag NPsbased SERS labels for Raman bioimaging.

Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

International audience; Nowadays, microfluidic 3D cell culture is widely used to mimic complex microtissue and dynamic environment, performing more realistic in vitro assays for drug testing. Herein, we developed a novel microfluidic platform for tumor microtissue culture, drug response analysis and versatile microscopic characterization. By reversibly bonding the chip, we go beyond the on/off chip tradeoff, which allows us to perform both fluorescence and SEM characterization of tumor microtissues on a simple platform. The microfluidic chip consists of spherical microwells connected via microchannels, bonded through a magnetic system. Colorectal cancer HT-29 cells were cultured as spherical microtissues on chip and their growth kinetics monitored. The cytotoxic activity of Camptothecin was evaluated by in situ live/dead fluorescence staining and quantification of morphology parameters. Finally, we demonstrated the possibility to collect the 3D tumor microtissues and characterize their surface damaged by the drug using scanning electron microscopy. This reversibly sealed microfluidic platform thus enables to grow sets of 3D tumor microtissues in a controlled dynamic microenvinroment, and subsequently to retrieve the 3D tumor microtissues after chemotherapeutic treatment for in-depth analysis.

Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

Bioanalysis: microfluidic-sorters to make better choices Droplet-based microfluidics provides new technologies for cell screening by steering cell-containing microdroplets of interest into different channels at the kilohertz frequency. Until now, in conventional microfluidic sorting, droplets are sorted using electric pulses to move them into one of the two channels. Jean–Christophe Baret from the CNRS and University of Bordeaux in France, Valérie Taly from the CNRS and University of Paris Descartes and Tobias Schneider from EPFL in Switzerland have now boosted dielectrophoretic droplet manipulation to allow for multiple selections in a single device. The teams show how emulsified dye-loaded microdroplets are sorted in five different channels depending on fluorescence intensity: a computer algorithm controls the application of electric pulses to direct the droplets into one of the five channels depending on the fluorescence signals. Numerical models unravel the effect of the dielectrophoretic actuation on the droplet trajectories and are now used to predict the sorting conditions for droplet volumes compatible with single-cell encapsulation.

Genetic markers are now widely used in the clinics, particularly in cancer patient management. Indeed, these tumor markers can help in the diagnosis and prognosis of the disease, and provide valuable information for treatment orientation in the context of personalized medicine. The presence of circulating cell-free tumor DNA (cftDNA) and thus of tumor markers in the blood can be considered to partly avoid the use of solid biopsies. The use of blood samples, as liquid biopsies, is less invasive and described as more representative of tumor heterogeneity. However, cftDNA can be found in blood in low proportion that can vary according to the nature and the progression of the tumor. For these reasons, the use of highly sensitive, specific and ideally quantitative methods for its detection are required. These requirements constituted until recently a technological limit, which now can be overcome thanks to digital PCR. This technology could now become a very efficient and non-invasive tool in oncology, complementary to conventional diagnostic techniques.

The BRAF

BACKGROUNDCirculating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored.METHODSWe analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up.RESULTSHypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution.CONCLUSIONSThese results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

International audience; Most cell studies are performed at a population level, relying on the assumption of a normal distribution of the function and fate of a cell among a population. However, technologies allowing single-cell analysis (SCA) have recently arisen and have led to increasing evidence of cell population heterogeneity and its importance. Tremendous amounts of new data could now be uncovered to redefine our understanding of cell omics. Microfluidics has emerged as a major technological player in this new era and is gradually increasing in use among biology laboratories, mainly due to the single-cell high-throughput handling solutions it offers. In this review, we assess its use and relevance for omics analysis at the single-cell level, with a specific focus on compartment-based microfluidic approaches.

International audience; Clusterin is a usually secreted glycoprotein with chaperone properties. Recently, it has been suggested that clusterin isoforms reside in the nuclear and cytosolic compartments of human cell types, where they can influence various cellular programs including DNA repair, transcription and apoptosis. Several mechanisms have been proposed to explain this atypical location, including alternative transcription initiation and alternative splicing. However, none of these have been unequivocally established as occurring in live cells. Here we provide direct experimental evidence that in live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)-associated protein degradation pathway and involves passage through the Golgi apparatus. We also report that the ER-associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin. The possible biological functions of these novel behaviours of clusterin are discussed.

