Profiling Of Extracellular Vesicles Research Articles

Protein profiling of extracellular vesicles from iPSC-derived astrocytes of patients with ALS/PDC in Kii peninsula.

Amyotrophic lateral sclerosis/Parkinsonism-dementia complex in Kii peninsula, Japan (Kii ALS/PDC), is an endemic neurodegenerative disease whose causes and pathogenesis remain unknown. However, astrocytes in autopsied cases of Kii ALS/PDC show characteristic lesions. In addition, relationships between extracellular vesicles (EVs) and neurodegenerative diseases are increasingly apparent. Therefore, we focused on proteins in EVs derived from Kii ALS/PDC astrocytes in the present study. Induced pluripotent stem cells (iPSCs) derived from three healthy controls (HCs) and three patients with Kii ALS/PDC were differentiated into astrocytes. EVs in the culture medium of astrocytes were collected and subjected to quantitative proteome analysis. Our proteome analysis reveals that EV-containing proteins derived from astrocytes of patients with Kii ALS/PDC show distinctive patterns compared with those of HCs. Moreover, EVs derived from Kii ALS/PDC astrocytes display increased proteins related to proteostasis and decreased proteins related to anti-inflammation. Proteins contained in EVs from astrocytes unveil protective support to neurons and may reflect the molecular pathomechanism of Kii ALS/PDC; accordingly, they may be potential biomarker candidates of Kii ALS/PDC.

The Adaptation of Botrytis cinerea Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process.

Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several sets of macromolecules implicated in cell communication. EVs have become a relevant topic in the study of pathogenic fungi due to their relationship with fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EVs since plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, EVs of Botrytis cinerea are little known, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the characterization of B. cinerea EVs proteome changes under two pathogenic conditions and the description of their potential role during the infective process. All the experimental procedure was conducted in B. cinerea growing in a minimal salt medium supplemented with glucose as a constitutive stage and deproteinized tomato cell walls (TCW) as a virulence inductor. The isolation of EVs was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EVs fractions were visualised by TEM using negative staining. Proteomic analysis of EVs cargo was addressed by LC-MS/MS. The methodology used allowed the correct isolation of B. cinerea EVs and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in morphology under both assayed conditions. GO analysis of EV proteins showed enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed the difference in EVs function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, among others. This work describes the first evidence of EVs protein cargo adaptation in B. cinerea, which seems to play an essential role in its infection process, sharing crucial functions with the conventional secretion pathways.

The Role of Autophagy as a Trigger of Post-Translational Modifications of Proteins and Extracellular Vesicles in the Pathogenesis of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by persistent joint inflammation, leading to cartilage and bone destruction. Autoantibody production is directed to post-translational modified (PTM) proteins, i.e., citrullinated or carbamylated. Autophagy may be the common feature in several types of stress (smoking, joint injury, and infections) and may be involved in post-translational modifications (PTMs) in proteins and the generation of citrullinated and carbamylated peptides recognized by the immune system in RA patients, with a consequent breakage of tolerance. Interestingly, autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy combines the autophagy machinery with the secretion of cellular content via extracellular vesicles (EVs). A role for exosomes in RA pathogenesis has been recently demonstrated. Exosomes are involved in intercellular communications, and upregulated proteins and RNAs may contribute to the development of inflammatory arthritis and the progression of RA. In RA, most of the exosomes are produced by leukocytes and synoviocytes, which are loaded with PTM proteins, mainly citrullinated proteins, inflammatory molecules, and enzymes that are implicated in RA pathogenesis. Microvesicles derived from cell plasma membrane may also be loaded with PTM proteins, playing a role in the immunopathogenesis of RA. An analysis of changes in EV profiles, including PTM proteins, could be a useful tool for the prevention of inflammation in RA patients and help in the discovery of personalized medicine.

Inaugurating High-Throughput Profiling of Extracellular Vesicles for Earlier Ovarian Cancer Detection.

