Associations of age and sex with characteristics of extracellular vesicles and protein-enriched fractions of blood plasma.

Extracellular vesicles (EVs) are nanosized particles that are released by various cell types and play vital roles in intercellular communication. They carry biological molecules reflecting the physiological and pathological states of their source cells and tissues, showing potential as biomarkers. However, the impact of demographic factors like age and sex on the properties of blood plasma EVs remains underexplored. This study aims to fill this gap by evaluating how these factors influence the particle count and proteomic profiles of plasma EV preparations and corresponding protein fractions. Plasma samples from 120 healthy volunteers were collected and pooled into six groups: young males (age: 27.6 ± 4.0), young females (27.4 ± 3.8), middle-aged males (48.8 ± 3.8), middle-aged females (48.9 ± 3.9), old males (69.3 ± 3.9), and old females (69.4 ± 4.3). EV- and protein-enriched fractions were separated by size-exclusion chromatography (SEC). Fractions were characterized for particle number concentration and protein composition to identify characteristics affected by age and biological sex. Plasma EVs and corresponding protein fractions exhibited distinct characteristics, with differential enrichment of markers related to EVs and other blood components, including lipoproteins. Proteomic profiles of both EVs and protein fractions displayed sex- and age-dependent differences. Differentially abundant proteins displayed functions previously identified in the context of aging and sex differences, highlighting their utility as biomarkers. Age and sex significantly affect the characteristics of plasma EVs and proteins, potentially influencing their efficacy and interpretation as biomarkers in clinical applications. This study lays the groundwork for detailed mechanistic research to understand how EVs mediate age- and sex-related effects in health.

Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses.

The individual detection of human immunodeficiency virus (HIV) virions and resolution from extracellular vesicles (EVs) during analysis is a difficult challenge. Infectious enveloped virions and nonviral EVs are released simultaneously by HIV-infected host cells, in addition to hybrid viral EVs containing combinations of HIV and host components but lacking replicative ability. Complicating the issue, EVs and enveloped virions are both delimited by a lipid bilayer and share similar size and density. The feature that distinguishes infectious virions from host and hybrid EVs is the HIV genomic RNA (gRNA), which allows the virus to replicate. Single-particle analysis techniques, which provide snapshots of single biological nanoparticles, could resolve infectious virions from EVs. However, current single-particle analysis techniques focus mainly on protein detection, which fail to resolve hybrid EVs from infectious virions. A method to simultaneously detect viral protein and internal gRNA in the same particle would allow resolution of infectious HIV from EVs and noninfectious virions. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor with single-molecule fluorescence in situ hybridization. Using SPIRFISH, we detect HIV-1 envelope protein gp120 and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNAs within EVs. This may have major utility for EV therapeutics, which are increasingly focused on EV-mediated RNA delivery. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles.

Aged fibroblast-derived extracellular vesicles promote angiogenesis in melanoma.

Advancing age is a negative prognostic factor for cutaneous melanoma. However, the role of extracellular vesicles (EVs) within the melanoma tumor microenvironment (TME) has remained unexplored in the context of aging. While the size and morphology of the EVs isolated from young vs. aged fibroblasts remained unaltered, the contents of the protein cargo were changed. Aging reduced the expression of the tetraspanin CD9 in both the dermal fibroblasts and released EVs. CD9 is a crucial regulator of EV cargo sorting. Modulating the CD9 expression in fibroblasts was sufficient to alter its levels in EVs. Mass spectrometry analysis of EVs released by CD9 knockdown (KD) vs. control cells revealed a significant increase in angiopoietin-like protein 2 (ANGPTL2), an angiogenesis promoter. Analysis of primary endothelial cells confirmed increased sprouting under CD9 KD conditions. Together, our data indicate that aged EVs play an important role in promoting a tumor-permissive microenvironment.

Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer.

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.

Urinary extracellular vesicle-derived miR-126-3p predicts lymph node invasion in patients with high-risk prostate cancer.

