Comparison of behavioral patterns of dairy cows with natural estrus and induced ovulation detected by an ear-tag based accelerometer.

Dairy farms face many challenges and changes. With increasing herd sizes and fewer farmers or employees per cow, new strategies to maintain or improve reproductive management are required. One of the major challenges is to detect cows in estrus and to estimate the perfect time for artificial insemination (AI). Several estrus and ovulation synchronization programs with timed AI as well as estrus detection aids, e.g., tail-paint, pedometer, accelerometer, and others are available. A combination of ovulation synchronization programs and technical solutions, however, has rarely been tested. This study was designed to gain insights into behavioral patterns of cows subjected to an Ovsynch program and to test if behavioral data could be used to optimize the timing of insemination within an Ovsynch program. In this study, we used an ear-tag based 3D-accelerometer system (SMARTBOW, Smartbow GmbH, Weibern, Austria) to generate data of behavioral patterns, i.e., rumination and activity. In Part 1 of this study, behavioral patterns during the peri-estrus period were compared between cows with physiological estrus and cows subjected to an Ovsynch protocol. On the day before estrus and on the day of estrus/AI, cows with natural estrus showed a clear drop in rumination and "inactivity" and an increase in "high activity", based on an algorithm of the accelerometer system, whereas, cows in the Ovsynch protocol showed only minor changes in behavioral patterns. In Part 2, we analyzed behavioral patterns between synchronized cows that became pregnant after AI and synchronized cows that remained open. As a result, no differences were detected between these two Ovsynch groups before AI. Thus, in this study we found no evidence that behavioral patterns can be used to improve conception rates within an Ovsynch protocol.

Pulsoximetric monitoring of fetal arterial oxygen saturation and fetal pulse at stage II of labor to predict acidosis in newborn Holstein Friesian calves.

During stage II of parturition, the bovine fetus is at risk of oxygen deficiency caused by insufficient gas exchange between the dam and the fetus. The early detection of this critical condition, followed by assistance at calving, can help to improve the vitality of the newborn calf, or even prevent it from being born dead. By using pulse oximetry, the arterial oxygen saturation, as well as the pulse rate, can be continuously and non-invasively measured. The aim of our study was to identify critical thresholds for the parameters 'arterial oxygen saturation (FSpO2)' and 'pulse rate (PR)' that indicate a severe postnatal risk for calves to suffer from acidosis. FSpO2 and PR from 40 bovine fetuses were recorded during the last 25 min of calving with a commercially available pulse oximeter (Radius-7, Masimo Corporation, Irvine, USA). The calves were tested immediately after birth for acidosis by analyzing their blood with a portable blood gas analyzer (VetScan iStat1, Abaxis Inc., Union City, USA). Retrospectively, the pulsoximetric data were scanned for predefined patterns. The validity of these patterns to predict acidosis in newborn calves was analyzed by using receiver operating characteristics (ROC) curve analyses. In general, PR was a stronger predictive parameter for acidosis than FSpO2, with the greatest area under the curve (AUC) for the PR criteria 'Pulse rate > 120 beats per minute (bpm) for at least 2 min', with an AUC of 0.764, in contrast to an AUC of 0.613 for the best FSpO2 criteria 'FSpO2 < 40% for at least 50% of the measurement'. Further studies should investigate whether vitality after calving can be improved and fetal death rate can be reduced when obstetric assistance is performed as soon as one of these criteria apply to the bovine fetus. For more practical implementation in the field, improvement of the device's hardware would be necessary.

Evaluation of an ear-attached accelerometer for detecting estrus events in indoor housed dairy cows.

