Detecting ovarian cancer using extracellular vesicles: Progress and possibilities

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumour growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so they represent an excellent potential source of novel biomarkers for the disease. In this review we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but a number of studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insight into the stage of the disease and predict the likely therapeutic outcome. There remain a number of challenges to the use of EVs as biomarkers, but through ongoing research and innovation in this exciting field there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome

In sickness and in health : The functional role of extracellular vesicles in physiology and pathology in vivo Part II: Pathology

It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change-size, number, and physicochemical composition-in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention.Peer reviewe

Long non-coding RNA dysregulation is a frequent event in non-small cell lung carcinoma pathogenesis

Background Long non-coding RNAs compose an important level of epigenetic regulation in normal physiology and disease. Despite the plethora of publications of lncRNAs in human cancer, the landscape is still unclear. Methods Microarray analysis in 44 NSCLC paired specimens was followed by qPCR-based validation in 29 (technical) and 38 (independent) tissue pairs. Cross-validation of the selected targets was achieved in 850 NSCLC tumours from TCGA datasets. Results Twelve targets were successfully validated by qPCR (upregulated: FEZF1-AS1, LINC01214, LINC00673, PCAT6, NUTM2A-AS1, LINC01929; downregulated: PCAT19, FENDRR, SVIL-AS1, LANCL1-AS1, ADAMTS9-AS2 and LINC00968). All of them were successfully cross-validated in the TCGA datasets. Abnormal DNA methylation was observed in the promoters of FENDRR, FEZF1-AS1, and SVIL-AS1. FEZF1-AS1 and LINC01929 were associated with survival in the TCGA set. Conclusions Our study provides through multiple levels of internal and external validation, a comprehensive list of dysregulated lncRNAs in NSCLC. We therefore envisage this dataset to serve as an important source for the lung cancer research community assisting future investigations on the involvement of lncRNAs in the pathogenesis of the disease and providing novel biomarkers for diagnosis, prognosis and therapeutic stratification

In sickness and in health : The functional role of extracellular vesicles in physiology and pathology in vivo Part I: Health and Normal Physiology

Previously thought to be nothing more than cellular debris, extracellular vesicles (EVs) are now known to mediate physiological and pathological functions throughout the body. We now understand more about their capacity to transfer nucleic acids and proteins between distant organs, the interaction of their surface proteins with target cells, and the role of vesicle-bound lipids in health and disease. To date, most observations have been made in reductionist cell culture systems, or as snapshots from patient cohorts. The heterogenous population of vesicles produced in vivo likely act in concert to mediate both beneficial and detrimental effects. EVs play crucial roles in both the pathogenesis of diseases, from cancer to neurodegenerative disease, as well as in the maintenance of system and organ homeostasis. This two-part review draws on the expertise of researchers working in the field of EV biology and aims to cover the functional role of EVs in physiology and pathology. Part I will outline the role of EVs in normal physiology.Peer reviewe

In sickness and in health : The functional role of extracellular vesicles in physiology and pathology in vivo Part II: Pathology

It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change-size, number, and physicochemical composition-in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention.Peer reviewe

Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals

In sickness and in health: The functional role of extracellular vesicles in physiology and pathology in vivo. Part 2

It is clear from Part I of this series that extracellular vesicles (EVs) play a critical role in maintaining the homeostasis of most, if not all, normal physiological systems. However, the majority of our knowledge about EV signalling has come from studying them in disease. Indeed, EVs have consistently been associated with propagating disease pathophysiology. The analysis of EVs in biofluids, obtained in the clinic, has been an essential of the work to improve our understanding of their role in disease. However, to interfere with EV signalling for therapeutic gain, a more fundamental understanding of the mechanisms by which they contribute to pathogenic processes is required. Only by discovering how the EV populations in different biofluids change—size, number, and physicochemical composition—in clinical samples, may we then begin to unravel their functional roles in translational models in vitro and in vivo, which can then feedback to the clinic. In Part II of this review series, the functional role of EVs in pathology and disease will be discussed, with a focus on in vivo evidence and their potential to be used as both biomarkers and points of therapeutic intervention

Investigations into the dynamic uptake of extracellular vesicles and their role in breast cancer metastasis after chemotherapy

