Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin-9 dependent mechanism.

Nasopharyngeal carcinoma-derived small extracellular vesicles (NPCSEVs) have an immunosuppressive impact on the tumour microenvironment. In this study, we investigated their influence on the generation of tolerogenic dendritic cells and the potential involvement of the galectin-9 (Gal9) they carry in this process. We analysed the phenotype and immunosuppressive properties of NPCSEVs and explored the ability of DCs exposed to NPCSEVs (NPCSEV-DCs) to regulate T cell proliferation. To assess their impact at the pathophysiological level, we performed real-time fluorescent chemoattraction assays. Finally, we analysed phenotype and immunosuppressive functions of NPCSEV-DCs using a proprietary anti-Gal9 neutralising antibody to assess the role of Gal9 in this effect. We described that NPCSEV-DCs were able to inhibit T cell proliferation despite their mature phenotype. These mature regulatory DCs (mregDCs) have a specific oxidative metabolism and secrete high levels of IL-4. Chemoattraction assays revealed that NPCSEVs could preferentially recruit NPCSEV-DCs. Finally, and very interestingly, the reduction of the immunosuppressive function of NPCSEV-DCs using an anti-Gal9 antibody clearly suggested an important role for vesicular Gal9 in the induction of mregDCs. These results revealed for the first time that NPCSEVs promote the emergence of mregDCs using a galectin-9 dependent mechanism and open new perspectives for antitumour immunotherapy targeting NPCSEVs.

A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Background: Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). Materials and methods: CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. Results: IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. Conclusion: We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

Transforming properties of MET receptor exon 14 skipping can be recapitulated by loss of the CBL ubiquitin ligase binding site.

MET is a receptor tyrosine kinase that is activated in many cancers through various mechanisms. MET exon 14 (Ex14) skipping occurs in 3% of nonsmall cell lung tumors. However, the contribution of the regulatory sites lost upon this skipping, which include a phosphorylated serine (S985) and a binding site for the E3 ubiquitin ligase CBL (Y1003), remains elusive. Sequencing of 2808 lung tumors revealed 71 mutations leading to MET exon 14 skipping and three mutations affecting Y1003 or S985. In addition, MET exon 14 skipping and MET Y1003F induced similar transcriptional programs, increased the activation of downstream signaling pathways, and increased cell mobility. Therefore, the MET Y1003F mutation is able to fully recapitulate responses induced by MET exon 14 skipping, suggesting that loss of the CBL binding site is the main contributor of cell transformation induced by MET Ex14 mutations.

A role for AKT1 in nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a highly regulated quality control mechanism through which mRNAs harboring a premature termination codon are degraded. It is also a regulatory pathway for some genes. This mechanism is subject to various levels of regulation, including phosphorylation. To date only one kinase, SMG1, has been described to participate in NMD, by targeting the central NMD factor UPF1. Here, screening of a kinase inhibitor library revealed as putative NMD inhibitors several molecules targeting the protein kinase AKT1. We present evidence demonstrating that AKT1, a central player in the PI3K/AKT/mTOR signaling pathway, plays an essential role in NMD, being recruited by the UPF3X protein to phosphorylate UPF1. As AKT1 is often overactivated in cancer cells and as this should result in increased NMD efficiency, the possibility that this increase might affect cancer processes and be targeted in cancer therapy is discussed.

2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations.

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2'-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.

MET amplification increases the metastatic spread of EGFR-mutated NSCLC.

BACKGROUND: Five to 20% of metastatic EGFR-mutated non-small cell lung cancers (NSCLC) develop acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI) through MET amplification. The effects of MET amplification on tumor and patient phenotype remain unknown. METHODS: We investigated,in vitro and in vivo, the impact of MET amplification on the biological properties of the HCC827 cell line, derived from an EGFR-mutated NSCLC. We further evaluated the time to new metastases after EGFR-TKI progression in EGFR-mutated NSCLC, exhibiting MET amplification or high MET overexpression. RESULTS: MET amplification significantly enhanced proliferation, anchorage independent growth, anoikis resistance, migration, and induced an epithelial to mesenchymal transition. In vivo, MET amplification significantly increased the tumor growth and metastatic spread. Treatment with a MET-TKI reversed this aggressive phenotype. We found that EGFR-mutated NSCLC patients exhibiting MET amplification on a re-biopsy, performed after EGFR-TKI progression, displayed a shorter time to new metastases after EGFR-TKI progression than patients with high MET overexpression but no MET amplification. CONCLUSION: MET amplification increases metastatic spread even in the context of an already pre-existing strong driver mutation such as EGFR mutation. These results prompt development of therapeutic strategies aiming at preventing emergence of MET amplification.

MAGP-1 and fibronectin control EGFL7 functions by driving its deposition into distinct endothelial extracellular matrix locations.

The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin-mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors, the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms [i.e., the repression of endothelial-derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway, and the expression of leukocyte adhesion molecules and of RHOA by endothelial cells], it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar-associated glycoprotein-1 (MAGP-1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Although EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP-1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions.