Precision dairy farming technologies have tremendous potential to improve and support farmers in herd management decisions, particularly in reproductive management. Nowadays, estrus detection in cows is challenging and several supporting tools are available. In this study, a 3D-accelerometer integrated into an ear-tag (SMARTBOW, Smartbow GmbH, Weibern, Austria) was used for the detection of cows in estrus. Movement pattern based on accelerometer data were analyzed and processed by algorithms and machine learning, resulting in estrus alerts. For the evaluation of the system, reproductive performance data of 579 estrus events of multiparous cows were used to retrospectively evaluate the accuracy of estrus alerts generated by the accelerometer-based system and the overall performance of the system. Estrus events were classified as 'gold standard' events, if an estrus followed by AI resulted in pregnancy, and as 'recorded estrus' events, if two estrus events with an interval of 18-25 d were in the herd records, independent of whether estrus was followed by AI or pregnancy. In total, 316 'gold standard' events were matched with estrus alerts generated by the accelerometer-based system, resulting in a sensitivity of 97%. Furthermore, 263 'recorded estrus' events were compared with correct or incorrect estrus alerts by the system. Sensitivity, specificity, positive and negative predictive values, accuracy, and error rate for 'recorded estrus' events were 97%, 98%, 96%, 94%, 96%, and 2%, respectively. In summary, the SMARTBOW system is suitable for an automated detection of estrus events of multiparous cows in indoor housed dairy cows.

The Multiple Sclerosis Severity Score: fluctuations and prognostic ability in a longitudinal cohort of patients with MS.

BACKGROUND: The Multiple Sclerosis Severity Score (MSSS), combining the Expanded Disability Status Scale (EDSS) and disease duration, attempts to stratify multiple sclerosis (MS) patients based on their rate of progression. Its prognostic ability in the individual patient remains unproven. OBJECTIVES: To assess the stability of MSSS within individual persons with MS in a longitudinal cohort, to evaluate whether certain factors influence MSSS variability, and to explore the ability of MSSS to predict future ambulatory function. METHODS: A single-center retrospective review was performed of patients following a single provider for at least 8 years. Mixed model regression modeled MSSS over time. A Kaplan-Meier survival plot was fitted, using change of baseline MSSS by at least one decile as the event. Cox modeling assessed the influence of baseline clinical and demographic factors on the hazard of changing MSSS by at least one decile. Linear models evaluated the impact of baseline EDSS, baseline MSSS, and other factors on the Timed 25-Foot Walk (T25FW). RESULTS: Out of 122 patients, 68 (55.7%) deviated from baseline MSSS by at least one decile. Final T25FW had slightly weaker correlation to baseline MSSS than to baseline EDSS, which was moderately strongly correlated with future log T25FW. CONCLUSION: Individual MSSS scores often vary over time. Clinicians should exercise caution when using MSSS to prognosticate.

Adaptation of two groups of Oenococcus oeni strains to red and white wines: the role of acidity and phenolic compounds.

AIMS: Oenococcus oeni is the lactic acid bacteria species which is the most adapted to wine. Recently, two groups of strains that form two genetic lineages were described in red and white Burgundy wines. The aim of this study was to analyse the phenotypes of these strains in order to determine how they have adapted specifically to either red or white wine. METHODS AND RESULTS: Four strains from each group were tested in grape must and in wines to evaluate their tolerance to pH and to phenolic compound content. White wine strains proved to be the most tolerant to low pH, both in grape must and in wine, whereas they were inhibited by the presence of grape tannins in wine. Red wine strains were more sensitive to acidity, but very resistant to phenolic compounds. CONCLUSIONS: The results suggest that pH and phenolic compounds drive strain selection at several stages of wine production. SIGNIFICANCE AND IMPACT OF THE STUDY: Although it is well known that O. oeni is well adapted to wine, this study shows that strains of some genetic lineages within this species have evolved to adapt better than others to specific types of wines.

Technical note: Evaluation of a wireless pulse oximeter for measuring arterial oxygen saturation and pulse rate in newborn Holstein Friesian calves.

