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Glioblastoma (GBM) is an aggressive brain tumor that is resistant to conventional radiation and cytotoxic chemotherapies. We hypothesize that brain tumor initiating cells (BTICs), a subpopulation of treatment-resistant cells with stem cell properties cause tumor relapse and a subset of neural stem cell genes regulate BTIC self-renewal, driving GBM recurrence. We adapted the existing treatment protocol for adults with primary GBM for in vivo treatment of immunocompromised mice engrafted with GBMs. Post-chemoradiotherapy, the recovered GFP+GBMs were profiled for self-renewal and expression of critical stem cell genes. Using invitro and invivo gain-of-function/loss-of-function experiments, we investigated the regulatory functions of Bmi1 in primary neural stem & progenitor cells (NSPCs) and GBM tumor formation. Finally, global RNA-Seq profiling was performed to understand the consequences of Bmi1 dysregulation on target gene expression. GBM cells showed an increase in Bmi1 levels post-chemoradiotherapy, suggesting the presence of a treatment-refractory BTICs. GFP+cells extracted from treated xenografts had higher self-renewal and BTIC marker expression. Although treated mice responded to therapy, we observed tumor relapse with increased Bmi1 expression. Knockdown of Bmi1 diminished self-renewal and proliferation of GBM cells and delayed tumorigenesis, highlighting a critical role for Bmi1 in tumor maintenance. Conversely, over-expressing Bmi1 in NSPCs failed to initiate tumor formation in vivo. Using high-throughput sequencing data, we generated a map of signaling pathways dysregulated in GBM that may lead to tumor recurrence. Our data confirms the existence of a rare treatment-refractory BTIC population with enhanced self-renewal capacity that escapes therapy and drives tumor relapse.
Medulloblastoma (MB) is the most common malignant pediatric brain tumour, and is categorized into four molecular subgroups, with Group 3 MB having the worst prognosis due to the highest rate of metastatic dissemination and relapse. In this work, we describe the epigenetic regulator Bmi1 as a novel therapeutic target for treatment of recurrent Group 3 MB. Through comparative profiling of primary and recurrent MB, we show that Bmi1 defines a treatment-refractory cell population that is uniquely targetable by a novel class of small molecule inhibitors. We have optimized an in vivo mouse-adapted therapy model that has the advantage of generating recurrent, human, treatment-refractory MBs. Our preliminary studies showed that although chemoradiotherapy administered to mice engrafted with human MB showed reduction in tumour size, Bmi1 expression was enriched in the post-treatment residual tumour. Furthermore, we found that knockdown of Bmi1 in human recurrent MB cells decreases proliferation and self-renewing capacities of MB cells in vitro as well as both tumour size and extent of spinal leptomeningeal metastases in vivo. Oral administration of a potent Bmi1 inhibitor, PTC 028, resulted in a marked reduction in tumour burden and an increased survival in treatment cohort. Bmi1 inhibitors showed high specificity for MB cells and spared normal human neural stem cells, when treated with doses relevant for MB cells. As Group 3 medulloblastoma is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious agent such as Bmi1 inhibitor could be rapidly transitioned to clinical trials.
Despite aggressive multimodal therapy, human glioblastoma (hGBM), a highly malignant grade IV astrocytic tumour, remains incurable and inevitably relapses. Recent data has implicated intratumoral heterogeneity as the driver of therapy resistance and tumour relapse in hGBM. Thus models that capture the evolving hGBM biology in response to chemoradiotherapy will allow for the identification of cellular pathways that govern GBM therapy failure. In this study, we have developed a novel model to profile the clonal evolution of treatment naïve brain tumour initiating cell (BTIC) enriched hGBMs through chemoradiotherapy using: stem cell assays, BTIC marker expression and transcriptome analysis, immunohistochemistry, and cellular DNA barcoding technology. We report that treatment of hGBM BTICs leads to increased self-renewal capacity and higher transcript expression of stem cell genes Bmi1 and Sox2. Based on global transcriptome analysis of the in vitro treated hGBM, we also identify a hyper-aggressive form of glioma. Using our therapy-adapted hGBM-mouse xenograft model, we discover that despite tumour regression and increased mouse survival post-therapy, tumour relapse remains inevitable. The treatment-refractory cells again have increased self-renewal capacity and higher expression of Bmi1 and Sox2. Furthermore, by combining cellular DNA barcoding technology, which barcodes hGBM at single cell resolution, with our novel in vitro and in vivo therapy models, we are able to determine whether a pre-existing or a therapy driven subpopulation(s) seeds hGBM tumour relapse. Profiling the dynamic nature of heterogeneous hGBM subpopulations through disease progression and treatment may lead to the identification of novel therapeutic targets for the treatment of recurrent hGBM.
