[Show abstract][Hide abstract] ABSTRACT: Over the past decade significant advances have been made in the fields of genomic and transcriptomic profiling, inspired by the advent of next-generation sequencing (NGS). Yet despite the considerable promise of these new technologies, uptake has been slow. The focus of this review is the use of next-generation transcriptomic analysis in the field of cancer endothelial biology, highlighting its advantages and a few of the disadvantages compared with current-generation technologies.
European Pharmaceutical Review 09/2015; 20(4):10-13.
[Show abstract][Hide abstract] ABSTRACT: Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.
[Show abstract][Hide abstract] ABSTRACT: We previously identified CLEC14A as a tumour endothelial marker. Here we show that CLEC14A is a regulator of sprouting angiogenesis in vitro and in vivo. Using a human umbilical vein endothelial cell spheroid-sprouting assay, we found CLEC14A to be a regulator of sprout initiation. Analysis of endothelial sprouting in aortic ring and in vivo subcutaneous sponge assays from clec14a(+/+) and clec14a(-/-) mice revealed defects in sprouting angiogenesis in CLEC14A-deficient animals. Tumour growth was retarded and vascularity reduced in clec14a(-/-) mice. Pull-down and co-immunoprecipitation experiments confirmed that MMRN2 binds to the extracellular region of CLEC14A. The CLEC14A-MMRN2 interaction was interrogated using mouse monoclonal antibodies. Monoclonal antibodies were screened for their ability to block this interaction. Clone C4, but not C2, blocked CLEC14A-MMRN2 binding. C4 antibody perturbed tube formation and endothelial sprouting in vitro and in vivo, with a similar phenotype to loss of CLEC14A. Significantly, tumour growth was impaired in C4-treated animals and vascular density was also reduced in the C4-treated group. We conclude that CLEC14A-MMRN2 binding has a role in inducing sprouting angiogenesis during tumour growth, which has the potential to be manipulated in future antiangiogenic therapy design.Oncogene advance online publication, 9 March 2015; doi:10.1038/onc.2015.34.
[Show abstract][Hide abstract] ABSTRACT: Background:
Lung cancer remains the leading cause of cancer-related death, largely owing to the lack of effective treatments. A tumour vascular targeting strategy presents an attractive alternative; however, the molecular signature of the vasculature in lung cancer is poorly explored. This work aimed to identify novel tumour vascular targets in lung cancer.
Enzymatic digestion of fresh tissue followed by endothelial capture with Ulex lectin-coated magnetic beads was used to isolate the endothelium from fresh tumour specimens of lung cancer patients. Endothelial isolates from the healthy and tumour lung tissue were subjected to whole human genome expression profiling using microarray technology.
Bioinformatics analysis identified tumour endothelial expression of angiogenic factors, matrix metalloproteases and cell-surface transmembrane proteins. Predicted novel tumour vascular targets were verified by RNA-seq, quantitative real-time PCR analysis and immunohistochemistry. Further detailed expression profiling of STEAP1 on 82 lung cancer patients confirmed STEAP1 as a novel target in the tumour vasculature. Functional analysis of STEAP1 using siRNA silencing implicates a role in endothelial cell migration and tube formation.
The identification of cell-surface tumour endothelial markers in lung is of interest in therapeutic antibody and vaccine development.
British Journal of Cancer 12/2014; 112(3). DOI:10.1038/bjc.2014.626 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Regulatory T cells (Treg) are enriched in human colorectal cancer (CRC) where they suppress anti-tumour immunity. The chemokine receptor CCR5 has been implicated in the recruitment of Treg from blood into CRC and tumour growth is delayed in CCR5-/- mice, associated with reduced tumour Treg infiltration.
Tissue and blood samples were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were phenotyped for chemokine receptors using flow cytometry. The presence of tissue chemokines was assessed. Standard chemotaxis and suppression assays were performed and the effects of CCR5 blockade were tested in murine tumour models.
Functional CCR5 was highly expressed by human CRC infiltrating Treg and CCR5(high) Treg were more suppressive than their CCR5(low) Treg counterparts. Human CRC-Treg were more proliferative and activated than other T cells suggesting that local proliferation could provide an alternative explanation for the observed tumour Treg enrichment. Pharmacological inhibition of CCR5 failed to reduce tumour Treg infiltration in murine tumour models although it did result in delayed tumour growth.