Genetic markers are now widely used in the clinics, particularly in cancer patient management. Indeed, these tumor markers can help in the diagnosis and prognosis of the disease, and provide valuable information for treatment orientation in the context of personalized medicine. The presence of circulating cell-free tumor DNA (cftDNA) and thus of tumor markers in the blood can be considered to partly avoid the use of solid biopsies. The use of blood samples, as liquid biopsies, is less invasive and described as more representative of tumor heterogeneity. However, cftDNA can be found in blood in low proportion that can vary according to the nature and the progression of the tumor. For these reasons, the use of highly sensitive, specific and ideally quantitative methods for its detection are required. These requirements constituted until recently a technological limit, which now can be overcome thanks to digital PCR. This technology could now become a very efficient and non-invasive tool in oncology, complementary to conventional diagnostic techniques.

Purpose: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis. Experimental Design: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction. Results: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors. Conclusions: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies. Clin Cancer Res; 21(5); 1087–97. ©2014 AACR.

La PCR (reaction de polymerisation en chaine) digitale represente un saut technologique dans la detection et la quantification de sequences rares d'ADN, comme certaines mutations specifiques des tumeurs. La detection de ces sequences est d'un grand interet pour la prise en charge clinique des patients. En effet, leur detection dans les tumeurs ou les fluides biologiques, comme le plasma ou l'urine, pourrait permettre d'adapter le traitement aux caracteristiques moleculaires precises de la tumeur et de limiter les risques de recidive. Apres une rapide presentation du contexte des recherches, l'article se penche sur le cancer et ses biomarqueurs, afin d'enchainer sur l'emergence de la PCR digitale, et enfin explore les perspectives et potentielles evolutions de la PCR digitale.

Polymerase chain reaction based techniques have been widely used in laboratory settings. Several applications in oncology, virology or prenatal diagnosis require highly sensitive detection methods, which cannot be achieved with conventional techniques. Digital PCR (dPCR) was developed from the association of PCR and limiting dilution procedures. It is based on the compartmentalization of DNA molecules in small volumes. Controlling the size and the content of each compartment is crucial to obtain a high sensitivity with a single molecule resolution. Microfluidics offers promising tools to isolate DNA fragments such as microdroplets, microchambers or microwells with volumes ranging from few picoliters to nanoliters. The review provides an overview of recent developments of microfluidics dPCR platforms and how this technology can influence the management of cancer patients.

International audience; Many methods have been developed for DNA integrity assessment including electrophoresis-based procedures, quantitative PCR, and, more recently, microfluidics-based procedures. DNA integrity evaluation can be employed for characterizing biological samples quality before extensive genomic analysis and also finds applications in reproductive medicine, prenatal diagnostics, or cancer research. In this chapter, we will focus on the assessment of DNA integrity in cancer research. In particular, we will present the application of the determination of DNA integrity for tracking of circulating tumor DNA. Finally, we will conclude by illustrating the potential innovative application of DNA integrity as a biomarker in clinical research, especially for prognostic purposes, patient follow-up, or early diagnosis.

International audience; Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides.

The authors have investigated a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This method may offer an alternative to gene therapy approaches. It consists in anchoring recombinant cytokines to the cell membrane. Attachment is mediated by the transmembrane domain of diphtheria toxin (T) genetically fused to the cytokine and is triggered by an acid pH pulse. The authors found that the fusion protein T-hIL-2 anchored to the surface of tumor cells retained its IL-2 activity while remaining exposed for several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective antitumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared with the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and the selection of transfected clones, it eliminates the safety problems connected with viral vectors, and it allows the control of the amount of cytokines delivered with the vaccine.

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.

Among bacterial protein toxins with an intracellular target, diphtheria toxin is one of the most studied. Since the first publication of its crystal structure in 1992, tremendous progress has been made describing the molecular events involved in its toxicity. However, the precise mechanism of translocation is not fully understood yet. The diphtheria toxin contains three structural domains, each carrying a distinct biological function implicated in the intoxication of the cell. The receptor-binding domain mediates the recognition of a specific receptor on the surface of targeted cells. This binding event allows the internalization of the toxin by the cells and its routing towards acidic intracellular compartments. The translocation (or transmembrane) domain, reacting to the low pH, penetrates the membrane and assists the transport of the catalytic domain through this membrane into the cytoplasm. There, the catalytic domain transfers an ADP-ribose from cytosolic NAD to its substrate, the elongation factor 2...