Detecting early cancer through liquid biopsy is challenging due to the lack of specific biomarkers for early lesions and potentially low levels of these markers. The current studysystematically develops an extracellular-vesicle (EV)-based test for early detection, specifically focusing on high-grade serous ovarian carcinoma (HGSOC). The marker selection is based on emerging insights into HGSOC pathogenesis, notably that it arises from precursor lesions within the fallopian tube. This work thus establishes murine fallopian tube (mFT) cells with oncogenic mutations and performs proteomic analyses on mFT-derived EVs. The identified markers are then evaluated with an orthotopic HGSOC animal model. In serially-drawn blood of tumor-bearing mice, mFT-EV markers increase with tumor initiation, supporting their potential use in early cancer detection. A pilot clinical study (n = 51) further narrows EV markers to five candidates, EpCAM, CD24, VCAN, HE4, and TNC. The combined expression of these markers distinguishes HGSOC from non-cancer with 89% sensitivity and 93% specificity. The same markers are also effective in classifying three groups (non-cancer, early-stage HGSOC, and late-stage HGSOC). The developed approach, for the first time inaugurated in fallopian tube-derived EVs, could be a minimally invasive tool to monitor women at high risk of ovarian cancer for timely intervention.

Extracellular Vesicles Isolated from Menstrual Blood-derived Mesenchymal Stem Cells in Regenerative Medicine

: Extracellular vesicles (EVs) are small particles ranging from 30 - 1000 nm released by different cells to mediate cell-cell communication. The content of these particles mirrors the cellular properties of their producing cells. The extracellular vesicles, including exosomes and microvesicles, could exert the same therapeutic effects as stem cell therapy. However, these stem cell-derived particles (cell-free derivatives) do not have concerns associated with stem cell-based therapies. Among various mesenchymal stem/stromal cells, Menstrual Blood Stem Cells (MenSCs) are known for their high proliferative activity, ease of harvesting, lack of moral dilemma, and high differentiation potential. Extracellular vesicles derived from MenSCs have widely been employed in various preclinical studies for regenerative purposes. This paper aims to provide an overview of the therapeutic utility of MenSCs-derived EVs in regenerative medicine. We also reviewed the current knowledge on the cellular profile, biogenesis, separation, and action mechanism of MenSCs-derived EVs in organ regeneration.

The lipid composition of extracellular vesicles: applications in diagnostics and therapeutic delivery.

Extracellular vesicles (EVs), including exosomes, with nanoscale sizes, biological origins, various functions, and unique lipid and protein compositions have been introduced as versatile tools for diagnostic and therapeutic medical applications. Numerous studies have reported the importance of the lipid composition of EVs and its influence on their mechanism of action. For example, changes in the lipidomic profile of EVs have been shown to influence the progression of various diseases, including ovarian malignancies and prostate cancer. In this review, we endeavored to examine differences in the lipid content of EV membranes derived from different cell types to characterize their capabilities as diagnostic tools and treatments for diseases like cancer and Alzheimer's disease. We additionally discuss designing functionalized vesicles, whether synthetically by hybrid methods or by changing the lipid composition of natural EVs. Lastly, we provide an overview of current and potential biomedical applications and perspectives on the future of this growing field.

Extracellular Vesicles Play a Central Role in Cerebral Venous Disease-Associated Brain Atrophy.

Cerebral venous abnormalities, distinct from traditional arterial diseases, have been linked to brain atrophy in a previous community-based cohort study, specifically in relation to the reduction of deep medullary veins (r-DMVs). To better understand the properties and biological functions of serum extracellular vesicles (EVs) in cerebral venous disease-associated brain atrophy, EVs are extracted from the serum of both participants with r-DMV and normal controls and analyzed their proteomic profiles using Tandem Mass Tag label quantitation analysis. Phenotypic experiments showed that EVs from individuals with r-DMVs are able to disrupt the normal functions of neurons, endothelial cells, and smooth muscle cells, and induce A1 reactive astrocytes. Additionally, this study provided a comprehensive characterization of the proteomic profile of DMV EVs and found that the collagen hydroxyproline is upregulated, while complement C3 is downregulated in the r-DMV group, suggesting that r-DMV may not be a simple pathological phenomenon and highlighting the potential involvement of EVs in the progression of brain atrophy in r-DMVs which has implications for the development of future therapeutic strategies.

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in Multiple sclerosis

BackgroundMultiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease’s pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease’s pathological processes. PurposeTo identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. ResultsHigher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen β chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. ConclusionNeural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MS for reflecting the disease state.

