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.

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.

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.

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 tumor 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 many studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insights into the stage of the disease and predict the likely therapeutic outcome. There remain many 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.

BackgroundCongenital infection of the fetus via trans-placental passage of pathogens can result in severe morbidity and mortality. Even without transmission to the fetus, infection of the placenta itself is associated with pregnancy complications including pregnancy loss and preterm birth. Placental macrophages, also termed Hofbauer cells (HBCs), are fetal-origin macrophages residing in the placenta that are likely involved in responding to placental infection and protection of the developing fetus. As HBCs are the only immune cell present in the villous placenta, they represent one of the final opportunities for control of infection and prevention of passage to the developing fetus.Objective and RationaleThe objective of this review was to provide a systematic overview of the literature regarding HBC responses during infection in pregnancy, including responses to viral, bacterial, and parasitic pathogens.MethodsPubMed and Scopus were searched on May 20th, 2021, with no limit on publication date, to identify all papers that have studied placental macrophages/Hofbauer cells in the context of infection. The following search strategy was utilized: (hofbauer* OR “hofbauer cells” OR “hofbauer cell” OR “placental macrophage” OR “placental macrophages”) AND [infect* OR virus OR viral OR bacteri* OR parasite* OR pathogen* OR LPS OR “poly(i:c)” OR toxoplasm* OR microb* OR HIV)].Outcomes86 studies were identified for review. This included those that investigated HBCs in placentas from pregnancies complicated by maternal infection and in vitro studies investigating HBC responses to pathogens or Pathogen-Associated Molecular Patterns (PAMPs). HBCs can be infected by a variety of pathogens, and HBC hyperplasia was a common observation. HBCs respond to pathogen infection and PAMPs by altering their transcriptional, translational and secretion profiles. Co-culture investigations demonstrate that they can replicate and transmit pathogens to other cells. In other cases, they may eliminate the pathogen through a variety of mechanisms including phagocytosis, cytokine-mediated pathogen elimination, release of macrophage extracellular traps and HBC-antibody-mediated neutralization. HBC responses differ across gestation and may be influenced by pre-existing immunity. Clinical information, including gestational age at infection, gestational age of the samples, mode of sample collection and pregnancy outcome were missing for the majority of studies.

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.

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Macrophages are important antigen presenting cells which can release extracellular vesicles (EVs) carrying functional cargo including non‐coding RNAs. Macrophages can be broadly classified into M1 ‘classical’ and M2 ‘alternatively‐activated’ macrophages. M1 macrophages have been linked with inflammation‐associated pathologies, whereas a switch towards an M2 phenotype indicates resolution of inflammation and tissue regeneration. Here, we provide the first comprehensive analysis of the small RNA cargo of EVs from human M1 and M2 primary macrophages. Using small RNA sequencing, we identified several types of small non‐coding RNAs in M1 and M2 macrophage EVs including miRNAs, isomiRs, tRNA fragments, piRNA, snRNA, snoRNA and Y‐RNA fragments. Distinct differences were observed between M1 and M2 EVs, with higher relative abundance of miRNAs, and lower abundance of tRNA fragments in M1 compared to M2 EVs. MicroRNA‐target enrichment analysis identified several gene targets involved in gene expression and inflammatory signalling pathways. EVs were also enriched in tRNA fragments, primarily originating from the 5′ end or the internal region of the full length tRNAs, many of which were differentially abundant in M1 and M2 EVs. Similarly, several other small non‐coding RNAs, namely snRNAs, snoRNAs and Y‐RNA fragments, were differentially enriched in M1 and M2 EVs; we discuss their putative roles in macrophage EVs. In conclusion, we show that M1 and M2 macrophages release EVs with distinct RNA cargo, which has the potential to contribute to the unique effect of these cell subsets on their microenvironment.