Phenotypic assays for Mycobacterium tuberculosis infection.

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry.

Developmental Expression of 4-Repeat-Tau Induces Neuronal Aneuploidy in Drosophila Tauopathy Models.

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. Here, we used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. Our results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, we observed a mitotic delay in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, we found a high level of aneuploidy in post-mitotic differentiated tissue. Finally, we showed that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, our results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development.

MET receptor variant R970C favors calpain-dependent generation of a fragment promoting epithelial cell scattering.

The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.

PEA3 transcription factors are downstream effectors of Met signaling involved in migration and invasiveness of Met-addicted tumor cells.

Various solid tumors including lung or gastric carcinomas display aberrant activation of the Met receptor which correlates with aggressive phenotypes and poor prognosis. Although downstream signaling of Met is well described, its integration at the transcriptional level is poorly understood. We demonstrate here that in cancer cells harboring met gene amplification, inhibition of Met activity with tyrosine kinase inhibitors or specific siRNA drastically decreased expression of ETV1, ETV4 and ETV5, three transcription factors constituting the PEA3 subgroup of the ETS family, while expression of the other members of the family were less or not affected. Similar link between Met activity and PEA3 factors expression was found in lung cancer cells displaying resistance to EGFR targeted therapy involving met gene amplification. Using silencing experiments, we demonstrate that the PEA3 factors are required for efficient migration and invasion mediated by Met, while other biological responses such as proliferation or unanchored growth remain unaffected. PEA3 overexpression or silencing revealed that they participated in the regulation of the MMP2 target gene involved in extracellular matrix remodeling. Our results demonstrated that PEA3-subgroup transcription factors are key players of the Met signaling integration involved in regulation of migration and invasiveness.

Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator.

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5' untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.

Role of VAMP3 and VAMP7 in the commitment of Yersinia pseudotuberculosis to LC3-associated pathways involving single- or double-membrane vacuoles.

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking autophagosome acidification. In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy. Here, we address the question of the membrane traffic during internalization of Yersinia investigating the role of vesicle- associated membrane proteins (VAMPs). First, we show that as in epithelial cells, Yersinia pseudotuberculosis replicates mainly in nonacidic LC3-positive vacuoles. Second, in these cells, we unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single membranes using correlative light-electron microscopy. Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3. Fourth, we show that VAMP7 knockdown alters LC3's association with single-and multimembrane-YCVs. Finally, in uninfected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher number of double-membrane, LC3-positive vesicles. Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion proteins involved in Yersinia pseudotuberculosis' intravesicular life cycle.

Activation of invariant Natural Killer T lymphocytes in response to the α-galactosylceramide analogue KRN7000 encapsulated in PLGA-based nanoparticles and microparticles.

Invariant Natural Killer T (iNKT) cells have potent immunostimulatory activities that could be exploited for human therapies. The high-affinity CD1d antigen α-galactosylceramide analogue KRN7000 (KRN) activates a cascade of anti-tumor effector cells and clinical studies have already had some initial success. To improve the efficacy of the treatment, strategies that aim to vectorize KRN would be valuable. In this study, we intended to characterize and compare the effect of KRN encapsulated in poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs, 90nm) and microparticles instead of macroparticles (MPs, 715nm) on the iNKT cell response. Our data show that whatever the size of the particles, vectorized KRN induced potent primary activation of iNKT cells in vitro and in vivo. We show that endocytosis of PLGA-based particles by dendritic cells is mediated by a clathrin-dependent manner and that this event is important to stimulate iNKT cells. Finally, we report that KRN vectorized in NPs and MPs exhibited different behaviours in vivo in terms of iNKT cell expansion and responsiveness to a recall stimulation. Collectively, our data validate the concept that KRN encapsulated in PLGA-based particles can be used as delivery systems to activate iNKT cells in vitro and in vivo.

Ploidy of cell-sorted trophic and cystic forms of Pneumocystis carinii.

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.

Photodynamic therapy with intratumoral administration of Lipid-Based mTHPC in a model of breast cancer recurrence.

BACKGROUND AND OBJECTIVES: Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer. MATERIALS AND METHODS: Mice were directly injected into the tumor (IT) with 25 microl of a Foslip suspension (0.15 mg/ml) and illumination (652 nm, 20 J/cm(2)) was performed at different time points with pathological assessment after 48 hours. In a parallel mice series plasma samples were obtained at different endpoints after IT Foslip injection for HPLC analysis and the tumors were subjected in toto to macrofluorescence imaging. Fluorescence polarization measurements were conducted in vitro to estimate the rate of sensitizer redistribution from liposomes. RESULTS: Optimal, albeit partial, cure rates were obtained at 24 hours post-sensitizer and uninistration. Inhomogeneous and weak fluorescence was observed at early time points and became maximal at 24 hours. Plasma levels of mTHPC increased until 15 hours. Fluorescence polarization measurements showed a slow sensitizer transfer from liposomes to model membranes. DISCUSSION AND CONCLUSION: The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response.