CCR5 inhibition does not mediate anti-tumour effects as a consequence of inhibiting Treg recruitment. Other mechanisms must be found to explain this effect. This has important implications for anti-CCR5 therapy in CRC.
British Journal of Cancer 11/2014; 112(2). DOI:10.1038/bjc.2014.572 · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor endothelial specific expression of Robo4 in adults identifies this plasma membrane protein as an anti-cancer target for immunotherapeutic approaches, such as vaccination. In this report, we describe how vaccination against Robo4 inhibits angiogenesis and tumor growth. To break tolerance to the auto-antigen Robo4, mice were immunised with the extracellular domain of mouse Robo4, fused to the Fc domain of human immunoglobulin within an adjuvant. Vaccinated mice show a strong antibody response to Robo4, with no objectively detectable adverse effects on health. Robo4 vaccinated mice showed impaired fibrovascular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The anti-tumor effect of Robo4 vaccination was present in CD8 deficient mice but absent in B cell or IgG1 knockout mice, suggesting antibody dependent cell mediated cytotoxicity as the anti-vascular/anti-tumor mechanism. Finally, we show that an adjuvant free soluble Robo4-carrier conjugate can retard tumor growth in carrier primed mice. These results point to appropriate Robo4 conjugates as potential anti-angiogenic vaccines for cancer patients.
[Show abstract][Hide abstract] ABSTRACT: We sought to determine a role for platelets in in vivo angiogenesis, quantified by changes in the capillary to fibre ratio (C∶F) of mouse skeletal muscle, utilising two distinct forms of capillary growth to identify differential effects. Capillary sprouting was induced by muscle overload, and longitudinal splitting by chronic hyperaemia. Platelet depletion was achieved by anti-GPIbα antibody treatment. Sprouting induced a significant increase in C∶F (1.42±0.02 vs. contralateral 1.29±0.02, P<0.001) that was abolished by platelet depletion, while the significant C∶F increase caused by splitting (1.40±0.03 vs. control 1.28±0.03, P<0.01) was unaffected. Granulocyte/monocyte depletion showed this response was not immune-regulated. VEGF overexpression failed to rescue angiogenesis following platelet depletion, suggesting the mechanism is not simply reliant on growth factor release. Sprouting occurred normally following antibody-induced GPVI shedding, suggesting platelet activation via collagen is not involved. BrdU pulse-labelling showed no change in the proliferative potential of cells associated with capillaries after platelet depletion. Inhibition of platelet activation by acetylsalicylic acid abolished sprouting, but not splitting angiogenesis, paralleling the response to platelet depletion. We conclude that platelets differentially regulate mechanisms of angiogenesis in vivo, likely via COX signalling. Since endothelial proliferation is not impaired, we propose a link between COX1 and induction of endothelial migration.
PLoS ONE 09/2014; 9(9):e107503. DOI:10.1371/journal.pone.0107503 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RhoJ is a RhoGTPase expressed in endothelial cells and tumour cells which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; siRNA-mediated knockdown of RhoJ increased focal adhesion disassembly time, while expression of an active mutant (daRhoJ) decreased it. Further, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast-2-hybrid, which depended on the Spa homology domain of GIT1. GIT1, GIT2, β-PIX and RhoJ all co-localised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of GIT1/2 or β-PIX phenocopying RhoJ knockdown. RhoJ knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give novel insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.
[Show abstract][Hide abstract] ABSTRACT: The vascular disruption, blood vessel loss and cavitation that occur at spinal cord injury (SCI) epicentres in mice and rats are different, but few studies have compared the acute SCI response in the two species. This is of interest since key elements of the rat SCI response are shared with humans. In this study, we investigated acute SCI responses and characterised changes in pro- and anti-angiogenic factors and matrix deposition in both species. Cavitation was absent in mouse but the area of the lesion site was 21- and 27-fold larger at 8 and 15 days post lesion (dpl), respectively, in the rat compared to intact control. The absence of wound cavitation in the mouse was correlated with increased levels of immunoreactive pro-angiogenic, pro-matrix and pro-wound healing factors, e.g. laminin, matrix metalloproteinase-1 (MMP-1) and vascular endothelial growth factor-A (VEGF-A) within the wound, which were 6.0-, 2.9-, and 2.8-fold, respectively, higher in the mouse compared to rats at 8 dpl. Increased axonal sparing was observed after DC injury, detected by higher levels of PKC-γ immunoreactivity in the dorsal column of mice compared to rats at both T7 and T9 spinal segments. Despite similar post SCI deficits in plantar heat tests at 2 hours after injury (1.4- and 1.6-fold lower than control mice and rats, respectively), by 7 days the magnitude of these responses were comparable to sham-treated controls in both species, while no post-SCI changes in Von Frey hair filament test response were observed in either species. We conclude that the more robust angiogenesis/wound healing response in the mouse attenuates post-injury wound cavitation. Although the spinal cord functions that were monitored post-injury were similarly affected in both species, we suggest that the quality of the angiogenesis/wound healing response together with the diminished lesion size seen after mouse SCI may protect against secondary axon damage and create an environment more conducive to axon sprouting/regeneration. These results suggest the potential therapeutic utility of manipulating the angiogenic response after human SCI.