Proteomic Profiling of Extracellular Vesicles Isolated from Plasma and Peritoneal Exudate in Mice Induced by Crotalus scutulatus scutulatus Crude Venom and Its Purified Cysteine-Rich Secretory Protein (Css-CRiSP).

Increased vascular permeability is a frequent outcome of viperid snakebite envenomation, leading to local and systemic complications. We reported that snake venom cysteine-rich secretory proteins (svCRiSPs) from North American pit vipers increase vascular permeability both in vitro and in vivo. They also induce acute activation of several adhesion and signaling molecules that may play a critical role in the pathophysiology of snakebites. Extracellular vesicles (EVs) have gained interest for their diverse functions in intercellular communication, regulating cellular processes, blood-endothelium interactions, vascular permeability, and immune modulation. They also hold potential as valuable biomarkers for diagnosing, predicting, and monitoring therapeutic responses in different diseases. This study aimed to identify proteins in peritoneal exudate and plasma EVs isolated from BALB/c mice following a 30 min post-injection of Crotalus scutulatus scutulatus venom and its purified CRiSP (Css-CRiSP). EVs were isolated from these biofluids using the EVtrap method. Proteomic analysis of exudate- and plasma-derived EVs was performed using LC-MS/MS. We observed significant upregulation or downregulation of proteins involved in cell adhesion, cytoskeleton rearrangement, signal transduction, immune responses, and vesicle-mediated transports. These findings suggest that svCRiSPs play a crucial role in the acute effects of venom and contribute to the local and systemic toxicity of snakebites.

Systematic Assessment of Small RNA Profiling in Human Extracellular Vesicles.

Extracellular vesicles (EVs) are produced and released by most cells and are now recognized to play a role in intercellular communication through the delivery of molecular cargo, including proteins, lipids, and RNA. Small RNA sequencing (small RNA-seq) has been widely used to characterize the small RNA content in EVs. However, there is a lack of a systematic assessment of the quality, technical biases, RNA composition, and RNA biotypes enrichment for small RNA profiling of EVs across cell types, biofluids, and conditions. We collected and reanalyzed small RNA-seq datasets for 2756 samples from 83 studies involving 55 with EVs only and 28 with both EVs and matched donor cells. We assessed their quality by the total number of reads after adapter trimming, the overall alignment rate to the host and non-host genomes, and the proportional abundance of total small RNA and specific biotypes, such as miRNA, tRNA, rRNA, and Y RNA. We found that EV extraction methods varied in their reproducibility in isolating small RNAs, with effects on small RNA composition. Comparing proportional abundances of RNA biotypes between EVs and matched donor cells, we discovered that rRNA and tRNA fragments were relatively enriched, but miRNAs and snoRNA were depleted in EVs. Except for the export of eight miRNAs being context-independent, the selective release of most miRNAs into EVs was study-specific. This work guides quality control and the selection of EV isolation methods and enhances the interpretation of small RNA contents and preferential loading in EVs.

MoS2-Plasmonic Nanocavities for Raman Spectra of Single Extracellular Vesicles Reveal Molecular Progression in Glioblastoma.

Extracellular vesicles (EVs) are continually released from cancer cells into biofluids, carrying actionable molecular fingerprints of the underlying disease with considerable diagnostic and therapeutic potential. The scarcity, heterogeneity and intrinsic complexity of tumor EVs present a major technological challenge in real-time monitoring of complex cancers such as glioblastoma (GBM). Surface-enhanced Raman spectroscopy (SERS) outputs a label-free spectroscopic fingerprint for EV molecular profiling. However, it has not been exploited to detect known biomarkers at the single EV level. We developed a multiplex fluidic device with embedded arrayed nanocavity microchips (MoSERS microchip) that achieves 97% confinement of single EVs in a minute amount of fluid (<10 μL) and enables molecular profiling of single EVs with SERS. The nanocavity arrays combine two featuring characteristics: (1) An embedded MoS2 monolayer that enables label-free isolation and nanoconfinement of single EVs due to physical interaction (Coulomb and van der Waals) between the MoS2 edge sites and the lipid bilayer; and (2) A layered plasmonic cavity that enables sufficient electromagnetic field enhancement inside the cavities to obtain a single EV level signal resolution for stratifying the molecular alterations. We used the GBM paradigm to demonstrate the diagnostic potential of the SERS single EV molecular profiling approach. The MoSERS multiplexing fluidic achieves parallel signal acquisition of glioma molecular variants (EGFRvIII oncogenic mutation and MGMT expression) in GBM cells. The detection limit of 1.23% was found for stratifying these key molecular variants in the wild-type population. When interfaced with a convolutional neural network (CNN), MoSERS improved diagnostic accuracy (87%) with which GBM mutations were detected in 12 patient blood samples, on par with clinical pathology tests. Thus, MoSERS demonstrates the potential for molecular stratification of cancer patients using circulating EVs.

