Assessing technical and biological variation in SWATH-MS-based proteomic analysis of chronic lymphocytic leukaemia cells.

Chronic lymphocytic leukaemia (CLL) exhibits variable clinical course and response to therapy, but the molecular basis of this variability remains incompletely understood. Data independent acquisition (DIA)-MS technologies, such as SWATH (Sequential Windowed Acquisition of all THeoretical fragments), provide an opportunity to study the pathophysiology of CLL at the proteome level. Here, a CLL-specific spectral library (7736 proteins) is described alongside an analysis of sample replication and data handling requirements for quantitative SWATH-MS analysis of clinical samples. The analysis was performed on 6 CLL samples, incorporating biological (IGHV mutational status), sample preparation and MS technical replicates. Quantitative information was obtained for 5169 proteins across 54 SWATH-MS acquisitions: the sources of variation and different computational approaches for batch correction were assessed. Functional enrichment analysis of proteins associated with IGHV mutational status showed significant overlap with previous studies based on gene expression profiling. Finally, an approach to perform statistical power analysis in proteomics studies was implemented. This study provides a valuable resource for researchers working on the proteomics of CLL. It also establishes a sound framework for the design of sufficiently powered clinical proteomics studies. Indeed, this study shows that it is possible to derive biologically plausible hypotheses from a relatively small dataset.

Mapping the Transcriptional and Fitness Landscapes of a Pathogenic E. coli Strain: The Effects of Organic Acid Stress under Aerobic and Anaerobic Conditions.

Several methods are available to probe cellular responses to external stresses at the whole genome level. RNAseq can be used to measure changes in expression of all genes following exposure to stress, but gives no information about the contribution of these genes to an organism's ability to survive the stress. The relative contribution of each non-essential gene in the genome to the fitness of the organism under stress can be obtained using methods that use sequencing to estimate the frequencies of members of a dense transposon library grown under different conditions, for example by transposon-directed insertion sequencing (TraDIS). These two methods thus probe different aspects of the underlying biology of the organism. We were interested to determine the extent to which the data from these two methods converge on related genes and pathways. To do this, we looked at a combination of biologically meaningful stresses. The human gut contains different organic short-chain fatty acids (SCFAs) produced by fermentation of carbon compounds, and Escherichia coli is exposed to these in its passage through the gut. Their effect is likely to depend on both the ambient pH and the level of oxygen present. We, therefore, generated RNAseq and TraDIS data on a uropathogenic E. coli strain grown at either pH 7 or pH 5.5 in the presence or absence of three SCFAs (acetic, propionic and butyric), either aerobically or anaerobically. Our analysis identifies both known and novel pathways as being likely to be important under these conditions. There is no simple correlation between gene expression and fitness, but we found a significant overlap in KEGG pathways that are predicted to be enriched following analysis of the data from the two methods, and the majority of these showed a fitness signature that would be predicted from the gene expression data, assuming expression to be adaptive. Genes which are not in the E. coli core genome were found to be particularly likely to show a positive correlation between level of expression and contribution to fitness.

The invasive proteome of glioblastoma revealed by laser-capture microdissection.

BACKGROUND: Glioblastomas are heterogeneous tumors composed of a necrotic and tumor core and an invasive periphery. METHODS: Here, we performed a proteomics analysis of laser-capture micro-dissected glioblastoma core and invasive areas of patient-derived xenografts. RESULTS: Bioinformatics analysis identified enriched proteins in central and invasive tumor areas. Novel markers of invasion were identified, the genes proteolipid protein 1 (PLP1) and Dynamin-1 (DNM1), which were subsequently validated in tumors and by functional assays. CONCLUSIONS: In summary, our results identify new networks and molecules that may play an important role in glioblastoma development and may constitute potential novel therapeutic targets.

Mapping the extracellular and membrane proteome associated with the vasculature and the stroma in the embryo.

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 for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the 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 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 might constitute the starting point for further developments.

The experimental renal cell carcinoma model in the chick embryo.

The clear cell subtype of renal carcinoma (CCRCC) is highly vascularized and despite a slow progression rate, it is potentially a highly aggressive tumor. Although a doubling of median progression-free survival in CCRCC patients treated by targeted therapies has been observed, the fact that tumors escape after anti-VEGF treatment suggests alternative pathways. The chick chorioallantoic membrane (CAM) is a well-established model, which allows in vivo studies of tumor angiogenesis and the testing of anti-angiogenic molecules. However, only a few data exist on CCRCC grafted onto CAM. We aimed to validate herein the CAM as a suitable model for studying the development of CCRCC and the interactions with the surrounding stroma. Our study uses both CCRCC cell lines and fresh tumor samples after surgical resection. We demonstrate that in both cases CCRCC can be grafted onto the CAM, to survive and to induce an angiogenic process. We further provide insights into the transcriptional regulation of the model by performing a differential analysis of tumor-derived and stroma-derived transcripts.

Gene expression analysis in human breast cancer associated blood vessels.