[Show abstract][Hide abstract] ABSTRACT: Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.
PLoS ONE 04/2014; 9(4):e95097. DOI:10.1371/journal.pone.0095097 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chimeric antigen receptor (CAR) therapy combines the specificity of a monoclonal antibody with the potent cytotoxic and immune regulatory activity of self-replicating T cells. This approach has shown remarkable efficacy in recent clinical trials targeting T cells to hematological cancers. More limited benefit has been reported with solid tumors, possibly because of lack of suitable target antigens, immune evasion mechanisms in the malignant cells and/or lack of T cell infiltration into the tumor tissue. An alternative approach that avoids some of these problems is to use a CAR that targets the tumor vasculature rather than the malignant cells directly.
CLEC14A is a glycoprotein recently identified to be selectively overexpressed on the surface of the tumor vasculature in a wide range of human cancers, including ovarian, liver, bladder, prostate, breast and kidney, and is therefore of considerable interest for tumor therapy. Here we explore for the first time the feasibility of targeting the tumor vasculature using CLEC14A-specific CARs. Initially we generated CARs from several CLEC14A-specific monoclonal antibodies and then expressed them in T cells. In vitro T cell functional assays demonstrated that these CARs can trigger potent antigen-specific activation including cytotoxicity in engineered T cells. These CARs recognize both human and mouse forms of CLEC14A, so safety/efficacy studies could be conducted using mouse models. Following infusion of escalating doses of engineered T cells into healthy mice, circulating CAR-T cells persisted for at least the next 5 weeks without signs of toxicity. To explore anti-tumor effects, Lewis lung carcinoma cells were subcutaneously implanted into C57BL6 mice and 4 days later mice received a single dose of CLEC14A-specific CAR-T cells. Our recent data demonstrate statistically significant inhibition of tumor growth in mice treated with CAR-T cells compared with mock-transduced T cells. The engineered cells also appeared to expand/accumulate at the tumor site. These data suggest CLEC14A can be safely and effectively targeted with CAR-T cells, potentially offering a potent and widely applicable cancer therapy.
Proceedings of the 105th Annual Meeting of the American Association for Cancer Research, San Diego, CA. Philadelphia (PA); 04/2014
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Hepatitis C virus (HCV) is a major cause of global morbidity, causing chronic liver injury that can progress to cirrhosis and hepatocellular carcinoma. The liver is a large and complex organ containing multiple cell types, including hepatocytes, sinusoidal endothelial cells (LSEC), Kupffer cells, and biliary epithelial cells. Hepatocytes are the major reservoir supporting HCV replication; however, the role of nonparenchymal cells in the viral lifecycle remains largely unexplored. LSEC secrete factors that promote HCV infection and transcript analysis identified bone morphogenetic protein 4 (BMP4) as a candidate endothelial-expressed proviral molecule. Recombinant BMP4 increased HCV replication and neutralization of BMP4 abrogated the proviral activity of LSEC-conditioned media. Importantly, BMP4 expression was negatively regulated by vascular endothelial growth factor A (VEGF-A) by way of a VEGF receptor-2 (VEGFR-2) primed activation of p38 MAPK. Consistent with our in vitro observations, we demonstrate that in normal liver VEGFR-2 is activated and BMP4 expression is suppressed. In contrast, in chronic liver disease including HCV infection where there is marked endothelial cell proliferation, we observed reduced endothelial cell VEGFR-2 activation and a concomitant increase in BMP4 expression.
These studies identify a role for LSEC and BMP4 in HCV infection and highlight BMP4 as a new therapeutic target for treating individuals with liver disease.