International audience; Erdheim-Chester disease (ECD) is a rare histiocytic disorder that is challenging to diagnose and treat. We performed molecular analysis of BRAF in the largest cohort of ECD patients studied to date followed by N/KRAS, PIK3CA, and AKT1 mutational analysis in BRAF wild-type patients. Forty-six of 80 (57.5%) of patients were BRAFV600E-mutant. NRAS mutations were detected in 3 of 17 ECD BRAFV600E wild-type patients. PIK3CA mutations (p.E542K, p.E545K, p.A1046T, and p.H1047R) were detected in 7 of 55 patients, 4 of whom also had BRAF mutations. Mutant NRAS was present in peripheral blood CD14(+) cells, but not lymphoid cells, from an NRASQ61R mutant patient. Our results underscore the central role of RAS-RAF-MEK-ERK activation in ECD and identify an important role of activation of RAS-PI3K-AKT signaling in ECD. These results provide a rationale for targeting mutant RAS or PI3K/AKT/mTOR signaling in the subset of ECD patients with NRAS or PIK3CA mutations.

Introduction Myelodysplastic syndromes (MDS) are phenotypically and genotypically heterogeneous diseases with several driver mutations, which are closely related to patient prognosis. Dynamic evolution of mutations reflects the selection of subclones during disease evolution until transformation in secondary acute myeloid leukemia. Thanks to increasing knowledge in gene functions, target drugs are now available in therapeutic. However, questions remain on the impact of such treatments on malignant cells. We have previously investigated the effects of lenalidomide on clonal evolution, by monitoring variant allele frequencies (VAF) using next generation sequencing (NGS) in non-del5q MDS patients (Chesnais et al, Blood 2015). Here, we present a rapid and ultra-sensitive method using picoliter-droplet digital PCR for mutation detection in MDS with ring sideroblasts (RS). Materials and Methods Bone marrow aspirates were obtained from MDS patients included at diagnosis in a multicentric observational trial (PHRC MDS-04, NCT02619565). Three cell lines (HL60, OCI-AML3, UKE-1) were also used to establish the specificity and the sensitivity of assays. Both frozen living cells and extracted DNA were used. Selected samples were screened for mutations in 39 genes by an NGS approach using a Personal Genome Machine® (PGM, ThermoFisher Scientific, Waltham, MA, USA). Primers and probes were designed for Taqman assays based on allelic discrimination of recurrent mutations found in DNMT3A, SF3B1, JAK2 and NRAS genes. For the detection of SF3B1 p.K700E mutation, 3 locked nucleic acids were notably added to the probes to improve specificity. Picoliter-droplet digital PCR was performed on RainDrop® Digital PCR System (RainDance™ Technologies). Results Allelic discrimination assays were validated on genomic DNA extracted from cell lines and patient samples harboring or not targeted mutations using the RainDance system. About 5.106 droplets were generated using RainDrop Source. Wild-type (WT) DNA was tested in order to assess false positive signals for each design, characterized by λFP (mean number of false positive signals), limit of blanck (LOB) and limit of detection (LOD) for all experiments. The limit of blanck (LOB) defined here the highest number of droplets corresponding to apparent droplets containing mutated amplicons while testing wild type DNA. The limit of detection (LOD) was the lower number of droplets which can be distinguish from LOB while testing DNA with very low concentration of mutant genome. All the designed assays were also strongly approved for linearity using mixtures of mutated and WT DNA from cell lines (0.01% to 100% mutated allele frequency). Specificity, linearity and sensibility of the selected assays were validated on genomic DNA. Later on, we investigate genomic DNA of 3 MDS patients with RS and harboring JAK2 and SF3B1 mutations. For these patients, we obtained comparable results using both NGS and picoliter-droplet digital PCR in term of mutant allele burden quantification. Moreover, a triplex assay allowing mutant allele discrimination in JAK2 and SF3B1 genes was established on these patients. Further analyses were conducted on living cells harboring JAK2 or NRAS mutations. This approach was first conducted using a "home made" microfluidic system based on the detection of fluorescent probes in living cells encapsulated into agarose beeds. We obtained specific fluorescent signals corresponding to the genotypes. In parallel, an alternative method based on the QX100™Droplet Digital™PCR system (Biorad) also demonstrated the feasibility of allelic discrimination in living cells. Experiments based on frozen cells of MDS patients are currently under investigation. Conclusion This study is the first application of multi-target digital PCR used to detect and quantify somatic mutations recurrently found in MDS. Analyses of the clonal architecture determined on living cells and its evolution upon treatment in MDS patients with RS by this approach will help us to investigate the monitoring of the therapeutic response. Our study supports a proof of principle for further large-scale analyses of MDS patients at diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.