Pulse oximetry is a well-established technique in human and veterinary medicine. In farm animals, it could also be a useful tool for the detection of critical conditions relating to oxygen supply and the cardiovascular system. Among other uses, an innovative application could be the monitoring of fetuses during birth. This could help in the early identification of critical situations and support farmers and veterinarians in their decision to start obstetric or life-support interventions. Until now, however, its use in ruminant medicine was still limited to experimental applications. The objective of this study was to evaluate the accuracy of the Radius-7 Wearable Pulse CO-Oximeter (Masimo Corporation, Irvine, CA) for monitoring vital parameters in newborn calves. All measurements were conducted on animals in the lying down position. The sensor of the pulse oximeter was placed in the interdigital space of the calves' front legs and fixed with a homemade latex hoof cover. The pulsoximetric measurements of arterial oxygen saturation (SpO2) in 40 newborn calves were compared with the corresponding results (SaO2) from a portable blood gas analyzer (VetScan iStat1, Abaxis Inc., Union City, CA), which served as the reference. For this, an arterial blood sample was taken from the medial intermediate branch of the caudal auricular artery. In addition, the pulse rate was measured in 10 calves aged between 0 and 7 d with the pulse oximeter and simultaneously with a heart rate belt (Polar Equine Belt, Polar Electro Oy, Kempele, Finland) to determine their level of agreement. Spearman correlation coefficient for oxygen saturation was 93.8% for the pulse oximeter and the blood gas analyzer, and 97.7% for the pulse rate measured with the pulse oximeter and the heart rate belt. Bland-Altman plots revealed an overestimation of SaO2 by 2.95 ± 6.39% and an underestimation of the pulse rate by -0.41 ± 3.18 beats per minute compared with the corresponding reference methods. In summary, the pulse oximeter is suitable for continuous monitoring of arterial oxygen saturation and pulse in newborn Holstein Friesian calves. For practical use, purpose-built technical equipment is required to attach the sensor to the calves' legs.

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition.

PURPOSE: The molecular mechanism of breast and/or ovarian cancer susceptibility remains unclear in the majority of patients. While germline mutations in the regulatory non-coding regions of BRCA1 and BRCA2 genes have been described, screening has generally been limited to coding regions. The aim of this study was to evaluate the contribution of BRCA1/2 non-coding variants. METHODS: Four BRCA1/2 non-coding regions were screened using high-resolution melting analysis/Sanger sequencing or next-generation sequencing on DNA extracted from index cases with breast and ovarian cancer predisposition (3926 for BRCA1 and 3910 for BRCA2). The impact of a set of variants on BRCA1/2 gene regulation was evaluated by site-directed mutagenesis, transfection, followed by Luciferase gene reporter assay. RESULTS: We identified a total of 117 variants and tested twelve BRCA1 and 8 BRCA2 variants mapping to promoter and intronic regions. We highlighted two neighboring BRCA1 promoter variants (c.-130del; c.-125C > T) and one BRCA2 promoter variants (c.-296C > T) inhibiting significantly the promoter activity. In the functional assays, a regulating region within the intron 12 was found with the same enhancing impact as within the intron 2. Furthermore, the variants c.81-3980A > G and c.4186-2022C > T suppress the positive effect of the introns 2 and 12, respectively, on the BRCA1 promoter activity. We also found some variants inducing the promoter activities. CONCLUSION: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

DNMT3A mutant transcript levels persist in remission and do not predict outcome in patients with acute myeloid leukemia.

We investigated the prognostic impact of minimal residual disease (MRD) monitoring in acute myeloid leukemia patients harboring DNA methyltransferase 3A-R882H/-R882C mutations (DNMT3Amut). MRD was determined by real-time quantitative PCR (RQ-PCR) in 1494 samples of 181 DNMT3Amut patients. At the time of diagnosis, DNMT3Amut transcript levels did not correlate with presenting clinical characteristics and concurrent gene mutations as well as the survival end points. In Cox regression analyses, bone marrow (BM) DNMT3Amut transcript levels (log10-transformed continuous variable) were not associated with the rate of relapse or death. DNMT3Amut transcript levels were significantly higher in BM than in blood after induction I (P=0.01), induction II (P=0.05), consolidation I (P=0.004) and consolidation II (P=0.008). With regard to the clinically relevant MRD time points, after two cycles of induction and at the end of therapy, DNMT3Amut transcript levels had no impact on the end point remission duration and overall survival. Of note, only a minority of the patients achieved RQ-PCR negativity, whereas most had constantly high DNMT3Amut transcript levels, a finding which is consistent with the persistence of clonal hematopoiesis in hematological remission.