#6120 MIRNA PROFILING OF URINARY EXTRACELLULAR VESICLES (EVS) IN TBMN AND CFHR5 NEPHROPATHY FOR THE IDENTIFICATION OF NOVEL DISEASE BIOMARKERS

Abstract Background and Aims Thin basement membrane nephropathy (TBMN) and complement factor-H related protein 5 (CFHR5) nephropathy are two of the most common renal monogenic diseases with high phenotypic heterogeneity that is only partly accounted for by locus and allelic factors. Intra-familial variability in the age of ESKD onset for some patients particularly exemplifies such highly variable phenotypes, suggesting a potential modifying role of additional factors such as non-coding regulatory RNAs. Given that miRNAs are key regulators of gene expression, miRNAs from urine-derived extracellular vesicles (EVs) were investigated for their potential use as biomarkers in TBMN and CFHR5 diagnosis and prognosis. Method MiRNA profiling of human urinary EVs was carried out by small RNA-sequencing in an initial discovery cohort of 54 patients with TBMN and 44 patients with CFHR5 nephropathy, each classified further into three subgroups based on disease severity (Grey-zone: eGFR &amp;gt;60 ml/min/1.73m2 and age &amp;lt;50 y/o; Mild: eGFR &amp;gt;60 ml/min/1.73m2 and age &amp;lt;50 y/o; Severe: eGFR &amp;lt;60 ml/min/1.73m2 independently of age). Differential expression of miRNAs from urine-derived EVs was compared with 30 age and sex matched healthy controls, whilst bioinformatic analysis was performed to delineate their associated mechanisms. Results Kidney-enriched candidate miRNA families were identified in urine-derived EVs for both TBMN and CFHR5 patients and further verified by RT-qPCR in the respective validation cohorts. Distinct miRNAs with significantly differentiated expression levels, compared to that of healthy control cohort, showed significant correlations with some of the evaluated clinical characteristics for both TBMN and CFHR5 patients suggesting a possible diagnostic and prognostic value on long follow-up. Finally, inverse correlations of miRNA expression with their predicted targeted genes identified dysregulated pathways and transcriptional networks for further investigation. Conclusion Collectively, distinct profiles of miRNAs from urine-derived EVs were identified in TBMN and CFHR5 nephropathy suggesting that a subset of differentially expressed miRNAs could serve as novel non-invasive biomarkers of disease progression and/or targets for the development of novel therapeutic approaches.

Towards optimised extracellular vesicle proteomics from cerebrospinal fluid

The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean ± standard deviation. Using proteomics, SEC fractions 3–5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4–5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6 ml, 3 ml, 1 ml, and 0.5 ml) to evaluate the effect on the proteomic profile. Even with a 0.5 ml starting volume, 743 ± 77 or 345 ± 88 proteins were identified depending on whether ‘matches between runs’ was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5 ml of canine CSF.