Angiogenesis is essential for solid tumour growth, whilst the molecular profiles of tumour blood vessels have been reported to be different between cancer types. Although presently available anti-angiogenic strategies are providing some promise for the treatment of some cancers it is perhaps not surprisingly that, none of the anti-angiogenic agents available work on all tumours. Thus, the discovery of novel anti-angiogenic targets, relevant to individual cancer types, is required. Using Affymetrix microarray analysis of laser-captured, CD31-positive blood vessels we have identified 63 genes that are upregulated significantly (5-72 fold) in angiogenic blood vessels associated with human invasive ductal carcinoma (IDC) of the breast as compared with blood vessels in normal human breast. We tested the angiogenic capacity of a subset of these genes. Genes were selected based on either their known cellular functions, their enriched expression in endothelial cells and/or their sensitivity to anti-VEGF treatment; all features implicating their involvement in angiogenesis. For example, RRM2, a ribonucleotide reductase involved in DNA synthesis, was upregulated 32-fold in IDC-associated blood vessels; ATF1, a nuclear activating transcription factor involved in cellular growth and survival was upregulated 23-fold in IDC-associated blood vessels and HEX-B, a hexosaminidase involved in the breakdown of GM2 gangliosides, was upregulated 8-fold in IDC-associated blood vessels. Furthermore, in silico analysis confirmed that AFT1 and HEX-B also were enriched in endothelial cells when compared with non-endothelial cells. None of these genes have been reported previously to be involved in neovascularisation. However, our data establish that siRNA depletion of Rrm2, Atf1 or Hex-B had significant anti-angiogenic effects in VEGF-stimulated ex vivo mouse aortic ring assays. Overall, our results provide proof-of-principle that our approach can identify a cohort of potentially novel anti-angiogenic targets that are likley to be, but not exclusivley, relevant to breast cancer.

Bioinformatic methods for finding differentially expressed genes in cDNA libraries, applied to the identification of tumour vascular targets.

The aim of this method is to guide a bench scientist to maximise cDNA library analyses to predict biologically relevant genes to pursue in the laboratory. Many groups have successfully utilised cDNA libraries to discover novel and/or differentially expressed genes in pathologies of interest. This is despite the high cost of cDNA library production using the Sanger method of sequencing, which produces modest numbers of expressed sequences compared to the total transcriptome. Both public and propriety cDNA libraries can be utilised in this way, and combining biologically relevant data can reveal biologically interesting genes. Pivotal to the quality of target identification are the selection of biologically relevant libraries, the accuracy of Expressed Sequence Tag to gene assignment, and the statistics used. The key steps, methods, and tools used to this end will be described using vascular targeting as an example. With the advent of next-generation sequencing, these or similar methods can be applied to find novel genes with this new source of data.

Functionally defining the endothelial transcriptome, from Robo4 to ECSCR.

We have applied search algorithms to expression databases to identify genes whose expression is restricted to the endothelial cell. Such genes frequently play a critical role in endothelial biology and angiogenesis. Two such genes are the roundabout receptor Robo4 and the ECSCR (endothelial-cell-specific chemotaxis regulator). Endothelial cells express both Robo1 and Robo4, which we have knocked down using siRNA (small interfering RNA) and then studied the effect in a variety of in vitro assays. Both Robo4 and Robo1 knockdown inhibited in vitro tube formation on Matrigel. Transfection of Robo4 into endothelial cells increased the number of filopodial extensions from the cell, but failed to do so in Robo1-knockdown cells. Separate immunoprecipitation studies showed that Robo1 and Robo4 heterodimerize. We conclude from this and other work that a heteroduplex of Robo1 and Robo4 signals through WASP (Wiskott-Aldrich syndrome protein) and other actin nucleation-promoting factors to increase the number of filopodia and cell migration. Knockdown of the transmembrane ECSCR protein in endothelial cells also reduced chemotaxis and impaired tube formation on Matrigel. Yeast two-hybrid analysis and immunoprecipitation studies showed that, in contrast with the roundabouts, ECSCR binds to the actin-modulatory filamin A. We conclude that all three of these genes are critical for effective endothelial cell migration and, in turn, angiogenesis.

A new procedure for determining the genetic basis of a physiological process in a non-model species, illustrated by cold induced angiogenesis in the carp.

