Extracellular vesicles mediate intercellular signaling and are potential sources of cancer biomarkers. Nawaz and colleagues describe the biogenesis of extracellular vesicles, and the methods available for their isolation and characterization. The authors also discuss current research into the identification of vesicle-derived biomarkers for cancers of the prostate, kidney and bladder.
Abstract
The knowledge gained from comprehensive profiling projects that aim to define the complex genomic alterations present within cancers will undoubtedly improve our ability to detect and treat those diseases, but the influence of these resources on our understanding of basic cancer biology is still to be demonstrated. Extracellular vesicles have gained considerable attention in past years, both as mediators of intercellular signaling and as potential sources for the discovery of novel cancer biomarkers. In general, research on extracellular vesicles investigates either the basic mechanism of vesicle formation and cargo incorporation, or the isolation of vesicles from available body fluids for biomarker discovery. A deeper understanding of the cargo molecules present in extracellular vesicles obtained from patients with urogenital cancers, through high-throughput proteomics or genomics approaches, will aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.
Key points
- Extracellular vesicles are small (40–5,000 nm diameter) membrane-bound vesicles that can be categorized into exosomes, microvesicles and apoptotic bodies according to their size, origin, morphology and mode of release
- Whereas the generation of exosomes involves endocytosis, formation of multivesicular bodies and subsequent membrane fusion, microvesicles are produced by membrane budding and apoptotic bodies result from membrane blebbing during apoptosis
- Over the past 10 years, various methodologies for the effective isolation of extracellular vesicles have been developed, including centrifugation, affinity capture, precipitation and the use of microfluidic devices
- Extracellular vesicle cargo is thought to reflect the cell-type of origin, suggesting it could be a promising source for the discovery of novel biomarkers
Urogenital cancers—cancers of the reproductive and renal organs—are major causes of morbidity and mortality worldwide.1, 2 The multistage, stochastic and heterogeneous nature of these malignancies, resulting from genetic and epigenetic modifications, poses a fundamental challenge to monitoring. Although surgical treatment and chemotherapy for urogenital cancers have improved in the last decade, the prognoses for these diseases remain poor, as existing tests are not sufficiently sensitive or specific to diagnose urogenital cancers at early stages, and none has been shown to significantly decrease overall mortality. Current diagnostic procedures include general examinations and biopsies, such as image-guided prostate biopsy,3 cystoscopy and transurethral resection of the bladder,4 nephrectomy and percutaneous renal tumour biopsies,5 all of which lack sensitivity and can be associated with significant health complications (for example, biopsies are invasive procedures associated with bleeding and risk of infections). Moreover, the location of urogenital cancers deep within the pelvic region makes them hard to access. Thus, in the absence of early symptoms, cancers are diagnosed at an advanced stage, by which time patients have poor outcomes and tumours have often metastasized.
Extracellular vesicles have gained considerable attention in the past 10 years as potential sources for biomarker discovery. These small (40–5000 nm diameter) membrane-bound vesicles are categorized into exosomes, microvesicles or ectosomes, apoptotic bodies6, 7, 8, 9, 10 or Golgi vesicles11 on the basis of their size, origin, morphology and mode of release. Well-known for biological effects, such as signalling and transfer of cargo, extracellular vesicles are secreted under various pathophysiologic conditions into the extracellular environment by a variety of cell types, promoting tumour progression, survival, invasion and angiogenesis,12, 13, 14, 15, 16, 17 as well as influencing the immune response, cell-to-cell communication, extracellular matrix degradation, coagulation, stem-cell renewal, cardiovascular functions and resistance to drugs (Figure 1).18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30
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| Figure 1: Classes of extracellular vesicles.
Extracellular vesicles comprise a heterogeneous mixture of exosomes, microvesicles and apoptotic bodies. The biogenesis of these three subtypes differs: microvesicles bud directly from the plasma membrane, whereas exosomes are formed by endocytosis and the subsequent formation of multivesicular bodies, and apoptotic bodies are formed as a consequence of apoptotic disintegration. Extracellular vesicles regulate numerous biological functions, such as cell-to-cell communication and horizontal transfer of cargo, and have been implicated in a number of biological pathways.
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Surprisingly, the biomolecular cargo of extracellular vesicles is stable in biological fluids and protected against exogenous RNases and proteases, owing to its encapsulation within membrane vesicles,23, 31, 32 or association with RNA-binding or DNA-binding proteins33, 34, 35 or lipoprotein complexes.36, 37 Thus, extracellular vesicles might be stable under adverse physical conditions, such as extremes in pH, long-term storage and multiple freeze–thaw cycles,33, 38 making them an appealing source for biomarker development.
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| Figure 2: Extracellular vesicle biogenesis.