Il est possible de stimuler la reponse immunitaire anticancereuse a l’aide de cytokines. Pour eviter les effets toxiques lies a leur administration par voie systemique, il est necessaire de concentrer les cytokines dans le microenvironnement tumoral. Afin de pallier les inconvenients des methodes actuelles, basees sur l’introduction de genes de cytokines dans les cellules tumorales, nous proposons une nouvelle approche. Celle-ci consiste a utiliser des cytokines recombinantes auxquelles est couplee une ancre membranaire derivee de la toxine diphterique. De l’interleukine-2 ainsi ancree a des cellules de lymphome et de melanome persiste a leur surface sans etre internalisee. L’injection de ces preparations cellulaires a des souris entraine une reponse immunitaire protectrice capable d’empecher ou de ralentir le developpement de la tumeur.

We have constructed a fusion protein, T-ZZ, in which the IgG-Fc binding protein ZZ was fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). While soluble at neutral pH, T-ZZ retained the capacity of the T domain to bind to phospholipid membranes at acidic pH. Once anchored to the membrane, the ZZ part of the protein was capable of binding mouse monoclonal or rabbit polyclonal IgG. Our results show that the T-ZZ protein can function as a pH sensitive membrane anchor for the linkage of IgG to the membrane of lipid vesicles, adherent and non-adherent cells.

Background Cell proliferation is a hallmark of cancer and depends on complex signaling networks that are chiefly supported by protein kinase activities. Therapeutic strategies have been used to target specific kinases but new methods are required to identify combined targets and improve treatment. Here, we propose a small interfering RNA genetic screen and an integrative approach to identify kinase networks involved in the proliferation of cancer cells. Results The functional siRNA screen of 714 kinases in HeLa cells identified 91 kinases implicated in the regulation of cell growth, most of them never being reported in previous whole-genome siRNA screens. Based on gene ontology annotations, we have further discriminated between two classes of kinases that, when suppressed, result in alterations of the mitotic index and provoke cell-cycle arrest. Extinguished kinases that lead to a low mitotic index mostly include kinases implicated in cytosolic signaling. In contrast, extinguished kinases that result in a high mitotic index mostly include kinases implicated in cell division. By mapping hit kinases in the PhosphPOINT phosphoprotein database, we generated scale-free networks consisting of 449 and 661 protein-protein interactions for kinases from low MI and high MI groups, respectively. Further analyses of the kinase interactomes revealed specific modules such as FER- and CRKL-containing modules that connect three members of the epidermal growth factor receptor (EGFR) family, suggesting a tight control of the mitogenic EGF-dependent pathway. Based on experimental studies, we confirm the involvement of these two kinases in the regulation of tumor cell growth. Conclusion Based on a combined approach of large kinome-wide siRNA screens and ontology annotations, our study identifies for the first time two kinase groups differentially implicated in the control of cell proliferation. We further demonstrate that integrative analysis of the kinase interactome provides key information which can be used to facilitate or optimize target design for new therapeutic strategies. The complete list of protein-protein interactions from the two functional kinase groups will provide a useful database for future investigations. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-1169) contains supplementary material, which is available to authorized users.

Poster

International audience; Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between -218 and -106 bp, which contains a particular heat-shock element, named CLE for 'clusterin element'. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1-HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity

Communiction orale présentée par Michel D.

Communication orale présentée par Loison F.

International audience; The authors have investigated a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This method may offer an alternative to gene therapy approaches. It consists in anchoring recombinant cytokines to the cell membrane. Attachment is mediated by the transmembrane domain of diphtheria toxin (T) genetically fused to the cytokine and is triggered by an acid pH pulse. The authors found that the fusion protein T-hIL-2 anchored to the surface of tumor cells retained its IL-2 activity while remaining exposed for several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective antitumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared with the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and the selection of transfected clones, it eliminates the safety problems connected with viral vectors, and it allows the control of the amount of cytokines delivered with the vaccine.