BACKGROUND: Physiological processes occur in many species for which there is yet no sequenced genome and for which we would like to identify the genetic basis. For example, some species increase their vascular network to minimise the effects of reduced oxygen diffusion and increased blood viscosity associated with low temperatures. Since many angiogenic and endothelial genes have been discovered in man, functional homolog relationships between carp, zebrafish and human were used to predict the genetic basis of cold-induced angiogenesis in Cyprinus Carpio (carp). In this work, carp sequences were collected and built into contigs. Human-carp functional homolog relationships were derived via zebrafish using a new Conditional Stepped Reciprocal Best Hit (CSRBH) protocol. Data sources including publications, Gene Ontology and cDNA libraries were then used to predict the identity of known or potential angiogenic genes. Finally, re-analyses of cold carp microarray data identified carp genes up-regulated in response to low temperatures in heart and muscle. RESULTS: The CSRBH approach outperformed all other methods and attained 8,726 carp to human functional homolog relationships for 16,650 contiguous sequences. This represented 3,762 non-redundant genes and 908 of them were predicted to have a role in angiogenesis. The total number of up-regulated differentially expressed genes was 698 and 171 of them were putatively angiogenic. Of these, 5 genes representing the functional homologs NCL, RHOA, MMP9, GRN and MAPK1 are angiogenesis-related genes expressed in response to low temperature. CONCLUSION: We show that CSRBH functional homologs relationships and re-analyses of gene expression data can be combined in a non-model species to predict genes of biological interest before a genome sequence is fully available. Programs to run these analyses locally are available from http://www.cbrg.ox.ac.uk/~jherbert/.

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains.

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.

ECSM2, an endothelial specific filamin a binding protein that mediates chemotaxis.

OBJECTIVE: We aimed to characterize the expression and function of a novel transcript that bioinformatics analysis predicted to be endothelial specific, called endothelial-specific molecule-2 (ECSM2). METHODS AND RESULTS: A full-length cDNA was isolated and predicted ECSM2 to be a putative 205-amino acid transmembrane protein that bears no homology to any known protein. Quantitative polymerase chain reaction analysis in vitro and in situ hybridization analysis in vivo confirmed ECSM2 expression to be exclusively endothelial, and localization to the plasma membrane was shown. Knockdown of ECSM2 expression in human umbilical vein endothelial cells using siRNA resulted in both reduced chemotaxis and impaired tube formation on matrigel, a solubilized basement membrane, both processes involved in angiogenesis. A yeast 2 hybrid analysis using the ECSM2 intracellular domain identified filamin A as an interacting protein. This interaction was confirmed by precipitation of filamin-A from endothelial cell lysates by a GST-tagged intracellular domain of ECSM2. CONCLUSIONS: This study is the first to characterize a novel cell surface protein ECSM2 that regulates endothelial chemotaxis and tube formation, and interacts with filamin A. These studies implicate a role for ECSM2 in angiogenesis via modulation of the actin cytoskeleton.

A novel method of differential gene expression analysis using multiple cDNA libraries applied to the identification of tumour endothelial genes.

BACKGROUND: In this study, differential gene expression analysis using complementary DNA (cDNA) libraries has been improved. Firstly by the introduction of an accurate method of assigning Expressed Sequence Tags (ESTs) to genes and secondly, by using a novel likelihood ratio statistical scoring of differential gene expression between two pools of cDNA libraries. These methods were applied to the latest available cell line and bulk tissue cDNA libraries in a two-step screen to predict novel tumour endothelial markers. Initially, endothelial cell lines were in silico subtracted from non-endothelial cell lines to identify endothelial genes. Subsequently, a second bulk tumour versus normal tissue subtraction was employed to predict tumour endothelial markers. RESULTS: From an endothelial cDNA library analysis, 431 genes were significantly up regulated in endothelial cells with a False Discovery Rate adjusted q-value of 0.01 or less and 104 of these were expressed only in endothelial cells. Combining the cDNA library data with the latest Serial Analysis of Gene Expression (SAGE) library data derived a complete list of 459 genes preferentially expressed in endothelium. 27 genes were predicted tumour endothelial markers in multiple tissues based on the second bulk tissue screen. CONCLUSION: This approach represents a significant advance on earlier work in its ability to accurately assign an EST to a gene, statistically measure differential expression between two pools of cDNA libraries and predict putative tumour endothelial markers before entering the laboratory. These methods are of value and available http://www.compbio.ox.ac.uk/data/diffex.html to researchers that are interested in the analysis of transcriptomic data.

Slits and Roundabouts in cancer, tumour angiogenesis and endothelial cell migration.

Angiogenesis describes the development of new blood vessels from pre-existing vessels. The hijacking of this physiological process by tumours allows them to develop their own supplies of nutrients and oxygen, enabling their growth and metastasis. A large body of literature has accumulated over the last 20 years relating to angiogenesis, including signalling pathways involved in this process. One such pathway uses Slit-Roundabout proteins that are implicated in the development of cancers and tumour angiogenesis. The Roundabout family of receptors are large, single-pass transmembrane cell surface receptors involved in directing cell migration in response to their cognate Slit ligands. Although best known for their role in neuronal development, Slits and Roundabouts have now been implicated in myogenesis, leukocyte chemotaxis and tumour angiogenesis, confirming that the Robo signalling pathway functions across multiple cell types. We review here the evidence for a role for Slits and Roundabouts in cancer. In particular, we focus on the role of Robo1 and Robo4 in tumour angiogenesis and discuss the signalling pathways downstream of these proteins mediating endothelial cell migration.