Extracellular vesicles as a source of prostate cancer biomarkers in liquid biopsies: a decade of research.

Prostate cancer is a global cancer burden and considerable effort has been made through the years to identify biomarkers for the disease. Approximately a decade ago, the potential of analysing extracellular vesicles in liquid biopsies started to be envisaged. This was the beginning of a new exciting area of research investigating the rich molecular treasure found in extracellular vesicles to identify biomarkers for a variety of diseases. Vesicles released from prostate cancer cells and cells of the tumour microenvironment carry molecular information about the disease that can be analysed in several biological fluids. Numerous studies document the interest of researchers in this field of research. However, methodological issues such as the isolation of vesicles have been challenging. Remarkably, novel technologies, including those based on nanotechnology, show promise for the further development and clinical use of extracellular vesicles as liquid biomarkers. Development of biomarkers is a long and complicated process, and there are still not many biomarkers based on extracellular vesicles in clinical use. However, the knowledge acquired during the last decade constitutes a solid basis for the future development of liquid biopsy tests for prostate cancer. These are urgently needed to bring prostate cancer treatment to the next level in precision medicine.

Extracellular Vesicle-Mediated Bone Remodeling and Bone Metastasis: Implications in Prostate Cancer.

Bone metastasis is the tendency of certain primary tumors to spawn and dictate secondary neoplasia in the bone. The process of bone metastasis is regulated by the dynamic crosstalk between metastatic cancer cells, cellular components of the bone marrow microenvironment (osteoblasts, osteoclasts, and osteocytes), and the bone matrix. The feed-forward loop mechanisms governs the co-option of homeostatic bone remodeling by cancer cells in bone. Recent developments have highlighted the discovery of extracellular vesicles (EVs) and their diverse roles in distant outgrowths. Several studies have implicated EV-mediated interactions between cancer cells and the bone microenvironment in synergistically promoting pathological skeletal metabolism in the metastatic site. Nevertheless, the potential role that EVs serve in arbitrating intricate sequences of coordinated events within the bone microenvironment remains an emerging field. In this chapter, we review the role of cellular participants and molecular mechanisms in regulating normal bone physiology and explore the progress of current research into bone-derived EVs in directly triggering and coordinating the processes of physiological bone remodeling. In view of the emerging role of EVs in interorgan crosstalk, this review also highlights the multiple systemic pathophysiological processes orchestrated by the EVs to direct organotropism in bone in prostate cancer. Given the deleterious consequences of bone metastasis and its clinical importance, in-depth knowledge of the multifarious role of EVs in distant organ metastasis is expected to open new possibilities for prognostic evaluation and therapeutic intervention for advanced bone metastatic prostate cancer.

Extracellular Vesicles in the Adaptive Process of Prostate Cancer during Inhibition of Androgen Receptor Signaling by Enzalutamide.

Current treatments for advanced prostate cancer focus on inhibition of the androgen receptor (AR) by androgen deprivation therapy (ADT). However, complex interactions mediated by tumor suppressors, oncogenes, aberrations of AR expression, or de novo androgen production have been shown to induce the adaptive response of prostate cancer, leading to the development of castration resistant prostate cancer. In this study, we report the effects of AR antagonist, enzalutamide on the protein contents of extracellular vesicles (EVs). EVs mediate cell-to-cell communication and increasing evidence shows the role of EVs in promoting cancer survival and metastasis. We found that treatment with enzalutamide alters the secretion of EVs, one of which is a plasma membrane calcium pump, ATP2B1/PMCA ATPase, as an AR-regulated EV protein. We highlight the networks of interactions between AR, Ca2+ , and ATP2B1, where the extracellular proteins thrombospondin-1, gelsolin, and integrinß1 were previously reported as regulators for cancer progression and metastasis, indicating the potential role of EV-derived proteins in mediating calcium homoeostasis under AR inhibition by enzalutamide. Our data further highlight the cross-talk between AR signaling and EV pathways in mediating resistance toward ADT.

Extracellular vesicles for personalized therapy decision support in advanced metastatic cancers and its potential impact for prostate cancer.

The use of circulating tumor cells (CTCs) and circulating extracellular vesicles (EVs), such as exosomes, as liquid biopsy-derived biomarkers for cancers have been investigated. CTC enumeration using the CellSearch based platform provides an accurate insight on overall survival where higher CTC counts indicate poor prognosis for patients with advanced metastatic cancer. EVs provide information based on their lipid, protein, and nucleic acid content and can be isolated from biofluids and analyzed from a relatively small volume, providing a routine and non-invasive modality to monitor disease progression. Our pilot experiment by assessing the level of two subpopulations of small EVs, the CD9 positive and CD63 positive EVs, showed that the CD9 positive EV level is higher in plasma from patients with advanced metastatic prostate cancer with detectable CTCs. These data show the potential utility of a particular EV subpopulation to serve as biomarkers for advanced metastatic prostate cancer. EVs can potentially be utilized as biomarkers to provide accurate genotypic and phenotypic information for advanced prostate cancer, where new strategies to design a more personalized therapy is currently the focus of considerable investigation.