Medulloblastoma (MB), the most common malignant pediatric brain tumor, is categorized into four molecular subgroups. Given the high rate of metastatic dissemination at diagnosis and recurrence in Group 3 MBs, these patients have the worst clinical outcome with a 5-year survivorship of approximately 50%. By adapting the existing COG (Children’s Oncology Group) Protocol for children with newly diagnosed high-risk MB, for treatment of immuno-deficient mice intracranially engrafted with human MB brain tumour initiating cells we aim to identify and characterize the treatment-refractory cell population in Group 3 MBs. Mice were sacrificed at multiple time points during the course of tumor development and therapy: (i) at engraftment; (ii) post-radiation; (iii) post-radiation and chemotherapy; and (iv) at MB recurrence. MB cell populations recovered separately from brains and spines were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy. We report a higher expression of CD133, Sox2 and Bmi1 in addition to increased self-renewal capacity following chemoradiotherapy treatment. The enrichment map constructed from global gene expression analysis showed an increase in pathways regulating self-renewal, DNA repair and chemoresistance post-therapy despite the apparent decrease in tumour size and vascularity. Additionally, from gene expression at MB recurrence, we identified a list of genes that negatively correlate with survival in patients diagnosed with Group 3 MB. A differential genomic profile of the “treatment-responsive” tumors against those that fail therapy may contribute to discovery of novel therapeutic approaches for the most aggressive subgroup of MB.
Brain Metastases (BM) represent a leading cause of cancer mortality. While metastatic lesions contain subclones derived from their primary lesion, their functional characterization has been limited by a paucity of preclinical models accurately recapitulating the stages of metastasis. This work describes the isolation of a unique subset of metastatic stem-like cells from primary human patient samples of BM, termed brain metastasis initiating cells (BMICs). Utilizing these BMICs we have established a novel patient-derived xenograft (PDX) model of BM that recapitulates the entire metastatic cascade, from primary tumor initiation to micro-metastasis and macro-metastasis formation in the brain. We then comprehensively interrogated human BM to identify genetic regulators of BMICs using in vitro and in vivo RNA interference screens, and validated hits using both our novel PDX model as well as primary clinical BM specimens. We identified SPOCK1 and TWIST2 as novel BMIC regulators, where in our model SPOCK1 regulated BMIC self-renewal and tumor initiation, and TWIST2 specifically regulated cell migration from lung to brain. A prospective cohort of primary lung cancer specimens was used to establish that SPOCK1 and TWIST2 were only expressed in patients who ultimately developed BM, thus establishing both clinical and functional utility for these gene products. This work offers the first comprehensive preclinical model of human brain metastasis for further characterization of therapeutic targets, identification of predictive biomarkers, and subsequent prophylactic treatment of patients most likely to develop BM. By blocking this process, metastatic lung cancer would effectively become a localized, more manageable disease.
Developmental time, fecundity, adult weight, numbers, and age distribution of Brevicoryne brassicae (L.) and leaf area and plant quality of Maris Kestrel kale, Brassica oleracea L., were determined at three aphid densities and two watering rates in a factorial field-cage experiment. The watering treatment had no effect on the aphid or plant response variates. The aphid density treatment significantly affected final aphid numbers (low < medium = high density), leaf area (high < low = medium density), average adult weight measured at the end of the experiment (high < low < medium density), and weight of the adults in the developmental time measurement (high < low = medium density). The results suggested a strong aphid–plant interaction, but at densities which rarely occur in the field. Leaf water/dry weight was used to assess plant quality, but it was not a good predictor of any of the aphid response variates. Developmental time decreased, and fecundity increased as adult aphid weight increased. Production of fourth-instar apterae decreased logarithmically as aphid density increased.
In two experiments with female Scottish Blackface or Border Leicester x Scottish Blackface sheep nine or ten animals were slaughtered in mid lactation and the remainder either in November at the time of the subsequent mating (Expt 1) or at weaning (Expt 2).
The sheep were infused periodically, including just before slaughter, with 100 μCi tritiated water (TOH) in order to measure total body water by dilution and to estimate body fat using the inverse relationship between the proportions of fat and water in the body. The accuracy of the methods was assessed when the sheep were slaughtered. In the ton lactating Scottish Blackface sheep of Expt 1 fat made up 11.5% of the total body weight with an S.D. of 8·38% whereas the 11 sheep slaughtered at mating were twice as fat (23·2, S.D. 4·01 %). The 25 Border Leicester × Blackface sheep were all thin, whether they were slaughtered in mid lactation (4·4, S.D. 2·56%) or at weaning (3·4, S.D. 2·81%).