To investigate extracellular vesicles (EVs), biomarkers for predicting lymph node invasion (LNI) in patients with high-risk prostate cancer (HRPCa), plasma, and/or urine samples were prospectively collected from 45 patients with prostate cancer (PCa) and five with benign prostatic hyperplasia (BPH). Small RNA sequencing was performed to identify miRNAs in the EVs. All patients with PCa underwent radical prostatectomy and extended pelvic lymph node dissection. Differentially expressed miRNAs were identified in patients with and without pathologically-verified LNI. The candidate miRNAs were validated in low-risk prostate cancer (LRPCa) and BPH. Four miRNA species (e.g., miR-126-3p) and three miRNA species (e.g., miR-27a-3p) were more abundant in urinary and plasma EVs, respectively, of patients with PCa. None of these miRNA species were shared between urinary and plasma EVs. miR-126-3p was significantly more abundant in patients with HR PCa with LNI than in those without (P = 0.018). miR-126-3p was significantly more abundant in the urinary EVs of patients with HRPCa than in those with LRPCa (P = 0.017) and BPH (P = 0.011). In conclusion, urinary EVs-derived miR-126-3p may serve as a good biomarker for predicting LNI in patients with HRPCa.

Characterization of spinal cord tissue-derived extracellular vesicles in neuroinflammation.

Extracellular vesicles (EVs) are released by all cells, can cross the blood-brain barrier, and have been shown to play an important role in cellular communication, substance shuttling, and immune modulation. In recent years EVs have shifted into focus in multiple sclerosis (MS) research as potential plasma biomarkers and therapeutic vehicles. Yet little is known about the disease-associated changes in EVs in the central nervous system (CNS). To address this gap, we characterized the physical and proteomic changes of mouse spinal cord-derived EVs before and at 16 and 25 days after the induction of experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory model of MS. Using various bioinformatic tools, we found changes in inflammatory, glial, and synaptic proteins and pathways, as well as a shift in the predicted contribution of immune and glial cell types over time. These results show that EVs provide snapshots of crucial disease processes such as CNS-compartmentalized inflammation, re/de-myelination, and synaptic pathology, and might also mediate these processes. Additionally, inflammatory plasma EV biomarkers previously identified in people with MS were also altered in EAE spinal cord EVs, suggesting commonalities of EV-related pathological processes during EAE and MS and overlap of EV proteomic changes between CNS and circulating EVs.

ELISA-based detection of immunoglobulins against extracellular vesicles in blood plasma.

Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable safety profiles and low immunogenicity make EVs an attractive alternative to synthetic nanoparticles. We recently showed that repeated intravenous administration of human cell-derived EVs into pig-tailed macaques unexpectedly elicited antibody responses after three or more injections. This coincided with decreasing EV circulation time, and may thus hamper successful EV-mediated cargo delivery into tissues. Here, we share the custom ELISA protocol that we used to measure such antibody responses. This protocol may help other researchers evaluate immune responses to EV-based therapies in preclinical studies.

Urinary extracellular vesicle-derived miR-126-3p predicts lymph node invasion in patients with high-risk prostate cancer.

To investigate extracellular vesicles (EVs) biomarkers for predicting lymph node invasion (LNI) in patients with high-risk prostate cancer (HRPCa), plasma and/or urine samples were prospectively collected from 45 patients with prostate cancer (PCa) and five with benign prostatic hyperplasia (BPH). Small RNA sequencing was performed to identify miRNAs in the EVs. All patients with PCa underwent radical prostatectomy and extended pelvic lymph node dissection. Differentially-expressed miRNAs were identified in patients with and without pathologically-verified LNI. The candidate miRNAs were validated in low-risk prostate cancer (LRPCa) and BPH. Four miRNA species (e.g. miR-126-3p) and three miRNA species (e.g. miR-27a-3p) were more abundant in urinary and plasma EVs, respectively, of patients with PCa. None of these miRNA species were shared between urinary and plasma EVs. miR-126-3p was significantly more abundant in patients with HR PCa with LNI than in those without (P = 0.018). miR-126-3p was significantly more abundant in the urinary EVs of patients with HRPCa than in those with LRPCa (P = 0.017) and BPH (P = 0.011). In conclusion, urinary EVs-derived miR-126-3p may serve as a good biomarker for predicting LNI in patients with HRPCa.

