Name: Blood and Urinary Vesicles in Health and Disease
Uploaded: 2013-09-13T00:00:00
Duration: 43 min 53 s
Description: Cells involved in pathophysiological processes shed sealed membranous vesicles called microvesicles and exosomes. The composition and count of vesicles in the blood and urine depend upon their cell of origin and the stimulus that triggers their production. Thus, extracellular vesicles may serve as potential biomarkers for diagnosis, prognosis, and management of ongoing disease processes.

SELECTBIO

Molecular Medicine Congress 2013

Cells involved in pathophysiological processes shed sealed membranous vesicles called microvesicles and exosomes. The composition and count of vesicles in the blood and urine depend upon their cell of origin and the stimulus that triggers their production. Thus, extracellular vesicles may serve as potential biomarkers for diagnosis, prognosis, and management of ongoing disease processes.


Blood and Urinary Vesicles in Health and Disease

The detection of circulating tumor cells in the blood (CTCs) is a common phenomenon seen in 10-20% of primary breast cancer patients. Recent studies have shown a prognostic potential of CTC in both primary and metastatic setting. The evaluation of CTC may become one of the crucial markers for prediction of survival and therapy monitoring.


Clinical Relevance of CTCs in Primary and Metastatic Breast Cancer

Since current assays cannot distinguish between apoptotic and viable DTCs/CTCs, it is now possible to apply a novel ELISPOT assay (designated ‘EPISPOT’) that detects proteins secreted/released/shed from single epithelial cancer cells. Cells are cultured for a short time on a membrane coated with antibodies that capture the secreted/released/shed proteins which are subsequently detected by secondary antibodies labeled with fluorochromes. In breast cancer, we measured the release of cytokeratin-19 (CK19) and mucin-1 (MUC1) and demonstrated that many patients harbored viable DTCs, even in patients with apparently localized tumors (stage M0 : 54%). Preliminary clinical data showed that patients with DTC-releasing CK19 have an unfavorable outcome. We also studied CTCs or CK19-releasing cells (CK19-RC) in the peripheral blood of M1 breast cancer patients and showed that patients with CK19-RC had a worse clinical outcome. In prostate cancer, we used prostate-specific antigen (PSA) secretion as the marker and found that a significant fraction of CTCs secreted fibroblast growth factor-2 (FGF-2), a known stem cell growth factor. 
More recently, in colon cancer, a considerable portion of viable CTCs detectable by the Epispot assay is trapped in the liver as the first filter organ in colon cancer patients. The enumeration of CK19-SC by the CK19-Epispot assay revealed viable CTCs in 65.9% and 55.4% (p=0.04) patients in mesenteric and peripheral blood, respectively, whereas CellSearch detected CTCs in 55.9% and 29.0% (p=0.0046) patients. In mesenteric blood, the number of CTC was significantly higher than in the peripheral blood. Our clinical data showed that localized colon cancer patients with a high level of CTCs have an unfavorable outcome (n=60).

In conclusion, the EPISPOT assay offers a new opportunity to detect and characterize viable DTCs/CTCs in cancer patients and it can be extended to a multi-parameter analysis revealing a CTC/DTC protein fingerprint.


Detection and Characterization of Viable Circulating Tumor Cells

Microscopic spread of cancer cells is usually undetected by current imaging technologies. Therefore, a variety of sensitive methods have been developed to detect circulating tumor cells (CTC) in the peripheral blood. The capture of CTC that are surrounded by millions of blood cells is based on the physical and/or biological properties of cancer cells. Thus far, only the automated CellSearchTM system has been cleared by the FDA. However, many assays including the CellSearchTM system rely on EpCAM for CTC capture and cytokeratins for CTC detection but these epithelial marker proteins can be downregulated during epithelial-mesenchymal transition. Besides assessing the clinical utility of CTC for assessment of prognosis, monitoring of CTC during and after systemic therapy can provide unique information for the clinical management of individual cancer patients. In particular, the molecular analysis of CTC as “liquid biopsy” will give new insights into therapeutic targets and the selection of tumor cells under specific therapies. In addition, molecular and functional characterization of CTC opens a new avenue for understanding metastatic spread of tumor cells with important implications for future therapies. Implementation of CTC analyses in clinical trials testing new anti-cancer agents as companion diagnostics will speed up the cumbersome and expensive drug validation process in oncology. 