The standard error of estimate of body water from TOH space in Expt 2 was 1·2 kg c.v. 2·8%) and lower in Expt 1.
The precision with which an animal could be weighed was very important in determining the accuracy with which body fat could be predicted from live weight and TOH space. In both experiments the standard error of estimate for body fat in lactating sheep was between 600 and 700 g, compared with 1·3–2·7 kg when body fat was predicted from live weight alone. These estimates were sufficiently accurate to be of value in following changes in body composition in live animals as their nutritional and physiological state alters and for comparing animals in groups where the average fatness is greater and the range wider than in the sheep used in Expt 2.
A photogrammetric stereo imaging system was used to capture 3-D models of live pigs, and quantitative shape measurements were extracted from cross sections of the models. Stereo images were captured of 32 pigs, divided into high-lysine and low-lysine diet groups, and 3-D models were built from the images. Each pig was imaged once per week for 14 weeks. After slaughter, 10 of the pigs were dissected for muscle and fat measurements. A sequence of algorithms was applied to the 3-D models: differential geometry to reveal surface curvature features and detect the spine; manual landmark placement; fitting a curve to the spine; determining the vertical axis of the body; placing a slice plane across the abdomen close to the P2 position; extracting a cross section; and fitting a shape model to the cross section. Differential geometry revealed many qualitative features of the musculature. The spine was a line of minimum curvature along the back. The high-lysine pigs had higher height-to-width ratios and flatter backs than the low-lysine pigs. The dissected total muscle mass had a -0·66 correlation with the flatness-of-back shape parameter, and a 0·64 correlation with weight.
Perennial ryegrass (Lolium perenne L.) is often subject to transient waterlogging during winter under dryland conditions and summer when flood-irrigated. Despite this, little is known about the physiological responses of perennial ryegrass genotypes to waterlogging. In a pot experiment, four perennial ryegrass genotypes with contrasting root growth characteristics were subjected to waterlogging for 0, 3, 7, 14, 21 or 28 days. The masses of roots and shoots of the genotype Aurora6 were not significantly (P>0·05) reduced by waterlogging, throughout the experiment. All other genotypes exhibited reductions in root and shoot biomass between 14 and 21 days after waterlogging was imposed. The masses of laminae and roots of susceptible genotypes were reduced by up to 70% after 28 days of waterlogging. Aurora6 was also able to maintain photosynthesis for longer into the waterlogging period. However, after 28 days of waterlogging, photosynthesis of all genotypes was reduced by 30–50%. The waterlogging tolerance of Aurora6 was not due to its relatively poor root growth, as its progeny (2178), which also had poor root growth under control conditions, was susceptible to waterlogging. These findings show that there is variation in physiological processes and herbage yield of perennial ryegrass under waterlogged conditions. The implications of these findings for the genetic improvement of waterlogging tolerance of perennial ryegrass are discussed.
The 3D shape of live animals plays an important role in achieving good husbandry and in selecting breeding stock. Many shape features are subtle and cannot be extracted from 2D images. With 3D data, it would be possible to extract cross-sectional areas and volumes, and to measure features such as the squareness of the back muscles, which are known indicators of lean muscle mass (Whittemore 1998). However, there is currently no simple method to measure 3D shape in live animals. In this work a system has been developed for freezing the motion of a pig using flash photography and processing the images to extract the 3D surface shape. The imaging system is based on stereo photogrammetry. Three stereo pods, each consisting of two digital cameras, were set up at perpendicular directions in order to cover the whole of a cuboidal imaging volume. The imaging volume dimensions were 1300 mm long × 900 mm high × 700 mm deep. The imaging system was calibrated prior to capturing the pig images. Multiresolution correlation-based stereo matching (Siebert and Urquhart, 1994) was used to establish correspondences between the left and right images in each stereo pair. The output of the stereo matching of each pod was a 2.5D range image. These range images were integrated into a 3D model.
The use of body tissue by dairy cows to support lactation is increasing, since selection has led to cows that can eat only around half of their incremental feed requirements per unit increase in genetic merit for milk production (Veerkamp et al., 1995). Continuing with this selection policy is likely to result in increasing use of body tissue to fuel milk production and to lead to thinner cows with associated health and fertility problems. This has created increasing interest in body condition scoring (CS) in dairy cows as both an important management tool and also for use in selection indices. The ability to automatically record CS would increase the use of this measure in farm management and enable large volumes of data to be collected for use in national evaluations.
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