Evolution of nasal and olfactory infection characteristics of SARS-CoV-2 variants.

SARS-CoV-2 infection of the upper airway and the subsequent immune response are early, critical factors in COVID-19 pathogenesis. By studying infection of human biopsies in vitro and in a hamster model in vivo, we demonstrated a transition in nasal tropism from olfactory to respiratory epithelium as the virus evolved. Analyzing each variant revealed that SARS-CoV-2 WA1 or Delta infect a proportion of olfactory neurons in addition to the primary target sustentacular cells. The Delta variant possessed broader cellular invasion capacity into the submucosa, while Omicron displayed enhanced nasal respiratory infection and longer retention in the sinonasal epithelium. The olfactory neuronal infection by WA1 and the subsequent olfactory bulb transport via axon were more pronounced in younger hosts. In addition, the observed viral clearance delay and phagocytic dysfunction in aged olfactory mucosa were accompanied by a decline of phagocytosis-related genes. Further, robust basal stem cell activation contributed to neuroepithelial regeneration and restored ACE2 expression postinfection. Together, our study characterized the nasal tropism of SARS-CoV-2 strains, immune clearance, and regeneration after infection. The shifting characteristics of viral infection at the airway portal provide insight into the variability of COVID-19 clinical features, particularly long COVID, and may suggest differing strategies for early local intervention.

Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs.

RNA viruses have been shown to express various short RNAs, some of which have regulatory roles during replication, transcription, and translation of viral genomes. However, short viral RNAs generated from SARS-CoV-1 and SARS-CoV-2 genomic RNAs remained largely unexplored, possibly due limitations of the widely used library preparation methods for small RNA deep sequencing and corresponding data processing. By analyzing publicly available small RNA sequencing datasets, we observed that human Calu-3 cells infected by SARS-CoV-1 or SARS-CoV-2 accumulate multiple previously unreported short viral RNAs. In addition, we verified the presence of the five most abundant SARS-CoV-2 short viral RNAs in SARS-CoV-2-infected human lung adenocarcinoma cells by quantitative PCR. Interestingly, the copy number of the observed SARS-CoV-2 short viral RNAs dramatically exceeded the expression of previously reported viral microRNAs in the same cells. We hypothesize that the reported SARS-CoV-2 short viral RNAs could serve as biomarkers for early infection stages due to their high abundance. Furthermore, unlike SARS-CoV-1, the SARS-CoV-2 infection induced significant (Benjamini-Hochberg-corrected p-value <0.05) deregulation of Y-RNA, transfer RNA, vault RNA, as well as more than 300 endogenous short RNAs that aligned predominantly to human protein-coding and long noncoding RNA transcripts. In particular, more than 20-fold upregulation of reads derived from Y-RNA (and several transfer RNAs) have been documented in RNA-seq datasets from SARS-CoV-2 infected cells. Finally, a significant proportion of short RNAs derived from full-length viral genomes also aligned to various human genome (hg38) sequences, suggesting opportunities to investigate regulatory roles of short viral RNAs during infection. Further characterization of the small RNA landscape of both viral and host genomes is clearly warranted to improve our understanding of molecular events related to infection and to design more efficient strategies for therapeutic interventions as well as early diagnosis.

International Society for Extracellular Vesicles Workshop. QuantitatEVs: multiscale analyses, from bulk to single extracellular vesicle.

The 'QuantitatEVs: multiscale analyses, from bulk to single vesicle' workshop aimed to discuss quantitative strategies and harmonized wet and computational approaches toward the comprehensive analysis of extracellular vesicles (EVs) from bulk to single vesicle analyses with a special focus on emerging technologies. The workshop covered the key issues in the quantitative analysis of different EV-associated molecular components and EV biophysical features, which are considered the core of EV-associated biomarker discovery and validation for their clinical translation. The in-person-only workshop was held in Trento, Italy, from January 31st to February 2nd, 2023, and continued in Milan on February 3rd with "Next Generation EVs", a satellite event dedicated to early career researchers (ECR). This report summarizes the main topics and outcomes of the workshop.