Metastatic prostate cancer-derived extracellular vesicles facilitate osteoclastogenesis by transferring the CDCP1 protein.

Bone metastases are still incurable and result in the development of clinical complications and decreased survival for prostate cancer patients. Recently, a number of studies have shown that extracellular vesicles (EVs) play important roles in tumour progression. Here, we show that EVs from metastatic prostate cancer cells promote osteoclast formation in the presence of receptor activator of NF-κB ligand (RANKL). EV characterization followed by functional siRNA screening identified CUB-domain containing protein 1 (CDCP1), a transmembrane protein, as an inducer of osteoclastogenesis. Additionally, CDCP1 expression on plasma-derived EVs was upregulated in bone metastatic prostate cancer patients. Our findings elucidate the effect of EVs from metastatic prostate cancer cells on osteoclast formation, which is promoted by CDCP1 located on EVs. Furthermore, our data suggested that CDCP1 expression on EVs might be useful to detect bone metastasis of prostate cancer.

MIBlood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.

Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor-dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre-analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood-EV), a tool to record and report information about pre-analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre-analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence-based optimization of pre-analytical protocols through meta-analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter-laboratory comparative EV studies, and (iv) improve the peer review process.

Cell culture-derived extracellular vesicles: Considerations for reporting cell culturing parameters.

Cell culture-conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM-derived EVs (CCM-EV). The CCM-EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell-specific recommendations may need to be established for non-mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM-EV research.

Extracellular vesicles in the development of organ-specific metastasis.

Distant organ metastasis, often termed as organotropic metastasis or metastatic organotropism, is a fundamental feature of malignant tumours and accounts for most cancer-related mortalities. This process is orchestrated by many complex biological interactions and processes that are mediated by a combination of anatomical, genetic, pathophysiological and biochemical factors. Recently, extracellular vesicles (EVs) are increasingly being demonstrated as critical mediators of bi-directional tumour-host cell interactions, controlling organ-specific infiltration, adaptation and colonization at the secondary site. EVs govern organotropic metastasis by modulating the pre-metastatic microenvironment through upregulation of pro-inflammatory gene expression and immunosuppressive cytokine secretion, induction of phenotype-specific differentiation and recruitment of specific stromal cell types. This review discusses EV-mediated metastatic organotropism in visceral (brain, lung, liver, and lymph node) and skeletal (bone) metastasis, and discusses how the pre-metastatic education by EVs transforms the organ into a hospitable, tumour cell-friendly milieu that supports the growth of metastatic cells. Decoding the organ-specific traits of EVs and their functions in organotropic metastasis is essential in accelerating the clinical application of EVs in cancer management.

Androgens alter the heterogeneity of small extracellular vesicles and the small RNA cargo in prostate cancer.

Proliferation and survival of prostate cancer cells are driven by the androgen receptor (AR) upon binding to androgen steroid hormones. Manipulating the AR signalling axis is the focus for prostate cancer therapy; thus, it is crucial to understand the role of androgens and AR on extracellular vesicle (EV) secretion and cargo. In this study, we report that plasma-derived circulating vesicles consisting of CD9 and double-positive for CD9 and Prostate Specific Membrane Antigen (PSMA) are increased in patients with advanced metastatic prostate cancer, whereas double positives for CD9 and CD63 small extracellular vesicles (S-EVs) are significantly higher in patients with localised prostate cancer. Androgen manipulation by dihydrotestosterone (DHT) and the clinical antagonist enzalutamide (ENZ) altered the heterogeneity and size of CD9 positive S-EVs in AR expressing prostate cancer cells, while assessment of the total number and protein cargo of total S-EVs was unaltered across different treatment groups. Furthermore, hormone stimulation caused strong and specific effects on the small RNA cargo of S-EVs. A total of 543 small RNAs were found to be regulated by androgens including miR-19-3p and miR-361-5p. Analysis of S-EVs heterogeneity and small RNA cargo may provide clinical utility for prostate cancer and be informative to understand further the mechanism of resistance to androgen targeted therapy in castration-resistant prostate cancer.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Secretory control: evidence for agonist regulation of post-fusion vesicle behaviour.

1. Here, we review recent work on vesicular secretion, with a focus on the control of post-fusion events as a means of regulating secretory output. 2. In the classical model of secretion, each fused vesicle releases the entirety of its content in an all-or-none manner. In this way, the secretory output of a cell is controlled by regulating the numbers of fused vesicles. The realisation that post-fusion events can control secretory output leads to a distinct model of partial release of vesicle content. 3. Recent work shows that post-fusion events are under cellular control. Further, new data from our laboratory demonstrates agonist-dependent regulation of fusion pore behaviour. 4. We conclude that post-fusion events are not epiphenomena, but are likely an important mechanism of secretory control.