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.

Cytokines have a strong potential for triggering anticancer immunity if released in the tumor microenvironment. Successful vaccines have been engineered using tumor cells genetically modified to secrete the cytokines. Unfortunately, this approach remains difficult and hazardous to perform in the clinic. We describe a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This consists in anchoring recombinant cytokines to the membrane of killed tumor cells. Attachment is mediated by a fragment of diphtheria toxin (T) genetically connected to the cytokine. It is triggered by an acid pH pulse. The method was applied to IL-2, a potent anti-tumor cytokine. IL-2 anchored to the surface of tumor cells by the T anchor retained its IL-2 activity and remained exposed several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective anti-tumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared to the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and eliminates the safety problems connected with viral vectors while allowing the control of the amount of cytokines delivered with the vaccine.

We investigated whether the structural and functional behaviors of two unrelated protein domains were modified when fused. The IgG-binding protein ZZ derived from staphylococcal protein A was fused to the N- and/or C-terminus of the diphtheria toxin transmembrane domain (T). T undergoes a conformational change from a soluble native state at neutral pH to a molten globule-like state at acidic pH, leading to its interaction with membranes. We found that this molten globule state was not connected to the GdnHCl-induced unfolding pathway of T. The pH-induced transition of T, and also the unfolding of T and ZZ at neutral and acidic pH, were unchanged whether the domains were isolated or fused. The position of ZZ, however, influenced the solubility of T near its pK i . SPR measurements revealed that T has a high affinity for membranes, isolated or within the fusion proteins (K D < 10 - 1 1 M). This work shows that in the case of T and ZZ, the fusion of protein domains with different stabilities does not alter the structural changes involved in folding and function. This supports the use of T as a soluble membrane anchor.

Alongside conventional chemotherapies, targeted treatments have significantly improved the survival of patients with metastatic colorectal cancer (mCRC) from 6 months with best supportive care treatment, to 24 months with a combination of conventional and targeted chemotherapies. However, patients with KRAS mutated tumors have little or no benefit from these anti-EGFR antibodies based therapies (Panitumumab, Cetuximab) as single agents or combined with chemotherapy. These findings led to an amendment of the Marketing Authorization of these drugs that are now restricted to patients with KRAS wild-type tumor. However, the response of these patients ranged from 40 to 60%. Recent works have demonstrated that secondary resistance to such therapies was associated with emergence of KRAS mutated subclones in KRAS wild-type patients at the diagnosis time. To understand the importance of KRAS mutant subclones at the diagnosis time and to evaluate the consequence of their presence in terms of management of mCRC patients, a highly sensitive and quantitative procedure is required. Droplet-based digital PCR has recently emerged as a highly sensitive and quantitative approach for rare sequence detection. A retrospective study was set up where tumor samples from 177 mCRC patients were analyzed using a multiplex highly sensitive droplet-based procedures and ultra-deep sequencing using a new droplet-based target enrichment. Results are compared with data obtained by conventional qPCR. Sixty-two patients were classified as responders according to RECIST criteria. All tumors (41) detected as positive using conventional procedures were also positive with our procedure. Among the samples detected as negative with conventional procedures, 23 presented a KRAS or a BRAF mutation only detected by droplet-based procedures and 6 presented additional subclones. For 20 patients, additional biopsy samples were available and presented comparable results. 167 samples were also submitted to deep sequencing and, for alleles detected at fractions superior to 1%, the mutational status of the sample were in agreement for 94% of the samples (157/167) and we observed a correlation with the observed fraction of mutant alleles with the two procedures (R2= 0.7). We observed an inverse correlation between the proportion of mutated DNA and the frequency of anti-EGFR responses (P< 0.0001). The mean percentage of mutated DNA was 0.45% and 12.7% for responders and non-responders respectively. Progression Free Survival of patients was significantly different depending on the percentage of KRAS mutated allele within the tumor. The Progression Free Survival of patients with tumor presenting less than 5% of mutated KRAS was comparable to the one of non-mutated patients. HRs were of 1.07 (CI95% 0.6-2, NS), 2.9 (CI95% 1.7-4.9, P Citation Format: Valerie Taly, Pierre Laurent-puig, Deniz Pekin, Corinne Normand, Steve K. Kotsopoulos, Philippe Nizard, Jeff Olson, Preethi Srinivasan, Delphine Le Corre, Xinyu Li, Qun Zhong, Darren R. Link, Olivier Bouché, Jean-François Emile, Bruno Landi, Valérie Boige, Brian J. Hutchison. Clinical significance of low frequency KRAS and BRAF subclones for advanced colon cancer management. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2820. doi:10.1158/1538-7445.AM2014-2820

