Exosomes : a source of novel disease biomarkers in bladder cancer

The major aim of this thesis was to perform the first ever proteomics study on bladder cancer exosomes. Initially, exosomes were isolated from urine specimens but hypervariable yields and poor sample quality made proteomics analysis challenging. As an alternative approach, exosomes were isolated from HT1376 bladder cancer cells. Exosomes were purified by ultracentrifugation on a sucrose cushion, and preparations verified as high quality by immunoblotting, flow cytometry and electron microscopy. For global proteomics analysis, the sample was solubilised using SDS and DTT and subjected to LC-MALDI-TOF/TOF MS. We identified 353 proteins with high confidence and 63 of these have not previously been identified in other proteomics studies on human exosomes. Overrepresentation analysis demonstrated that the proteome was consistent with that of other exosomes with significant overlap with exosomes of carcinoma origin. Comparisons with the Gene Ontology database also highlighted strong associations with carcinoma of the bladder and other sites. A GeneGo generated protein interaction network highlighted c-Myc as a major node of protein interaction within this dataset. Several MS-identified proteins were confirmed as genuinely exosomally expressed using a combination of immunoblotting, flotation on continuous sucrose gradients, and flow cytometry. Expression was also verified in exosomes from a variety of sources, including urine. The data will aid our understanding of exosome biogenesis and function and may inform the development of urine exosome-based clinical tools in bladder cancer.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Exosomes: a source of novel disease biomarkers in bladder cancer.

The major aim of this thesis was to perform the first ever proteomics study on bladder cancer exosomes. Initially, exosomes were isolated from urine specimens but hypervariable yields and poor sample quality made proteomics analysis challenging. As an alternative approach, exosomes were isolated from HT1376 bladder cancer cells. Exosomes were purified by ultracentrifugation on a sucrose cushion, and preparations verified as high quality by immunoblotting, flow cytometry and electron microscopy. For global proteomics analysis, the sample was solubilised using SDS and DTT and subjected to LC-MALDI-TOF/TOF MS. We identified 353 proteins with high confidence and 63 of these have not previously been identified in other proteomics studies on human exosomes. Overrepresentation analysis demonstrated that the proteome was consistent with that of other exosomes with significant overlap with exosomes of carcinoma origin. Comparisons with the Gene Ontology database also highlighted strong associations with carcinoma of the bladder and other sites. A GeneGo generated protein interaction network highlighted c-Myc as a major node of protein interaction within this dataset. Several MS-identified proteins were confirmed as genuinely exosomally expressed using a combination of immunoblotting, flotation on continuous sucrose gradients, and flow cytometry. Expression was also verified in exosomes from a variety of sources, including urine. The data will aid our understanding of exosome biogenesis and function and may inform the development of urine exosome-based clinical tools in bladder cancer

Cerebrospinal fluid extracellular vesicle enrichment for protein biomarker discovery in neurological disease; multiple sclerosis

The discovery of disease biomarkers, along with the use of “liquid biopsies” as a minimally invasive source of biomarkers, continues to be of great interest. In inflammatory diseases of the central nervous system (CNS), cerebrospinal fluid (CSF) is the most obvious biofluid source. Extracellular vesicles (EVs) are also present in CSF and are thought to be potential “biomarker treasure chests”. However, isolating these CSF-derived EVs remains challenging. This small-scale pilot study developed and tested a protocol to enrich for CSF-EVs, both in relapsing remitting multiple sclerosis (RRMS) CSF and controls. These were subsequently compared, using an aptamer based proteomics array, SOMAscan™. EVs were enriched from RRMS patient (n = 4) and non-demyelinating control (idiopathic intracranial hypertension (IIH) (n = 3)) CSF using precipitation and mini size-exclusion chromatography (SEC). EV-enriched fractions were selected using pre-defined EV characteristics, including increased levels of tetraspanins. EVs and paired CSF were analysed by SOMAscan™, providing relative abundance data for 1128 proteins. CSF-EVs were characterised, revealing exosome-like features: rich in tetraspanins CD9 and CD81, size ~100 nm, and exosome-like morphology by TEM. Sufficient quantities of, SOMAscan™ compatible, EV material was obtained from 5 ml CSF for proteomics analysis. Overall, 348 and 580 proteins were identified in CSF-EVs and CSF, respectively, of which 50 were found to be significantly (t-test) and exclusively enriched in RRMS CSF-EVs. Selected proteins, Plasma kallikrein and Apolipoprotein-E4, were further validated by western blot and appeared increased in CSF-EVs compared to CSF. Functional enrichment analysis of the 50 enriched proteins revealed strong associations with biological processes relating to MS pathology and also extracellular regions, consistent with EV enrichment. This pilot study demonstrates practicality for EV enrichment in CSF derived from patients with MS and controls, allowing detailed analysis of protein profiles that may offer opportunities to identify novel biomarkers and therapeutic approaches in CNS inflammatory disease

Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array

Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen–antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios

Ready-made chromatography columns for extracellular vesicle isolation from plasma

Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc.) that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio). The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma