po 290 etv7 regulates breast cancer stem cells content and chemoresistance

Introduction Cancer stem cells (CSCs) are considered the population of cells within the tumour able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy. ETV7 is a poorly studied transcription factor member of ETS large family, known to be an interferon-stimulated gene. It has been recently found over-expressed in breast cancer (BC), with higher expression levels in the more aggressive BC subtypes. In this work, we investigated the effects of ETV7 increased expression on breast CSCs population and resistance to chemotherapy in BC cells. Material and methods We generated MCF7 and T47D BC-derived cells stably over-expressing ETV7 and obtained ETV7 KO in MDA-MB-231 BC cells using CRISPR/Cas9 technology. We analysed breast CSCs content via CD44/CD24 staining and FACS analysis, as well as mammospheres formation assay. We measured expression of ABC transporters and anti-apoptotic proteins via RT-qPCR and western blot. We finally assessed sensitivity to Doxorubicin and 5-Fluorouracil (5-FU) via MTT assay and AnnexinV/PI staining at FACS. Results and discussions We observed that the expression of ETV7 could be induced by various stimuli, particularly by chemotherapeutic drugs able to induce DNA damage. We then analysed the impact of ETV7 expression on the sensitivity to Doxorubicin and 5-FU and we could observe a significantly decreased sensitivity to these drugs upon ETV7 over-expression. We could also appreciate an increase in ABC transporters and BCL2 anti-apoptotic protein expression following ETV7 over-expression. We further observed that alteration of ETV7 expression could significantly affect the population of breast cancer stem cells (CD44+/CD24low cells) in different BC cell lines. Conclusion We propose a novel role for ETV7 in breast cancer stem cells plasticity and associated resistance to conventional chemotherapy. We finally suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens with the aim of avoiding resistance and relapse in breast cancer

Synergistic NGF/B27 Gradients Position Synapses Heterogeneously in 3D Micropatterned Neural Cultures

Native functional brain circuits show different numbers of synapses (synaptic densities) in the cerebral cortex. Until now, different synaptic densities could not be studied in vitro using current cell culture methods for primary neurons. Herein, we present a novel microfluidic based cell culture method that combines 3D micropatterning of hydrogel layers with linear chemical gradient formation. Micropatterned hydrogels were used to encapsulate dissociated cortical neurons in laminar cell layers and neurotrophic factors NGF and B27 were added to influence the formation of synapses. Neurotrophic gradients allowed for the positioning of distinguishable synaptic densities throughout a 3D micropatterned neural culture. NGF and B27 gradients were maintained in the microfluidic device for over two weeks without perfusion pumps by utilizing a refilling procedure. Spatial distribution of synapses was examined with a pre-synaptic marker to determine synaptic densities. From our experiments, we observed that (1) cortical neurons responded only to synergistic NGF/B27 gradients, (2) synaptic density increased proportionally to synergistic NGF/B27 gradients; (3) homogeneous distribution of B27 disturbed cortical neurons in sensing NGF gradients and (4) the cell layer position significantly impacted spatial distribution of synapses

Density of Gr1-positive myeloid precursor cells, p-STAT3 expression and gene expression pattern in canine mammary cancer metastasis

The very recent studies on human and mice models have indicated an important role of myeloid precursor cells (progenitors or not fully differentiated cells that express the Gr1 antigen also called Gr1-positive myeloid suppressor cells) in the tumor progression and metastasis. They are thought to suppress the immune system and promote angiogenesis via Signal transducer and activator of transcription 3 (STAT3) activation. As of now there is no data available on the correlation of Gr1-positive cell number, phosphorylated STAT3 (p-STAT3) expression and cancer ability to metastasis. Thus, we counted the myeloid precursor cell number and analyzed p-STAT3 expression in 50 canine mammary tumors that gave local/distant metastases and did not metastasize. We showed that the number of Gr1-positive cells and p-STAT3 expression are significantly higher (p < 0.001) in the metastatic tumors than in the non-metastatic ones. We also observed higher expression of p-STAT3 in the canine mammary cancer cell lines with metastatic potential than in other cell lines (p < 0.001). Moreover, the number of myeloid precursors and p-STAT3 expression in metastatic tumors correlate strongly. The tumor infiltrating myeloid precursor cells may invigorate the STAT3 activity (probably via vascular endothelial growth factor – VEGF) that contributes to the tumor angiogenesis and furthermore tumor`s ability to metastasize. The analysis of gene expression in canine mammary cancer cell lines with metastatic potential indicated that semaphorin 3B (SEMA3B) and neuropilin receptors (NRP) may also be important elements in this process. Thus, we discuss the possible interactions within the tumor that may be required for cancer metastatis

RD-Connect, NeurOmics and EURenOmics: collaborative European initiative for rare diseases

Functional Genomics of Muscle, Nerve and Brain Disorder

Modulation of cognition and neuronal plasticity in gain- and loss-of-function mouse models of the schizophrenia risk gene Tcf4

The transcription factorTCF4was confirmed in several large genome-wide association studies as one of the most significant schizophrenia (SZ) susceptibility genes. Transgenic mice moderately overexpressingTcf4in forebrain (Tcf4tg) display deficits in fear memory and sensorimotor gating. As second hit, we exposedTcf4tg animals to isolation rearing (IR), chronic social defeat (SD), enriched environment (EE), or handling control (HC) conditions and examined mice with heterozygous deletion of the exon 4 (Tcf4Ex4 delta(+/-)) to unravel gene-dosage effects. We applied multivariate statistics for behavioral profiling and demonstrate that IR and SD cause strong cognitive deficits ofTcf4tg mice, whereas EE masked the genetic vulnerability. We observed enhanced long-term depression inTcf4tg mice and enhanced long-term potentiation inTcf4Ex4 delta(+/-)mice indicating specific gene-dosage effects.Tcf4tg mice showed higher density of immature spines during development as assessed by STED nanoscopy and proteomic analyses of synaptosomes revealed concurrently increased levels of proteins involved in synaptic function and metabolic pathways. We conclude that environmental stress and Tcf4 misexpression precipitate cognitive deficits in 2-hit mouse models of relevance for schizophrenia