Adapter dimer contamination in sRNA‐sequencing datasets predicts sequencing failure and batch effects and hampers extracellular vesicle‐sRNA analysis

AbstractSmall RNA (sRNA) profiling of Extracellular Vesicles (EVs) by Next‐Generation Sequencing (NGS) often delivers poor outcomes, independently of reagents, platforms or pipelines used, which contributes to poor reproducibility of studies. Here we analysed pre/post‐sequencing quality controls (QC) to predict issues potentially biasing biological sRNA‐sequencing results from purified human milk EVs, human and mouse EV‐enriched plasma and human paraffin‐embedded tissues. Although different RNA isolation protocols and NGS platforms were used in these experiments, all datasets had samples characterized by a marked removal of reads after pre‐processing. The extent of read loss between individual samples within a dataset did not correlate with isolated RNA quantity or sequenced base quality. Rather, cDNA electropherograms revealed the presence of a constant peak whose intensity correlated with the degree of read loss and, remarkably, with the percentage of adapter dimers, which were found to be overrepresented sequences in high read‐loss samples. The analysis through a QC pipeline, which allowed us to monitor quality parameters in a step‐by‐step manner, provided compelling evidence that adapter dimer contamination was the main factor causing batch effects. We concluded this study by summarising peer‐reviewed published workflows that perform consistently well in avoiding adapter dimer contamination towards a greater likelihood of sequencing success.

Surface protein profiling of milk and serum extracellular vesicles unveils body fluid-specific signatures

Cell-derived extracellular vesicles (EVs) are currently in the limelight as potential disease biomarkers. The promise of EV-based liquid biopsy resides in the identification of specific disease-associated EV signatures. Knowing the reference EV profile of a body fluid can facilitate the identification of such disease-associated EV-biomarkers. With this aim, we purified EVs from paired human milk and serum samples and used the MACSPlex bead-based flow-cytometry assay to capture EVs on bead-bound antibodies specific for a certain surface protein, followed by EV detection by the tetraspanins CD9, CD63, and CD81. Using this approach we identified body fluid-specific EV signatures, e.g. breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, as well as body fluid-specific markers associated to immune cells and stem cells. Interestingly, comparison of pan-tetraspanin detection (simultaneous CD9, CD63 and CD81 detection) and single tetraspanin detection (detection by CD9, CD63 or CD81) also unveiled body fluid-specific tetraspanin distributions on EVs. Moreover, certain EV surface proteins were associated with a specific tetraspanin distribution, which could be indicative of the biogenesis route of this EV subset. Altogether, the identified body fluid-specific EV profiles can contribute to study EV profile deviations in these fluids during disease processes.

Improved isolation of extracellular vesicles by removal of both free proteins and lipoproteins.

Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery.

Extracellular vesicles in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity.

Metabolomic investigation of urinary extracellular vesicles for early detection and screening of lung cancer

Lung cancer is a prevalent cancer type worldwide that often remains asymptomatic in its early stages and is frequently diagnosed at an advanced stage with a poor prognosis due to the lack of effective diagnostic techniques and molecular biomarkers. However, emerging evidence suggests that extracellular vesicles (EVs) may promote lung cancer cell proliferation and metastasis, and modulate the anti-tumor immune response in lung cancer carcinogenesis, making them potential biomarkers for early cancer detection. To investigate the potential of urinary EVs for non-invasive detection and screening of patients at early stages, we studied metabolomic signatures of lung cancer. Specifically, we conducted metabolomic analysis of 102 EV samples and identified metabolome profiles of urinary EVs, including organic acids and derivatives, lipids and lipid-like molecules, organheterocyclic compounds, and benzenoids. Using machine learning with a random forest model, we screened for potential markers of lung cancer and identified a marker panel consisting of Kanzonol Z, Xanthosine, Nervonyl carnitine, and 3,4-Dihydroxybenzaldehyde, which exhibited a diagnostic potency of 96% for the testing cohort (AUC value). Importantly, this marker panel also demonstrated effective prediction for the validation set, with an AUC value of 84%, indicating the reliability of the marker screening process. Our findings suggest that the metabolomic analysis of urinary EVs provides a promising source of non-invasive markers for lung cancer diagnostics. We believe that the EV metabolic signatures could be used to develop clinical applications for the early detection and screening of lung cancer, potentially improving patient outcomes.

Proteomic profiling of extracellular vesicles in synovial fluid and plasma from Oligoarticular Juvenile Idiopathic Arthritis patients reveals novel immunopathogenic biomarkers.