Extracellular vesicles (EVs) are small lipid bound spherical structures released by almost all types of cells. They are mediators of intercellular communication, however, the exact mechanisms of EV-cell interactions and uptake have not been fully characterised. EVs play key roles in cancer including all the stages of metastasis. Breast cancer is amongst the most common types of cancer for women and the primary cause of cancer related death. The high mortality rates are due to the metastatic spread of cancer cells and tumour recurrence after therapy. Studies have proposed chemotherapy to be a double-edged sword, which in some cases can promote metastasis. Here, it was hypothesised that EVs from chemotherapy treated cells were able to induce metastasis-related capacities to naïve cells. The aims of this project were: a) to explore the dynamics and specificity of EV uptake by recipient cells, and b) to investigate the effect of chemotherapy induced intercellular communication via EVs on breast cancer metastasis. To study EV uptake, different methods of EV labelling were tested. It was observed that PKH26 labelling may result in false-positive signals due to micelle formation. EV labelling with CFSE also produced non-specific effects, while dual labelling with both PKH26 and CFSE resulted in EV populations with heterogeneous labelling. EVs bearing a fluorescently tagged EV marker (mEmerald-CD81) were preferential for experiments investigating EV dynamics, as labelling appeared to be more specific than the dyes previously tested. Using this type of labelled EVs, it was shown that EV uptake differs across cell lines and the EV origin affects docking on the plasma membrane. Using a range of uptake inhibitors, it was observed that cholesterol may be involved in early EV-plasma membrane interactions, thus affecting EV uptake. Single molecule tracking revealed two patterns of such interactions: short and fast, and long and slow. To study the effect of EVs after chemotherapy, docetaxel and mitomycin C, two anti-cancer agents used for the treatment of breast cancer, were employed. Chemotherapy-treated cells released significantly higher numbers of EVs compared to non-treated cells. EVs from chemotherapy-treated cells did not induce any pro-metastatic effects (increased motility and invasiveness, EMT, and ability to adhere to endothelial cells), although changes in the glycosylation patterns of recipient cells were observed. Overall, these results: highlight the variability amongst different EV labelling methods; suggest a role for cholesterol in early EV-membrane interactions and specificity of uptake; and indicate that chemotherapy-derived EVs may confer glycosylation changes but not pro-metastatic effects in recipient cells

Circulating extracellular vesicles in healthy and pathological pregnancies: A scoping review of methodology, rigour and results

Abstract Extracellular vesicles (EVs) play a crucial role in pregnancy, revealed by the presence of placental‐derived EVs in maternal blood, their in vitro functionality, and their altered cargo in pregnancy pathologies. These EVs are thought to be involved in the development of pregnancy pathologies, such as pre‐eclampsia, pre‐term birth, and fetal growth restriction, and have been suggested as a source of biomarkers for gestational diseases. However, to accurately interpret their function and biomarker potential, it is necessary to critically evaluate the EV isolation and characterization methodologies used in pregnant cohorts. In this systematic scoping review, we collated the results from 152 studies that have investigated EVs in the blood of pregnant women, and provide a detailed analysis of the EV isolation and characterization methodologies used. Our findings indicate an overall increase in EV concentrations in pregnant compared to non‐pregnant individuals, an increased EV count as gestation progresses, and an increased EV count in some pregnancy pathologies. We highlight the need for improved standardization of methodology, greater focus on gestational changes in EV concentrations, and further investigations into the functionality of EVs. Our review suggests that EVs hold great promise as diagnostic and translational tools for gestational diseases. However, to fully realize their potential, it is crucial to improve the standardization and reliability of EV isolation and characterization methodologies, and to gain a better understanding of their functional roles in pregnancy pathologies

A practical toolkit to study aspects of the metastatic cascade in vitro

While metastasis – the spread of cancer from the primary location to distant sites in the body – remains the principle cause of cancer death, it is incompletely understood. It is a complex process, requiring the metastatically successful cancer cell to negotiate a formidable series of interconnected steps, which are described in this paper. For each step, we review the range of in vitro assays that may be used to study them. We also provide a range of detailed, step-by-step protocols that can be undertaken in most modestly-equipped laboratories, including methods for converting qualitative observations into quantitative data for analysis. Assays include: (1) a gelatin degradation assay to study the ability of endothelial cells to degrade extracellular matrix during tumour angiogenesis; (2) the morphological characterisation of cells undergoing epithelial-mesenchymal transition (EMT) as they acquire motility; (3) a ‘scratch’ or ‘wound-healing’ assay to study cancer cell migration; (4) a transwell assay to study cancer cell invasion through extracellular matrix; and (5) a static adhesion assay to examine cancer cell interactions with, and adhesion to, endothelial monolayers. This toolkit of protocols will enable researchers who are interested in metastasis to begin to focus on defined aspects of the process. It is only by further understanding this complex, fascinating and clinically relevant series of events that we may ultimately devise ways of better treating, or even preventing, cancer metastasis. The assays may also be of more broad interest to researchers interested in studying aspects of cellular behaviour in relation to other developmental and disease processes