Impaired Spleen Formation Perturbs Morphogenesis of the Gastric Lobe of the Pancreas

Despite the extensive use of the mouse as a model for studies of pancreas development and disease, the development of the gastric pancreatic lobe has been largely overlooked. In this study we use optical projection tomography to provide a detailed three-dimensional and quantitative description of pancreatic growth dynamics in the mouse. Hereby, we describe the epithelial and mesenchymal events leading to the formation of the gastric lobe of the pancreas. We show that this structure forms by perpendicular growth from the dorsal pancreatic epithelium into a distinct lateral domain of the dorsal pancreatic mesenchyme. Our data support a role for spleen organogenesis in the establishment of this mesenchymal domain and in mice displaying perturbed spleen development, including Dh +/−, Bapx1−/− and Sox11−/−, gastric lobe development is disturbed. We further show that the expression profile of markers for multipotent progenitors is delayed in the gastric lobe as compared to the splenic and duodenal pancreatic lobes. Altogether, this study provides new information regarding the developmental dynamics underlying the formation of the gastric lobe of the pancreas and recognizes lobular heterogeneities regarding the time course of pancreatic cellular differentiation. Collectively, these data are likely to constitute important elements in future interpretations of the developing and/or diseased pancreas

Utility of Pathology Imagebase for Standardization of Prostate Cancer Grading

Aims: Despite efforts to standardise grading of prostate cancer, even among experts there is still a considerable variation in grading practices. In this study we describe the use of Pathology Imagebase, a novel reference image library, for setting an international standard in prostate cancer grading. Methods and results: The International Society of Urological Pathology (ISUP) recently launched a reference image database supervised by experts. A panel of 24 international experts in prostate pathology reviewed independently microphotographs of 90 cases of prostate needle biopsies with cancer. A linear weighted kappa of 0.67 (95% confidence interval = 0.62-0.72) and consensus was reached in 50 cases. The interobserver weighted kappa varied from 0.48 to 0.89. The highest level of agreement was seen for Gleason score (GS) 3 + 3 = 6 (ISUP grade 1), while higher grades and particularly GS 4 + 3 = 7 (ISUP grade 3) showed considerable disagreement. Once a two-thirds majority was reached, images were moved automatically into a public database available for all ISUP members at www.isupweb.org. Non-members are able to access a limited number of cases. Conclusions: It is anticipated that the database will assist pathologists to calibrate their grading and, hence, decrease interobserver variability. It will also help to identify instances where definitions of grades need to be clarified

Epigenomic perturbation of novel EGFR enhancers reduces the proliferative and invasive capacity of glioblastoma and increases sensitivity to temozolomide

Background: Glioblastoma (GB) is the most aggressive of all primary brain tumours and due to its highly invasive nature, surgical resection is nearly impossible. Patients typically rely on radiotherapy with concurrent temozolomide (TMZ) treatment and face a median survival of ~ 14 months. Alterations in the Epidermal Growth Factor Receptor gene (EGFR) are common in GB tumours, but therapies targeting EGFR have not shown significant clinical efficacy. Methods: Here, we investigated the influence of the EGFR regulatory genome on GB cells and identified novel EGFR enhancers located near the GB-associated SNP rs723527. We used CRISPR/Cas9-based approaches to target the EGFR enhancer regions, generating multiple modified GB cell lines, which enabled us to study the functional response to enhancer perturbation. Results: Epigenomic perturbation of the EGFR regulatory region decreases EGFR expression and reduces the proliferative and invasive capacity of glioblastoma cells, which also undergo a metabolic reprogramming in favour of mitochondrial respiration and present increased apoptosis. Moreover, EGFR enhancer-perturbation increases the sensitivity of GB cells to TMZ, the first-choice chemotherapeutic agent to treat glioblastoma. Conclusions: Our findings demonstrate how epigenomic perturbation of EGFR enhancers can ameliorate the aggressiveness of glioblastoma cells and enhance the efficacy of TMZ treatment. This study demonstrates how CRISPR/Cas9-based perturbation of enhancers can be used to modulate the expression of key cancer genes, which can help improve the effectiveness of existing cancer treatments and potentially the prognosis of difficult-to-treat cancers such as glioblastoma

