MOMENDO project deliverables
The page lists 19 deliverables related to the research project "MOMENDO".
List of Deliverables
| title and desprition | type | last update |
|---|---|---|
Effect and molecular mechanism of Syndecan-1 inhibition on endometriosisThe impact of shRNA-mediated knockdown of Syndecan-1 will be assessed in the in vivo endometriosis models available to partner CONICET. WWU and KI in cooperation with CONICET will employ several in vitro assays of endometriotic stem cell phenotypic analysis, including flow cytometric side population analysis, measurement of ALDH activity, qPCR and FACS analysis of stemness-associated cell surface markers, colony formation and sphere formation assays. The impact of Syndecan-1 and the HA-CD44 assays will be studied in the endometriotic cell line 12Z, and in patient-derived primary endometriotic stroma cells by siRNA knockdown and 4-MU inhibition. TaqMan low density array analysis will be used by WWU to screen for affected pathways, and targets will be confirmed by qPCR and Western blotting.Sphere formation assays, as well as lineage-specific differentiation assays will be performed in the experimental systems subjected to Syndecan-1 shRNA treatment. Results of TaqMan low density array analysis on identified targets will be confirmed by these functional analyses. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Evaluation of anti-endocrine therapy in vitro and in vivoTo evaluate the contribution of endometriotic-derived estrogens to ovarian epithelial carcinomas onset and development in vivo an experimental mouse model harbouring the two pathologic entities will be developed by inducing ovarian epithelial carcinomas in immunosuppressed mice with experimental endometriosis.The specific effects exerted by the estrogens secreted by patients-derived endometriotic tissue explants culture on cellular proliferation, migration, matrix invasion, survival and differential gene expression will be determined in human endometrioid and clear cell-derived carcinoma cell lines which express both estrogen receptors, one of them, or none. Finally, in all experimental models, the blockage of estrogens will be carried out to elucidate if this strategy has a potential therapeutic value. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
In vivo and in vitro models of inflammation-associated painUEDIN and partner AQUILA will explore how the peripheral inflammatory environment in endometriosis causes pain. The team has previously used a mouse model of endometriosis using syngeneic menstrual endometrial tissue introduced into the peritoneum of immunocompetent mice to understand the origin of macrophages present in endometriosis lesions (66) and to demonstrate a role for estradiol in regulating both neuroangiogenesis (67) and neuroinflammation (Greaves et al, in press) in endometriosis. UEDIN will use these mice to explore the dynamics of macrophages present in endometriosis lesions and how these cells may contribute to the inflammatory milieu. Analysis techniques will include confocal and multi-photon intra-vital imaging, gene expression analysis and behavioural assessments. We will use in vitro systems to explore how the peripheral inflammatory environment observed in endometriosis influences specific cell types (macrophages and sensory neurons). Analysis techniques will include fluorescent activated cell sorting (FACS), gene expression analysis, enzyme linked immunosorbent assay (ELISA) and calcium assays. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Molecular role and therapeutic potential of HA in endometriosisCONICET has demonstrated that inhibition of HA synthesis using 4-methylumbelliferone (4-MU) at low doses, lead to a significant reduction of the vascularized area and functional microvessel density as revealed by in vivo imaging in a mouse model of endometriosis, which was supported by a diminished CD31 staining (Olivares et al, manuscript under revision). We will induce peritoneal endometriosis in BALB/c mice treated with 4-MU and evaluate its effect on endometriotic lesions regarding attachment, proliferation and apoptosis, as well as on angiogenesis and inflammation markers.We will study the role of CD44 and HA on endometriosis by performing the following experiments: 1- cell proliferation and apoptosis evaluation of endometrial stromal and epithelial cells obtained from endometriosis patients and controls treated with 4-MU in vitro; 2- validation of the results obtained on primary cell cultures on Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Effect of herbal medicine on inflammation, angiogenesis and apoptotic resistanceCONICET has previously demondtrated a beneficial therapeutic effect of the natural compounds Wogonin (WG), an active constituent of Chinese Herbal Medicine, and two of the main antioxidant compounds found in rosemary extract: CA and RA, on the development of experimental endometriosis both in vitro and in vivo. CA, RA and WG inhibited significantly cell proliferation in human endometrial stromal cell line T-HESC, and in primary cultures. In a Balb/c-based murine model, these compounds diminished significantly the development of endometriotic-like lesions, reducing considerably its size. Moreover, all compounds decreased endometriotic epithelial and stromal cell proliferation evaluated by immunohistochemistry for PCNA Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Therapeutic effect of SARMSLead participant UEDIN has identified androgen target genes in primary endometrial stromal cells and obtained evidence that AR agonists can