Advances in Extracellular Vesicle Research Over the Past Decade: Source and Isolation Method are Connected with Cargo and Function.

The evolution of extracellular vesicle (EV) research has introduced nanotechnology into biomedical cell communication science while recognizing what is formerly considered cell "dust" as constituting an entirely new universe of cell signaling particles. To display the global EV research landscape, a systematic review of 20 364 original research articles selected from all 40 684 EV-related records identified in PubMed 2013-2022 is performed. Machine-learning is used to categorize the high-dimensional data and further dissected significant associations between EV source, isolation method, cargo, and function. Unexpected correlations between these four categories indicate prevalent experimental strategies based on cargo connectivity with function of interest being associated with certain EV sources or isolation strategies. Conceptually relevant association of size-based EV isolation with protein cargo and uptake function will guide strategic conclusions enhancing future EV research and product development. Based on this study, an open-source database is built to facilitate further analysis with conventional or AI tools to identify additional causative associations of interest.

RNA landscapes of brain and brain-derived extracellular vesicles in simian immunodeficiency virus (SIV) infection and central nervous system pathology.

INTRODUCTION: Brain tissue-derived extracellular vesicles (bdEVs) act locally in the central nervous system (CNS) and may indicate molecular mechanisms in HIV CNS pathology. Using brain homogenate (BH) and bdEVs from a simian immunodeficiency virus (SIV) model of HIV disease, we identified RNA networks in SIV infection and neuroinflammation. METHODS: Postmortem occipital cortex samples were obtained from uninfected controls and SIV-infected subjects (acute and chronic phases with or without CNS pathology (SIV encephalitis). bdEVs were separated and characterized per international consensus guidelines. RNAs from bdEVs and BH were sequenced and qPCR-amplified to detect levels of small RNAs (sRNAs, including microRNAs (miRNAs)) and longer RNAs including messenger RNAs (mRNAs) and circular RNAs (circRNAs). RESULTS: Dysregulated RNAs in BH and bdEVs were identified in acute and chronic infection with pathology groups, including mRNAs, miRNAs, and circRNAs. Most dysregulated mRNAs in bdEVs reflected dysregulation in source BH. These mRNAs are disproportionately involved in inflammation and immune responses. Based on target prediction, several circRNAs that were differentially abundant in source tissue might be responsible for specific differences in sRNA levels in bdEVs during SIV infection. CONCLUSIONS: RNA profiling of bdEVs and source tissues reveals potential regulatory networks in SIV infection and SIV-related CNS pathology.

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.

An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood.

Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture-derived EVs are administered intravenously to Macaca nemestrina (pig-tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 h post-intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig-tailed macaques. Using this ex vivo system, we found that labelled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV-blood cell interactions.

Toward a human brain extracellular vesicle atlas: Characteristics of extracellular vesicles from different brain regions, including small RNA and protein profiles.

Extracellular vesicles (EVs) are released from different cell types in the central nervous system (CNS) and play roles in regulating physiological and pathological functions. Although brain-derived EVs (bdEVs) have been successfully collected from brain tissue, there is not yet a "bdEV Atlas" of EVs from different brain regions. To address this gap, we separated EVs from eight anatomical brain regions of a single individual and subsequently characterized them by count, size, morphology, and protein and RNA content. The greatest particle yield was from cerebellum, while the fewest particles were recovered from the orbitofrontal, postcentral gyrus, and thalamus regions. EV surface phenotyping indicated that CD81 and CD9 were more abundant than CD63 in all regions. Cell-enriched surface markers varied between brain regions. For example, putative neuronal markers NCAM, CD271, and NRCAM were more abundant in medulla, cerebellum, and occipital regions, respectively. These findings, while restricted to tissues from a single individual, suggest that additional studies are warranted to provide more insight into the links between EV heterogeneity and function in the CNS.

Surfaceome analysis of extracellular vesicles from senescent cells uncovers uptake repressor DPP4.

Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.