Additional file 1 of Epigenomic perturbation of novel EGFR enhancers reduces the proliferative and invasive capacity of glioblastoma and increases sensitivity to temozolomide

Additional file 1: Supplementary Figure 1. Recruitment of dCas9-KRAB repressor complex leads to enrichment of H3K9me3 at specic targeted sites in U251 cells. A-F, Bar charts depicting the enrichment of H3K9me3, as determined by ChIP-qPCR, at each genomic region (i.e., CE5B, CE6B, CE8, Promoter) and in each of the EGFR enhancer-repressed lines: iCE5B (B), iCE6B (C), iCE5B+6B (D) and iCE8 (E), alongside the iPromoter (F) and control line (A). Supplementary Figure 2. CRISPRi of EGFR enhancer CE5B+6B in Human Glioblastoma cell line U3013 downregulates EGFR gene expression, reduces cell proliferation rate, and sensitises cells to temozolomide (TMZ). A, EGFR gene expression as determined by RT-qPCR. B, Proliferation rates of the iCE5B+6B and promoter-repressed U3013 cell lines determined by live-cell imaging. Images were acquired every 4 hours and proliferation was determined by automatic cell count. Data is normalised to t=0 and presented as mean ± SEM (n=3). Statistical significance was assessed by unpaired t test (* P < 0.05, ** P < 0.01). C-D, Proliferation rates of control U3013 and iCE5B+6B enhancer-repressed U3013 cells determined by live-cell imaging upon treatment with 125μM TMZ in comparison with the DMSO-treated control. Images were acquired every 4 hours and proliferation was determined by automatic cell count. Data is normalised to t=0h and represented as mean ± SEM (n=4). P values were determined by unpaired t test. Supplementary Figure 3. CRISPR/Cas9-mediated deletion of EGFR enhancers in U251 cells downregulates EGFR gene expression and affects cell proliferation rates. A, Genotyping PCR of the EGFR enhancer-deleted cell lines (ΔCE5B, ΔCE6B, ΔCE5B+6B, ΔCE8) alongside the Δ Promoter and empty vector control lines (left), and schematic outline of the PCR genotyping strategy (right). Note that the wild-type CE5B+6B allele is too large to be amplied under these conditions. Full uncropped gel image presented in Supplementary Figure 6. B, EGFR gene expression levels relative to HPRT in EGFR enhancer-deleted cell lines as determined by RT-qPCR assays. Data is represented as mean ± SEM (n=3). Statistical significance as assessed by unpaired t test with Welch’s correction (* P < 0.05, ** P < 0.01). C, Cropped western blots showing EGFR protein expression and normalised to GAPDH protein levels (full uncropped blots in Supplementary Figure 6). D, Proliferation rates of cell lines carrying EGFR enhancer deletions or promoter deletions as determined by live-cell imaging, and in comparison to the empty vector control line. Images were acquired every 4 hours and proliferation was determined by automatic cell count. Data is normalised to t= 0h and plotted as mean ± SEM (n=3). Supplementary Figure 4. CRISPRi of the EGFR enhancer CE5B+6B and promoter compromises the invasive capacity of U251 cells. A, Representative images of the fusion between the fetal brain spheroids and the GFP+ tumour spheres generated from control, iCE5B+6B and iPromoter lines. Scale bars 750µm (0h) and 300µm (2-24h). B, Quantication of fusion events across a pool of independent tumour sphere/brain spheroid confrontation assays (n=10). C, Quantication of invasion frequency between the GFP+ tumour spheres and brain spheroids between 0h and 96h. D, Representative images of invasion/non-invasion between GFP+ tumour spheres and brain spheroids. Scale bar 100µm. Arrowheads point to areas of invasion. E-F, Line plots comparing the rate at which the respective cell lines migrate either from media containing 1% FBS to 1% FBS (no-chemoattractant negative control) or from 1% FBS to 10% FBS (chemoattractant condition). Migration was assessed by live-cell imaging taking images every hour and migration rate was determined by automatic quantification of the area of migrated cells. Data is represented as mean ± SEM (n=3). Supplementary Figure 5. Expression of key metabolic genes in the EGFR enhancer-repressed iCE5B+6B cell line. A, Results from qPCR analysis where certain metabolic genes are upregulated in the iCE5B+6B line relative to the control. Gene expression levels are relative to HPRT. Supplementary Figure 6. Uncropped western blot and gel images. A-B, Western blot on repressed cell lines (A) and deletion cell lines (B) detecting EGFR and GAPDH expression. Cropped images presented in Figure 1G and Supplementary Figure 3C, respectively. C, Uncropped genotyping gel from enhancer-deletion U251 cell lines. Cropped image presented in Supplementary Figure 3A