Pathogenesis of Viral Hepatitis-Induced Chronic Liver Disease: Role of Extracellular Vesicles.

Extracellular vesicles are encapsulated lipid nanoparticles secreted by a variety of cell types in living organisms. They are known to carry proteins, metabolites, nucleic acids, and lipids as their cargoes and are important mediators of intercellular communication. The role of extracellular vesicles in chronic liver disease has been reported. Chronic liver disease such as viral hepatitis accounts for a significant mortality and morbidity burden worldwide. Hepatic fibrosis has been commonly associated with the chronic form of viral hepatitis, which results in end-stage liver disease, including cirrhosis, liver failure, and carcinoma in some patients. In this review, we discuss the potential role of extracellular vesicles in mediating communication between infectious agents (hepatitis B and C viruses) and host cells, and how these complex cell-cell interactions may facilitate the development of chronic liver disease. We will further discuss how understanding their biological mechanism of action might be beneficial for developing therapeutic strategies to treat chronic liver disease.

The future of Extracellular Vesicles as Theranostics - an ISEV meeting report.

The utilization of extracellular vesicles (EVs) in clinical theranostics has rapidly advanced in the past decade. In November 2018, the International Society for Extracellular Vesicles (ISEV) held a workshop on "EVs in Clinical Theranostic". Here, we report the conclusions of roundtable discussions on the current advancement in the analysis technologies and we provide some guidelines to researchers in the field to consider the use of EVs in clinical application. The main challenges and the requirements for EV separation and characterization strategies, quality control and clinical investigation were discussed to promote the application of EVs in future clinical studies.

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids ("liquid biopsies"). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.

Essentials of extracellular vesicles: posters on basic and clinical aspects of extracellular vesicles.

The past decade has witnessed an exponential development in the field of extracellular vesicles. Sporadic observations have reached a critical level and the scientific community increasingly recognizes the potential biomedical significance of these subcellular structures present in all body fluids as significant components of the cellular secretome. The Educational Committee of the International Society for Extracellular Vesicles prepared two posters ("Basic aspects of extracellular vesicles" and "Clinical aspects of extracellular vesicles") to provide essential pieces of information on extracellular vesicles at glance for anyone not familiar with the field.

Towards mechanisms and standardization in extracellular vesicle and extracellular RNA studies: results of a worldwide survey.

The discovery that extracellular vesicles (EVs) can transfer functional extracellular RNAs (exRNAs) between cells opened new avenues into the study of EVs in health and disease. Growing interest in EV RNAs and other forms of exRNA has given rise to research programmes including but not limited to the Extracellular RNA Communication Consortium (ERCC) of the US National Institutes of Health. In 2017, the International Society for Extracellular Vesicles (ISEV) administered a survey focusing on EVs and exRNA to canvass-related views and perceived needs of the EV research community. Here, we report the results of this survey. Overall, respondents emphasized opportunities for technical developments, unraveling of molecular mechanisms and standardization of methodologies to increase understanding of the important roles of exRNAs in the broader context of EV science. In conclusion, although exRNA biology is a relatively recent emphasis in the EV field, it has driven considerable interest and resource commitment. The ISEV community looks forward to continuing developments in the science of exRNA and EVs, but without excluding other important molecular constituents of EVs.

Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer.

Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC-MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression.

Dynamic mechanism for the serpin loop insertion as revealed by quantitative kinetics.

The serpin conformational change by insertion of the reactive center loop into beta-sheet A plays a central role in multiple physiological consequences such as serine proteinase inhibition, latency and serpinopathic polymerization. To study the dynamic mechanism for the loop insertion, a novel kinetic method was established utilizing the ovalbumin mutant R339T/A352R; the loop insertion progressed after the cleavage of P1-P1' (Arg352-Ser353) by trypsin was quenched at pH 8 and 0.5 degrees C, and different conformers were quantified by separation using ion-exchange HPLC. The apparent first-order rate constant k(app) determined for various R339T/A352R derivatives differing in conformational stability was greatly increased by lowering the pH. The pH-dependence of k(app) indicated that the protonation of side-chain(s) with a pK(a) value of around 4.6 is a pre-requisite for the loop insertion. The theoretical rate constant k for the protonated form calculated from k(app) was highly variable, depending on the ovalbumin derivative; structural modifications that give increased mobility to helix F and the sheet-A half (s3A/s2A/s1A) resulted in a striking increase in the loop insertion rate constant k. The k values were determined at different temperatures for all the ovalbumin derivatives, and DeltaH(double dagger) and DeltaS(double dagger) values for the loop insertion reaction were determined according to the transition theory. The formation of the transition state was highly endothermic with minor entropy gain, requiring a DeltaG(double dagger) larger than 18 kcal/mol, which can offset the hydrogen-bond cleavages between s3A and s5A. These results are consistent with the transition state with an opened sheet A and altered orientation of helix F.