Measurement of ß-hydroxybutyrate in capillary blood obtained from an ear to detect hyperketonemia in dairy cows by using an electronic handheld device.

The primary objective of the present study was to test whether capillary blood obtained by puncturing the skin of an ear with a minimal invasive lancet technique is able to detect hyperketonemia (HYK) in dairy cows. Furthermore, test characteristics of a new available handheld device, the FreeStyle Precision Neo (FSP-Neo, Abbott GmbH & Co. KG, Wiesbaden, Germany) for determination of ß-hydroxybutyrate (BHB) concentrations in bovine blood were evaluated by comparing the measurements with a laboratory reference. The BHB concentration was determined with the FSP-Neo device in 720 capillary blood samples from 3 different sampling sites (left, right ear, and repeated measurement) and in 240 samples from a coccygeal vessel. The concentration of BHB in serum harvested from the coccygeal blood samples was analyzed at the laboratory and was used as reference. The Spearman correlation coefficient (ρs) between the BHB concentrations in capillary blood measured with the handheld device and the reference test was between 0.76 and 0.81. Using capillary blood, the mean ± standard deviation BHB difference compared with the reference test was 0.20±0.47 mmol/L for all 3 sampling locations at the ears. The receiver operating characteristic analyses for the FSP-Neo device resulted in an optimized threshold for the detection of subclinical ketosis (SCK) in capillary blood of 1.3 mmol/L (left and right ear) and 1.2 mmol/L (repeated measurements). Applying these adjusted threshold sensitivities (Se) for all 3 capillary sampling sites at the ear were 100%, and specificities (Sp) ranged between 93 and 94%. Hence, we conclude that all sampling locations were suitable to identify cows suffering from SCK. The reference test compared with BHB measurements in coccygeal blood resulted in a ρs of 0.92 with a mean ± standard deviation of 0.02±0.21 mmol/L. The receiver operating characteristic analyses for the FSP-Neo device resulted in an optimized threshold for the detection of SCK in coccygeal blood of 1.1 mmol/L, with a corresponding Se and Sp of 100 and 95%, respectively. Because capillary blood is easily achievable from an ear, particularly if animals are fixed in headlocks for routine checkups, this technique is considered as an additional minimally invasive method for the identification of dairy cows suffering from HYK.

Preoperative anemia is associated with adverse outcome in patients with urothelial carcinoma of the bladder following radical cystectomy.

PURPOSE: Radical cystectomy (RC) can be associated with significant blood loss, whereas many patients are presenting with anemia preoperatively. To date, there is a lack of data addressing the impact of preoperative anemia (PA) on survival of patients undergoing RC for urothelial carcinoma of the bladder (UCB). METHODS: This retrospective multicenter study includes 684 patients with UCB undergoing RC with pelvic lymph node dissection. The median follow-up was 50 (IQR 29,78) months. Anemia was defined in line with the WHO classification (hemoglobin (Hb): male ≤13 g/dL, female ≤12 g/dL) and based on contemporary gender- and age-adjusted classification (Hb: white male aged <60 years: ≤13.7 g/dL; ≥60 years: ≤13.2 g/dL; white female of all ages ≤12.2 g/dL). Univariable and multivariable Cox regression analyses were used to assess the effects of PA on oncological outcomes. RESULTS: A total of 269 (39.3 %) and 302 (44.2 %) patients were anemic according to the WHO classification versus contemporary classification. Age, increased ECOG performance status, advanced tumor stages, lymph node metastasis, positive surgical margin and anemia were associated with disease recurrence (DR), cancer-specific mortality (CSM) and all-cause mortality (ACM). In multivariable analysis, anemia was an independent predictor of DR, CSM and ACM (WHO and/or contemporary classification). Blood transfusion was significantly associated with ACM in both classifications of anemia. CONCLUSIONS: PA is significantly associated with worse oncological outcome in patients undergoing RC. Based on the additional unfavorable influence of blood transfusion, this emphasizes the importance of early diagnosis and correction of anemia and implementation of alternative methods of blood volume management.