oppose oestrogen-dependent proliferation in this cell type (55). (i) In vitro cell systems developed in Edinburgh using 3D culture scaffolds will be used to evaluate the impact of a range of selective androgen receptor modulators (SARMs) alone, and in combination with E2, on cell behavior (e.g. proliferation, apoptosis, migration) and gene expression. A range of analysis methods will be used to stratify the response to different SARMs including analysis using pathway focused RT² Profiler PCR Arrays (e.g. AR signaling targets, EMT, cell cycle), qRTPCR, reporter assays, Westerns, immunohistochemistry). To test whether SARMs are capable of activating the mouse Ar we will quantify their ability to induce expression of a luciferase reporter gene in female ‘androgen reporter mice’ (68). Uterine tissues will be retrieved from the mice following an injection of BrDu and both Ar expression and cell proliferation determined. We will assess the impact of SARMs identified as having the most potent impact on uterine cells using our in house model of mouse endometriosis (66): endpoints will include lesion size and pain scores. In addition, UEDIN will identify DCA-responsive proteins/functional pathways as future candidate targets for therapeutic development. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
CSC marker analysis and Nanostic analysis in patient tissuesTo further investigate CSCs/ESCs, KI will explore CSC markers in endometrial /endometriotic samples in a large number (n=50) of endometriosis patients to identify endometriosis patients with high risk for EAOEC and develop a prophylactic strategy. This study will be assisted by partners CONICET and WWU. Proliferative phase endometrial and endometriotic samples will be collected at the time of surgery from women undergoing surgery for endometriosis. The expression levels of CSC markers will be quantified by real time PCR and protein quantification setup Wes.This work will be complemented by a studying the whole transcriptome (mRNA, miRNA) and by epigenetic profiling of endometrial /endometriotic samples from women with endometriosis expressing cancer stem cells to understand the molecular mechanism and find new markers to develop prophylactic measures. For this, next generation RNA sequencing with illumina platform available at KI will be used. Differential expression between the groups will be compared using bioinformatics platform, Ingenuity Pathway Analysis (IPA) at KI. This study will give knowledge on functional and non-functional genes and its association with signalling pathways, which have carcinogenic linkage. Genome wide scanning for epigenetic profile in endometrium and endometrioma will be studied using high throughput meDI . These results will be analysed together with transcriptome data using IPA. Nanostic atomic force microscopy to characterize enSC with the expression of CSC to screen endometrium of high risk patients for EAOEC by partner KI will be done with partner Serend-IP. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Nocireceptive neuronal marker studyCHARITE will characterize tissues of more than 120 endometriosis patients and controls for the appearance of nociceptive innervation with the classical markers for nociceptive neurons substance P and TRPV1. The cellular / tissue phenotype will be correlated to a pain score with patients’ pain questionnaires. Using a screening assay allowing to quantify neuronal outgrowth of rodent cultured neurons, sensory neurons will be incubated with with patients’ peritoneal fluid and neurite outgrowth will be quantified. Furthermore, by fluorescence microscopy, the cellular outgrowth phenotype in vitro will be determined as a readout for cells potentially generating nociceptive neurons.The RNA and protein expression of 22 gene candidates, specifically expressed in nociceptive neurons, for neuronal outgrowth after treatment with patients’ peritoneal fluid in rat dorsal root ganglia neurons will be studied to characterise the molecular phenotype.Candidate gene expression will be independelty confirmed in already available patient tissue and/or modulated by specific cellular and animal experiments (65) leading to the development of novel therapeutic opportunities for the treatment, preventions, and understanding of endometriosis related pelvic pain (WP2). Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Impact of HA-CD44 inhibition on an endometriotic stem cell phenotypeExtending the analyses described in Deliverable 19, KI will perform sphere formation assays, as well as lineage-specific differentiation assays in the experimental systems subjected to 4MU inhibition. Results of TaqMan low density array analysis on identified targets will be confirmed by these functional analyses. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Project meetings and workshopsA kickoff meeting of MOMENDO will by organized by WWU and will take place in Muenster, Germany.Workshops will ensure further communication between participants and Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Impact of miRNA expression on an invasive phenotypeEndometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA and analyzed for changes in invasive cell behaviour by matrigel invasion chamber assays or video microscopy.Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA. Changes in predicted target gene expression potentially linked to the invasive phenotype will be monitored by qPCR, Western Blotting and confocal immunofluorescence microscopy (WWU, CONICET). Regulation of selected targets will be confirmed by 3’UTR luciferase reporter assays and plasmid-based complementation analysis using target constructs devoid of the endogenous 3’UTR (WWU).Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA.Altered signal transduction processes will be analyzed by phosphokinase array screening (R&D systems) and phosphokinase blotting (WWU, CONICET). Finally, we will employ proprietary nanoscale topography analysis (nanostic TM) provided by SEREND-IP to determine cell surface properties (filopodia formation, cell shape, etc.) in cooperation with partners WWU and CONICET. This technique has the potential to develop a marker-free diagnostic tool for invasive endometriotic cell behavious based on quantitative atomic force microscopy. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Pathogenetic role of the NFkB pathway following iron overloadWe will study study endometrial epithelial and stromal cells NF-B-dependent responses to iron overload as a mechanism promoting endometriosis origin, development and maintenance: assessing endometrial stromal and epithelial cell cultures in response to iron overload, evaluating p65-NF-B activation, inflammatory NF-B-dependent cytokines expression (IL-1, IL-8), cell proliferation and apoptosis. Furthermore, by using intracellular iron chelators, this project aims to suggest new drugs for the prevention and treatment of endometriosis: to determine if the iron chelators deferoxamine and deferasirox are able to prevent iron overload-induced changes in endometrial stromal and epithelial cell cultures. In cooperation with partner WWU, UNIVERSIDAD DE CHILE will aim to discover the main function(s) of the alternative NF-kB pathway in human endometrial cells. This will be achieved by silencing RelB expression by using small interfering RNA (siRNA) in endometrial stromal and epithelial cell cultures, by assessing the protein expression of proinflammatory cytokines in response to RelB silencing in endometrial cells using ELISA-based methodology, and by assessing cell proliferation and apoptosis in response to RelB silencing in endometrial cells. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
NFkB pathway analysisUNIVERSIDAD DE CHILE will study NfKB-dependent signaling both in rodent primary neurons exposed to peritoneal fluid of endometriosis patients with CHARITE, and in primary endometrial stroma cells of endometriosis patients and controls. Methods include p65-NF-B activation assays (TransAM ® kit (Active Motif Europe)) ELISAs for inflammatory NF-B-dependent cytokine expression (IL-1, IL-8), and proliferation and apoptosis assays following siRNA-mediated knockdown of components of the NFkB, Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Induced differentiation of ESCs and CSCs as a therapeutic approachThe notch and CD44 pathways will be targeted using in vitro cell culture systems after inhibition with gamma secretase inhibitors, Musashi-1 and -2 siRNA, or 4MU (see WP3) respectively, in cooperation with partners KI, WWU and CONICET. A phenotypic stem cell analysis (see WP3), apoptosis and cell viability assays, as well as sphereformation assays will be utilized as readouts. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Identification and functional analysis of pain-related microRNAsIn cooperation with partner CHARITE, WWU and CONICET, we will investigate Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Role of NFkB-dependent microRNAs in endometriotic cell behaviourTo identify novel downstream regulators of this pathway in endometriosis, partners WWU and UNIVERSIDAD DE CHILE will investigate if activation of this pathway in endometriotic stroma cells leads to an upregulation of miR-9/9* and miR146a, reported to be regulated by NFkB in other systems (73-75). The functional consequences of NFkB-dependent miRNA regulation on invasive behaviour and a stem cell phenotype of these cells will be investigated analogously to WP3 and WP6, and potential targets responsible for the phenotype will be identified following bioinformatic targtet prediction analysis and functional characterisation after transfection with miRNA precursor molecules (see WP6). Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Atomic force microscopyThe impact of SARM treatment on the invasive phenotype of endometriotic cells will be evaluated by quantitative atomic force microscopy by partner SERENDIP. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |
Webpage and Social MediaWWU will furthermore be the lead organizer of Results Dissemination via a Website and Facebook site of the project. Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Websites, patent fillings, videos etc. | 2020-04-06 |
Therapeutic effects of DCAWe will determine whether dichloroacetate (DCA) can alter the peritoneal environment and halt the growth of endometriosis lesions in women with endometriosis. We will evaluate the impact of DCA in an in-vivo model of endometriosis on (a) glycolysis/lactate production in the peritoneal mesothelium (b) endometriosis lesion growth (c) endometriosis-associated pain behaviour. The effect of DCA will also be tested in vitro, on CSC spheroids generated from endometrium/endometrioma, in collaboration with our KI partner (see WP5). In addition, UEDIN and AQUILA will identify DCA-responsive proteins/functional pathways as future candidate targets for therapeutic development. The effect of Dichloroacetate on a putative stem cell phenotype will be tested in vitro, on CSC spheroids generated from endometrium/endometrioma (see WP5). Programme: H2020-EU.1.3.3. - Topic(s): MSCA-RISE-2015 |
Documents, reports | 2020-04-06 |