[Show abstract][Hide abstract] ABSTRACT: The behavior of vascular endothelial cells is greatly altered in sites of pathological angiogenesis, such as a developing tumour or atherosclerotic plaque. Until recently it was thought that this was largely due to abnormal chemical signalling, i.e. endothelial cell chemotransduction, at these sites. However, we now demonstrate that the shear stress intensity encountered by endothelial cells can have a profound impact on their gene expression and behaviour. We review the growing body of evidence suggesting that mechanotransduction, too, is a major regulator of pathological angiogenesis. This fits with the evolving story of physiological angiogenesis, where a combination of metabolic and mechanical signalling is emerging as the probable mechanism by which tight feedback regulation of angiogenesis is achieved in vivo. This article is protected by copyright. All rights reserved.
Microcirculation (New York, N.Y.: 1994) 01/2014; DOI:10.1111/micc.12119 · 2.57 Impact Factor
European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 12/2013; 40(2). DOI:10.1016/j.ejso.2013.11.025 · 3.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kinesin motor proteins exert essential cellular functions in all eukaryotes. They control mitosis, migration and intracellular transport through interaction with microtubules. Small molecule inhibitors of the mitotic kinesin KiF11/Eg5 are a promising new class of anti-neoplastic agents currently evaluated in clinical cancer trials for solid tumors and hematological malignancies. Here we report induction of Eg5 and four other mitotic kinesins including KIF20A/Mklp2 upon stimulation of in vivo angiogenesis with vascular endothelial growth factor-A (VEGF-A). Expression analyses indicate up-regulation of several kinesin-encoding genes predominantly in lymphoblasts and endothelial cells. Chemical blockade of Eg5 inhibits endothelial cell proliferation and migration in vitro. Mitosis-independent vascular outgrowth in aortic ring cultures is strongly impaired after Eg5 or Mklp2 protein inhibition. In vivo, interfering with KIF11/Eg5 function causes developmental and vascular defects in zebrafish and chick embryos and potent inhibition of tumor angiogenesis in experimental tumor models. Besides blocking tumor cell proliferation, impairing endothelial function is a novel mechanism of action of kinesin inhibitors.
[Show abstract][Hide abstract] ABSTRACT: Anticancer therapeutics have historically been targeted against malignant cells directly. These approaches often have limited efficacy, particularly in advanced disease, due to poor drug infiltration into the tumour. In recent years increasing interest has been focused on the development of alternative targeted therapies, which inhibit tumour development by disrupting the stromal cells that support it. This chapter explores the development of tumour vascular targeting therapies, the successes and setbacks and the encouraging potential of this approach to potentiate 11 the effect of other anti-tumour therapeutics.
Cancer Targeted Drug Delivery an Elusive Dream, 1st edited by Bae YH, Mrsny RJ, Park K, 07/2013: chapter 3: pages 59-95; Springer New York., ISBN: ISBN 978-1-4614-7876-8
[Show abstract][Hide abstract] ABSTRACT: In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in-vivo, we developed a biotinylation technique in the chicken embryo combined with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumours growing on the chorioallantoic membrane or after induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in-vivo biotinylation, in combination with a robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that may constitute the starting point for further developments.
[Show abstract][Hide abstract] ABSTRACT: Background
The development of disease-modifying therapies for Alzheimer’s disease is hampered by our lack of understanding of the early pathogenic mechanisms and the lack of early biomarkers and risk factors.
We have documented the expression pattern of mTOR regulated genes in the frontal cortex of Alzheimer’s disease patients. We have also examined the functional integrity of mTOR signaling in peripheral lymphocytes in Alzheimer’s disease patients relative to healthy controls.
In the brain mTOR is seen to control molecular functions related to cell cycle regulation, cell death and several metabolic pathways. These downstream elements of the mTOR signaling cascade are deregulated in the brain of Alzheimer’s disease patients well before the development of pathology. This dysregulation of the mTOR downstream signaling cascade is not restricted to the brain but appears to be systemic and can be detected in peripheral lymphocytes as a reduced Rapamycin response.
The dysfunction of the signaling pathways downstream of mTOR may represent a risk factor for Alzheimer’s disease and is independent of the ApoE status of the patients.
We have also identified the molecular substrates of the beneficial effects of Rapamycin on the nervous system. We believe that these results can further inform the development of clinical predictive tests for the risk of Alzheimer’s disease in patients with mild cognitive impairment.