Clinical course in multiple sclerosis patients presenting with a history of progressive disease.

OBJECTIVES: Determine the likelihood of worsening clinical status in the near-term course of progressive MS and evaluate the predictive validity of our diagnostic impression of progressive forms of MS. METHODS: Retrospective review of charts from 175 patients seen between 2000 and 2007 who were diagnosed with either primary or secondary progressive multiple sclerosis. Data extracted included demographic factors, neurological examination findings to determine EDSS, timed 25 foot walk (T25FW) when available, duration of symptoms, clinical course as documented on initial visit, and history of disease-modifying agent (DMA) use. Significant change in EDSS was defined as a change of one point or more from initial to final clinical evaluation. Significant change in T25FW was defined as a ±20% difference from baseline. RESULTS: Of the 175 charts reviewed, 35 patients met criteria and had sufficient documentation to allow for EDSS abstraction. Twenty-four patients (68.6%) showed no significant change in EDSS from baseline while eleven patients (31.4%) worsened and none improved. For those patients that had T25FW data available, 6 out of 20 (30%) patients worsened while 11 (55%) showed no change. Three patients (15%) improved. CONCLUSION: In this observational study at a tertiary care MS center, patients classified as progressive MS did not progress as often, or as rapidly, as previous studies have suggested. Greater than two-thirds of patients in this cohort, did not increase 1 step on the EDSS.

Fulvestrant induces resistance by modulating GPER and CDK6 expression: implication of methyltransferases, deacetylases and the hSWI/SNF chromatin remodelling complex.

BACKGROUND: Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance. METHODS: Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated. RESULTS: Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations. CONCLUSION: The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.

Assessment of the respiratory sensitization potential of proteins using an enhanced mouse intranasal test (MINT).

There remains a need for a simple and predictive animal model to identify potential respiratory sensitizers. The mouse intranasal test (MINT) was developed to assess the relative allergic potential of detergent enzymes, however, the experimental endpoints were limited to evaluation of antibody levels. The present study was designed to evaluate additional endpoints (serum and allergic antibody levels, pulmonary inflammation and airway hyperresponsiveness (AHR)) to determine their value in improving the predictive accuracy of the MINT. BDF1 mice were intranasally instilled on days 1, 3, 10, 17 and 24 with subtilisin, ovalbumin, betalactoglobulin, mouse serum albumin or keyhole limpet hemocyanin; challenged with aerosolized methacholine or the sensitizing protein on day 29 to assess AHR, and sacrificed on day 29 or 30. Under the conditions of this study, evaluation of AHR did not improve the predictive power of this experimental model. Allergic antibody responses and IgG isotype characterization proved to be the most sensitive and reliable indicators of the protein allergenic potential with BAL responses providing additional insight. These data highlight that the evaluation of the respiratory sensitization potential of proteins can be best informed when multiple parameters are evaluated and that further improvements and refinements of the assay are necessary.

In vitro inhibition of breast cancer spheroid-induced lymphendothelial defects resembling intravasation into the lymphatic vasculature by acetohexamide, isoxsuprine, nifedipin and proadifen.

BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

Bay11-7082 inhibits the disintegration of the lymphendothelial barrier triggered by MCF-7 breast cancer spheroids; the role of ICAM-1 and adhesion.

BACKGROUND: Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics. METHODS: Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs. RESULTS: Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively. CONCLUSION: Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells.

BACKGROUND: The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts. METHODS: To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs. RESULTS: We show that tumour spheroids generate 'circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 µM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general. INTERPRETATION: These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell-cell contacts, migration, and the generation of CCID.