International audience; We have shown previously that the diphtheria toxin transmembrane domain (T) may function as a membrane anchor for soluble proteins fused at its C-terminus. Binding to membranes is triggered by acidic pH. Here, we further characterized this anchoring device. Soluble proteins may be fused at the N-terminus of the T domain or at both extremities, without modifying its membrane binding properties. This allows one to choose the orientation of the protein to be attached to the membrane. Maximum binding to the cell surface is reached within 1 h. Anchoring occurs on cells previously treated with proteinase K, suggesting that T interacts with the lipid phase of the membrane without the help of cell surface proteins. Binding does not permeabilize cells or affect cell viability, despite the fact that it permeabilizes liposomes and alters their structure. When attached to L929 fibroblasts, the proteins are not internalized and remain displayed at their surface for more than 24 h. When bound to K562 myeloid cells, the molecules are internalized and degraded. Thus, depending on the cell type, soluble proteins may be anchored to the surface of cells by the T domain for an extended time or directed towards an internalization pathway.

We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.

KRAS mutations are associated with an absence of response to EGFR-targeting treatments in colorectal cancer. These therapies are restricted to patients with a KRAS wild type tumor but only 50% of these patients present an objective response. It has been recently demonstrated that secondary resistance was associated with appearance of a KRAS mutation. The presence of a low fraction of KRAS-mutated cells within tumors, not detectable by commonly used procedures, could explain these false negative and these secondary resistances. To understand the importance of mutated subclones in the apparition of resistance to therapy targeting EGFR, a retrospective study including 95 patients with a metastatic colorectal cancer have been performed. All these patients, refractory to irinotecan were treated either with a combination of anti-EGFR and irinotecan or by anti-EGFR in monotherapy. These patients consisted of 54 men and 41 women with an average age of 62 years ± 11. Thirty-six patients were classified as responders according to RECIST criteria and 59 as non responders. Within this historitical serie, 27 patients were previously characterized as bearing a KRAS mutation by bulk Taqman analysis. DNA extracted from the tumors was analyzed by multiplex droplet-based microfluidics. This method, by segregating individual DNA molecules within million of aqueous microdroplets acting as independent microreactors, allows to perform precise quantitative experiments, for the 7 most frequent mutations of KRAS. All tumors reported as bearing a mutated KRAS were detected as positives in droplets with proportions of mutated DNA from 10 to 64% (mean of 34,7% ± 17,9%). Two of them (KRAS G12D) were bearing additional subclones (KRAS G12V; KRAS G12S) detected only in droplets. With the 68 negative tumors, 18 were found positive in droplets with KRAS proportions from 0,013% to 86% (mean of 8.6% ± 20). Only 4 patients had a proportion of mutated DNA higher than 10% (86%, 24.5%, 12.7% and 10%), all presented low quantities of amplifiable DNA. We observed an inverse correlation between the proportion of mutated DNA and the frequency of anti-EGFR responses (P< 0.03). The percentage of mutated DNA was 11.6% and 28,4% for responders and non-responders respectively. Survival free progression of patients was significantly different depending on the level of mutated DNA within the tumor. The survival free progression of patients with tumor presenting less than 5% of mutated KRAS was identical to the one of non mutated patients with both technics. HRs were of 0.54±0.2 NS, 3.1±0.9, (P Citation Format: Deniz Pekin, Corinne Normand, Steve Kotsopoulos, Li Xinyu, Leonor Benhaim, O. Bouche, T. Lecomte, D. Le Corre, T. Hor, Z. El Harrak, P. Nizard, D. LINK, B. HUTCHISON, P. LAURENT-PUIG, Valerie Taly. Detection and quantification of minoritary subclones of KRAS in metastatic colorectal cancers by digital microfluidics: therapeutic implications. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4211. doi:10.1158/1538-7445.AM2013-4211