Extracellular vesicles originate through different mechanisms. a | Exosomes initiate as intraluminal vesicles that are formed by endocytosis in response to pathogens, ligands or other stimuli; these endocytic vesicles mature to early endosomes, and then into late endosomes, or MVBs. Following the ubiquitin-dependent interactions with ESCRT complexes, MVBs can be sorted for lysosomal degradation or they can fuse with the plasma membrane and be released as exosomes. ALIX binds to MVB cargo, preventing lysosomal degradation and favouring exosomal release. Rab GTPases regulate MVB fusion with the plasma membrane and release of exosomes. b | Microvesicles are formed by the outward budding and fission of plasma membrane lipid microdomains, which is controlled by regulatory proteins and cytoskeleton elements, that promote membrane curvature at ceramide-enriched domains (blue bars), resulting in microvesicle budding. After synthesis in the ER, protein cargo is transported to the Golgi apparatus, modified and packaged into small vesicles secreted as transport Golgi vesicles. c | Cells undergoing apoptotic disaggregation produce large membrane blebs, known as apoptotic bodies or apoptosomes. Abbreviations: ALIX, ALG-2-interacting protein X; ER, endoplasmic reticulum; ESCRT, endosomal sorting complexes required for transport; MVB, multivesicular body; TSG101, tumour susceptibility gene 101 protein.
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Several reports indicate that cancer cells release more extracellular vesicles than normal cells,17, 39, 40 and that the biomolecular cargo (that is, proteins, nucleic acids and lipids) is reflective of the cell of origin.41, 42 Consequently, knowledge about the content of extracellular vesicles derived from tumour cells with differing stages of aggression could be used to establish new diagnostic approaches using patient-derived vesicles from body fluids. The detection of biomarkers in body fluids has major advantages over the use of tissue markers, which most often require invasive biopsies that can be difficult to perform and potentially dangerous. Urine-based tests, in particular, could offer attractive approaches for large-scale screening, as large amounts of urine can be collected longitudinally. Ultimately, discriminating between cargoes associated with extracellular vesicles in body fluids using proteomic and genomic profiling approaches could provide insight into disease staging. An important first step is to develop sensitive, rapid and highly effective strategies to enable the collection of extracellular vesicles, and to adapt standardized procedures for routine clinical diagnostic application.
In this Review, we provide a comprehensive overview of the roles of extracellular vesicles in the most common urogenital cancers (prostate, kidney and bladder). This includes a detailed overview of the current knowledge of the different classes of extracellular vesicles, their biogenesis, potential biological functions and available technologies for isolation and downstream analyses. Existing knowledge regarding the cancer-specific biology of extracellular vesicles, and their potential use as vehicles for biomarker discovery, are reviewed and discussed.
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| Figure 3: Multistep validation of biomarkers from extracellular vesicles.
A potential flowchart for the validation and clinical implementation of biomarkers based on extracellular vesicles. The flowchart shows a step-by-step process by which the profiling and discovery of exosomal cargo molecules could ultimately be translated into a clinically applicable biomarker signature. At each step defined goals and criteria must be met in order to proceed to the next level.163
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Conclusions
Despite the considerable research efforts applied to cancer biomarker discovery, a deficit of reliable markers to facilitate early detection, accurate prognosis and reliable prediction of response to treatment still remains. The quest for clinically relevant biomarkers for urogenital cancers remains an unmet challenge. Ongoing efforts for the identification of biomarkers include a variety of profiling technologies that are aimed at the discovery of genetic/epigenetic, proteomic and lipidomic alterations. Although lipidomic analyses of extracellular vesicles are still relatively rare, we expect that the recently revived interest in cancer metabolism will result in an increase in such studies in the future.160
The identification of molecular signatures in biological fluids could create 'liquid biopsies', which would effectively overcome many of the challenges associated with traditional tissue sampling (such as invasiveness and tumour heterogeneity). In this regard, the analysis of extracellular vesicles derived from body fluids could offer an especially attractive source of biomarkers, since these vesicles are thought to reflect the molecular composition of the secreting cell. In urogenital cancers, increased levels of extracellular vesicles during tumorigenesis might serve as indicators for disease surveillance. Interestingly, molecular cargoes, such as nucleic acids and proteins, seem to be selectively sorted into extracellular vesicles, as recently shown for neural precursor cells exposed to proinflammatory cytokines,161 and these regulated mechanisms in cancer cells can provide access to a tumour-specific repertoire. For instance, several proteomic studies have revealed the presence of urinary extracellular vesicles that contain candidate proteins unique to cancer types that include a broad range of urogenital diseases.25, 26, 29, 102, 110, 112, 147, 157, 162 Such tumour-specific vesicles are easily captured from urine using established isolation procedures, which enables repeated tissue sampling.
One of the current challenges for the implementation of biomarkers based on extracellular vesicles in clinical practice is the development of isolation and detection methods that are compatible with current practices (Figure 3). The development of robust techniques and sensitive capture platforms that use readily accessible body fluids, particularly urine, could offer novel approaches for disease staging and diagnosis. Current efforts to systematically catalogue the nucleic acid, protein and lipid constituents of extracellular vesicles isolated from richly annotated clinical samples could ultimately help in developing sensitive and selective capture platforms directed towards specific extracellular vesicle subpopulations. Advances in next-generation sequencing and mass-spectrometry-based proteomics and metabolomics are likely to enable appropriate candidates to be established in the near future.
Muhammad Nawaz,1, Giovanni Camussi,2, Hadi Valadi,3, Irina Nazarenko,4, Kari Xiaoqin Wang,3, n Ekström,3, Simona Principe, Neelam Shah,6, Naeem M. Ashraf,7, Farah Fatima,1, Luciano Neder1, & Thomas Kislinger5,
Nature Reviews Urology | Review
Nature Reviews Urology (
Published online 18 November 2014
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