Rewiring of the promoter-enhancer interactome and regulatory landscape in glioblastoma orchestrates gene expression underlying neurogliomal synaptic communication

Chromatin organization controls transcription by modulating 3D-interactions between enhancers and promoters in the nucleus. Alterations in epigenetic states and 3D-chromatin organization result in gene expression changes contributing to cancer. Here, we map the promoter-enhancer interactome and regulatory landscape of glioblastoma, the most aggressive primary brain tumour. Our data reveals profound rewiring of promoter-enhancer interactions, chromatin accessibility and redistribution of histone marks in glioblastoma. This leads to loss of long-range regulatory interactions and overall activation of promoters, which orchestrate changes in the expression of genes associated to glutamatergic synapses, axon guidance, axonogenesis and chromatin remodelling. SMAD3 and PITX1 emerge as major transcription factors controlling genes related to synapse organization and axon guidance. Inhibition of SMAD3 and neuronal activity stimulation cooperate to promote proliferation of glioblastoma cells in co-culture with glutamatergic neurons, and in mice bearing patient-derived xenografts. Our findings provide mechanistic insight into the regulatory networks that mediate neurogliomal synaptic communication

Differences in bronchodilating potency of salbutamol in Turbuhaler as compared with a pressurized metered-dose inhaler formulation in patients with reversible airway obstruction

Two studies are presented, with the aim of establishing the dose potency ratio for salbutamol given via Turbuhaler and via a pressurized metered-dose inhaler (pMDI). Both studies were of a double-blind, randomized design. Outpatients with mild-to-moderate chronic reversible airway obstruction were given single doses of salbutamol administered via Turbuhaler and via pMDI. Efficacy and safety variables were measured before and during 6 h after each dose. The first study was a four-way crossover study including 12 patients. The salbutamol doses given were: 50, 100 and 2x100 microg via Turbuhaler and 2x100 microg via pMDI (Ventolin). The study showed that 2x100 microg of salbutamol inhaled via Turbuhaler is more potent than 2x100 microg salbutamol inhaled via a pMDI, and that 100 microg salbutamol via Turbuhaler is at least as potent as 2x100 microg salbutamol inhaled via a pMDI. The second study including 50 patients was a placebo-controlled five-way crossover, study. Two doses of salbutamol via Turbuhaler, 50 and 2x100 microg, and via pMDI, 100 and 2x200 microg, were given. There was a dose-dependent response in forced expiratory volume in one second (FEV1) for both inhalers. Adjusted for differences in baseline FEV1 values, the estimated relative dose potency for Turbuhaler versus pMDI was 1.98:1 (95% confidence interval 12-3.2). These studies showed that the same bronchodilating effect can be achieved when half the dose of salbutamol given via a conventional pressurized metered-dose inhaler is given via Turbuhaler

Improving signal detection in emission optical projection tomography via single source multi-exposure image fusion

Abstract We demonstrate a technique to improve structural data obtained from Optical Projection Tomography (OPT) using Image Fusion (IF) and contrast normalization. This enables the visualization of molecular expression patterns in biological specimens with highly variable contrast values. In the approach, termed IF-OPT, different exposures are fused by assigning weighted contrasts to each. When applied to projection images from mouse organs and digital phantoms our results demonstrate the capability of IF-OPT to reveal high and low signal intensity details in challenging specimens. We further provide measurements to highlight the benefits of the new algorithm in comparison to other similar methods