Detection and Clinical Implications of Circulating Tumor Cells in Cancer Patients

Stress on blood: formation of extracellular vesicles.  Blood cells respond to stress with release of extracellular vesicles. In particular red blood cells are prone to vesicle release upon a range of stress signals which can range from mechanical stress (e.g. during cardiopumonary bypass procedures) or chemical stress after pharmaceutical intervention (e.g. Taxol).  These vesicles carry phosphatidylserine which is thrombogeneic, and carry iron/heme which can cause oxidative stress.



Exosomes and Microvesicles in the Circulation

"Exosomes are 30-100nm nanovesicular bodies released from endosomes in a wide variety of cells. They are currently the subject of intense study, being increasingly recognised as playing multiple roles in intracellular communication and immune regulation. As well as displaying membrane proteins betraying their cellular origin, exosomes have now been shown to carry micro- and mRNA. It is increasingly accepted that exosomes are implicated and appear in a multitude of pathological conditions and show much promise as diagnostics for many different diseases such as cancer, heart disease, diabetes, Alzheimer’s, pre-eclampsia, etc. However, to date developments in this area have been constrained by limitations in the technology available for their measurement.. NTA allows, however, for the first time, exosomes to both be visualized, counted and sized in real time and with a rapid, robust and cost effective methodology. Importantly, through the use of antibody-mediated fluorescent labeling, specific phenotypes of exosomal subpopulations indicative of specific diseases can be detected in complex body fluid backgrounds.  We will show results gained recently from the use of NTA in the diagnosis of pre-eclampsia and will review other multiple studies in which NTA has been most lately used to speciate and enumerate these deeply sub-micron structures.
"


Exosomes Detection and Speciation by Nanoparticle Tracking Analysis  (NTA) – Potential Biomarkers for a Broad Range of Diseases

The application of a range of proteomics strategies has resulted in the discovery of very large numbers of potential new protein biomarkers but few have reached clinical utility for (a) understanding the molecular mechanisms of disease pathogenesis, (b) identifying new therapeutic targets, or (c) implementing biomarker based personalised treatment.    Some of the challenges faced in the protein biomarker development process and potential solutions will be described from our recent studies on prostate cancer and psoriatic arthritis. In these on-going collaborative projects we are developing a ‘clinical question focused’  pipeline for candidate protein biomarker assembly and validation of 'protein biomarker signatures' by multiple reaction monitoring (MRM). The potential of this approach for the delivery of more personalised patient treatment will be discussed.  

Multiplexed Measurement of Disease Biomarkers by Mass Spectometry (MRM) for Predictive and Prognostic Purposes  

The technique of Nanoparticle Tracking Analysis from NanoSight Ltd is a recently developed method which is being rapidly adopted for the analysis and counting of exosomes and microvesicles. Based on a unique microscopical laser illumination configuration, this technique allows biological nanoparticles in a sample to be simultaneously but individually visualised, sized (through video analysis of their Brownian motion) and counted in real time and in complex backgrounds. Furthermore, it is capable of detecting and analysing fluorescently labelled exosomes and microvesicles allowing for phenotyping of subpopulations of such structures in clinical samples.  The technique will be described and selected examples will be shown from the 250+ papers describing the use of NTA in the routine analysis of biological samples of clinical significance in a wide range of applications concentrating on the use of exosomes and microvesicles in diagnosis and therapeutics.


Nanoparticle Tracking Analysis – A Novel Technique for Exosomes Research

The adoption of molecular diagnostics of infectious diseases and antibiotic resistance is well on the rise, serving an ever increasing demand for rapid results which can be obtained at the point of care. The advent of next generation sequencing technologies is now set to revolutionise these applications in infectious disease diagnostics, outbreak tracking and personalised medicine.

The Promise of Next Generation Sequencing for Infectious Disease Diagnostics

SELECTBIO has been tracking the qualitative and quantitative trends and opportunities in the exosomes and microvesicles marketplace. &nbsp;In this presentation, Enal Razvi provide an up-to-date snapshot of this evolving marketplace excerpted from SELECTBIO's various market reports on this topic. &nbsp;Presented are technologies for exosome analysis, segmentation of the exosome research market, market penetrance of various approaches for exosome study, disciplines/disease areas where exosome research is currently being carried out, and sizes of some of the market segments in this space—both from a basic research and a translational standpoint.

Blood and Urinary Vesicles in Health and Disease

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September 13, 2013

Molecular Medicine Congress 2013

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