Cancer Cell Expression of Autotaxin Controls Bone Metastasis Formation in Mouse through Lysophosphatidic Acid-Dependent Activation of Osteoclasts

Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorptive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models.Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to immunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis.Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast differentiation. Therefore, targeting the autotaxin/LPA track emerges as a potential new therapeutic approach to improve the outcome of patients with bone metastases

CoNIC Challenge: Pushing the Frontiers of Nuclear Detection, Segmentation, Classification and Counting

Nuclear detection, segmentation and morphometric profiling are essential in helping us further understand the relationship between histology and patient outcome. To drive innovation in this area, we setup a community-wide challenge using the largest available dataset of its kind to assess nuclear segmentation and cellular composition. Our challenge, named CoNIC, stimulated the development of reproducible algorithms for cellular recognition with real-time result inspection on public leaderboards. We conducted an extensive post-challenge analysis based on the top-performing models using 1,658 whole-slide images of colon tissue. With around 700 million detected nuclei per model, associated features were used for dysplasia grading and survival analysis, where we demonstrated that the challenge's improvement over the previous state-of-the-art led to significant boosts in downstream performance. Our findings also suggest that eosinophils and neutrophils play an important role in the tumour microevironment. We release challenge models and WSI-level results to foster the development of further methods for biomarker discovery

Studies of GPR147 homo- and hetero-dimerization with GPR54 and GnRH-R by fluorescence imaging as FRET, BiFC and fluorescence anisotropy, in living cells

Studies of GPR147 homo- and hetero-dimerization with GPR54 and GnRH-R by fluorescence imaging as FRET, BiFC and fluorescence anisotropy, in living cells. 2. Journées du GdR 3606 Repr

GPR147 dimerization studies by fluorescence imaging in living cells

The GPR147, also known as Neuropeptide FF1 Receptor (Bonini et al., 2000), is a seven-transmembrane G protein-coupled receptor (GpCR) that binds the Gonadotropin inhibitory hormone (GnIH) or the RFamide-related peptide-3 (RfRP3) peptide (Smith & Clarke, 2010).These ligands play a role in the control of reproduction in mammals and birds. The GPR147 is expressed in neurons (i.e GnRH neurons) and takes part with other GpCR, as GnRHr and GPR54 (GnRH and Kiss peptide receptor respectively), in the regulation of the hypothalamic-pituitary-gonadal axe. We have generated plasmids to express all these receptors as fusion fluorescent proteins and studied the homodimerization of GPR147 and the heterodimerization with GPR54 and GnRHr receptors, by FRET, BiFC and in the current approach by Fluorescence anisotropy

Steroid hormones regulate sperm-oviduct interactions in the bovine

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro, the ovarian steroids 17beta-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that: (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32 to 47% of bound spermatozoa from BOEC; this sperm releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to then released from BOEC by the action of P4 induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization

Porcine oviductal extracellular vesicles interact with gametes and regulate sperm motility and survival

International audienceOnce in the female reproductive tract, spermatozoa undergo several modifications to acquire their complete fertilizing ability. Interactions between the oviductal fluid (OF) and gametes contribute to a successful fertilization. Recently, oviductal extracellular vesicles have been identified as an important part of the OF but their interactions with gametes are not fully understood. In the present study, we aim at determining the patterns of interactions between porcine oviductal extracellular vesicles (poEVs) and gametes (spermatozoa and oocytes). Moreover, we evaluate the effect of poEVs on sperm survival and motility to better understand the mechanisms by which poEVs modulate the processes leading to fertilization. Evaluation of poEVs uptake by spermatozoa showed that poEVs bind to spermatozoa in a time and dose dependent manner. Co-incubation of spermatozoa with poEVs (0.2 mg/mL) increased fresh and frozen sperm survival after 6 and 17 h, respectively. By contrast, poEVs supplementation reduced the total and progressive sperm motility after 2 h. Additionally, we demonstrated that poEVs interacted with the cumulus cells, zona pellucida (ZP) and oocyte, being able to cross the ZP. Besides, we showed that poEVs delivered their cargo into the oocyte, by the transfer of OVGP1 protein. In conclusion, our results demonstrated that poEVs are able to interact with both gametes. Besides, the findings from the present study showed that poEVs may participate in maintaining sperm viability and reducing motility, functions associated with the oviduct sperm reservoir. Although further investigations are needed, our results indicate that poEVs can be a potential tool to improve sperm life span during sperm handling and enhance IVF outcomes

Visualization of mouse neuronal ganglia infected by Herpes Simplex Virus 1 (HSV-1) using multimodal non-linear optical microscopy.

International audienceHerpes simplex virus 1 (HSV-1) is a neurotropic virus that causes skin lesions and goes on to enter a latent state in neurons of the trigeminal ganglia. Following stress, the virus may reactivate from latency leading to recurrent lesions. The in situ study of neuronal infections by HSV-1 is critical to understanding the mechanisms involved in the biology of this virus and how it causes disease; however, this normally requires fixation and sectioning of the target tissues followed by treatment with contrast agents to visualize key structures, which can lead to artifacts. To further our ability to study HSV-1 neuropathogenesis, we have generated a recombinant virus expressing a second generation red fluorescent protein (mCherry), which behaves like the parental virus in vivo. By optimizing the application of a multimodal non-linear optical microscopy platform, we have successfully visualized in unsectioned trigeminal ganglia of mice both infected cells by two-photon fluorescence microscopy, and myelinated axons of uninfected surrounding cells by coherent anti-Stokes Raman scattering (CARS) microscopy. These results represent the first report of CARS microscopy being combined with 2-photon fluorescence microscopy to visualize virus-infected cells deep within unsectioned explanted tissue, and demonstrate the application of multimodal non-linear optical microscopy for high spatial resolution biological imaging of tissues without the use of stains or fixatives

Giant lipid vesicles under electric field pulses assessed by non invasive imaging

We present experimental results regarding the effects of electric pulses on giant unilamellar vesicles (GUVs). We have used phase contrast and coherent anti-Stokes Raman scattering (CARS) microscopy as relevant optical approaches to gain insight into membrane changes under electropermeabilization. No addition of exogenous molecules (lipid analogue, fluorescent dye) was needed. Therefore, experiments were performed on pure lipid systems avoiding possible artefacts linked to their use. Structural membrane changes were assessed by loss of contrast inside the GUVs due to sucrose and glucose mixing. Our observations, performed at the single vesicle level, indicate these changes are under the control of the number of pulses and field intensity. Larger number of pulses enhances membrane alterations. A threshold value of the field intensity must be applied to allow exchange of molecules between GUVs and the external medium. This threshold depends on the size of the vesicles, the larger GUVs being affected at lower electric field strengths than the smaller ones. Our experimental data are well described by a simple model in which molecule entry is driven by direct exchange. The CARS microscopic study of the effect of pulse duration confirms that pulses, in the ms time range, induce loss of lipids and membrane deformations facing the electrodes

Versatile Cellular Uptake Mediated by Catanionic Vesicles: Simultaneous Spontaneous Membrane Fusion and Endocytosis

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Progesterone induces sperm release from oviductal epithelial cells by modifying sperm proteomics, lipidomics and membrane fluidity

International audienceThe sperm reservoir is formed after insemination in mammals, allowing sperm storage in the oviduct until their release. We previously showed that physiological concentrations of progesterone (P4) trigger in vitro the sperm release from bovine oviductal epithelial cells (BOECs), selecting a subpopulation of spermatozoa with a higher fertilizing competence. Here, by using Western-Blot, confocal microscopy and Intact Cell MALDI-TOF-Mass Spectrometry strategies, we elucidated the changes derived by the P4-induced release on sperm cells (BOEC-P4 spz). Our findings show that, compared to controls, BOEC-P4 spz presented a decrease in the abundance of Binder of Sperm Proteins (BSP) - 3 and - 5, suggesting one mechanism by which spermatozoa may detach from BOECs, and thus triggering the membrane remodeling with an increase of the sperm membrane fluidity. Furthermore, an interesting number of membrane lipids and proteins were differentially abundant in BOEC-P4 spz compared with controls