New early low-invasive biomarkers are demanded for the management of Oligoarticular Juvenile Idiopathic Arthritis (OJIA), the most common chronic pediatric rheumatic disease in Western countries and a leading cause of disability. A deeper understanding of the molecular basis of OJIA pathophysiology is essential for identifying new biomarkers for earlier disease diagnosis and patient stratification and to guide targeted therapeutic intervention. Proteomic profiling of extracellular vesicles (EVs) released in biological fluids has recently emerged as a minimally invasive approach to elucidate adult arthritis pathogenic mechanisms and identify new biomarkers. However, EV-prot expression and potential as biomarkers in OJIA have not been explored. This study represents the first detailed longitudinal characterization of the EV-proteome in OJIA patients. Fourty-five OJIA patients were recruited at disease onset and followed up for 24 months, and protein expression profiling was carried out by liquid chromatography-tandem mass spectrometry in EVs isolated from plasma (PL) and synovial fluid (SF) samples. We first compared the EV-proteome of SF vs paired PL and identified a panel of EV-prots whose expression was significantly deregulated in SF. Interaction network and GO enrichment analyses performed on deregulated EV-prots through STRING database and ShinyGO webserver revealed enrichment in processes related to cartilage/bone metabolism and inflammation, suggesting their role in OJIA pathogenesis and potential value as early molecular indicators of OJIA development. Comparative analysis of the EV-proteome in PL and SF from OJIA patients vs PL from age/gender-matched control children was then carried out. We detected altered expression of a panel of EV-prots able to differentiate new-onset OJIA patients from control children, potentially representing a disease-associated signature measurable at both the systemic and local levels with diagnostic potential. Deregulated EV-prots were significantly associated with biological processes related to innate immunity, antigen processing and presentation, and cytoskeleton organization. Finally, we ran WGCNA on the SF- and PL-derived EV-prot datasets and identified a few EV-prot modules associated with different clinical parameters stratifying OJIA patients in distinct subgroups. These data provide novel mechanistic insights into OJIA pathophysiology and an important contribution in the search of new candidate molecular biomarkers for the disease.

Abstract 5311: Lipidomic profiling of extracellular vesicles derived from cancer cell lines: Lipid species as potential biomarkers and cellular uptake enhancers

Abstract Introduction: Extracellular vesicles (EVs) are lipid bilayer-made particles shed by cells to the extracellular space. They carry different cargo proteins, nucleic acids, and other metabolites. EVs play a role in disseminating cancer to distant organs by communicating with the tumor microenvironment to prepare the metastatic niche and also through horizontal transfer of oncogenic traits to recipient cells. The EV surface, which includes proteins and lipids, plays a role in organotropism and cellular uptake. While proteins have been extensively characterized, lipids have not been explored sufficiently. This work aims to evaluate EV lipids as potential biomarkers and their role in enhancing cellular uptake. Methods: To detect which lipid species (LS) were differentially expressed, we used two cell models of liver metastatic cells: colorectal cancer (HT29) and uveal melanoma (MP41, MP46, MEL 270, OMM 2.5) cell lines. Colon (CCD18-Co) and fibroblast (BJ) immortalized non-cancerous cells were used as controls. EVs were isolated from culture media by ultrafiltration using 100 kDa units filters. Lipids were extracted by methyl-tert-butyl ether for high-throughput lipidomics. High-resolution ‘shotgun’ mass spectrometry was performed. Data was analyzed using LipidView software (SCIEX), and the lipid % normalized was reported. MarkerView (SCIEX) was used to perform Principal Component Analysis. The LS segregating cancerous vs. non-cancerous cells were identified. To evaluate the influence on cellular uptake, we used liposomes as EV models with lipid formulations containing the segregating LS to compare them with naturally occurring lipids and EVs using Incucyte live cell imaging. Results: We identified four LS that segregated EV subpopulations. PE 34:1 and PS 36:1 divided cancerous vs non-cancerous cells, uveal melanoma cells were segregated by PE 36:2, and normal colon cells were segregated by LPC 18:0. We validated the effect of PS 36:1 in cellular uptake by producing liposomes with a lipid formulation resembling the lipid profile of naturally occurring EVs lipid profile with an artificially high DOPS concentration (17% of the total molar ratio). We determined that human hepatocytes preferentially internalized liposomes made of naturally occurring EVs, followed by the ones with a high concentration of DOPS and lastly by a control EV lipid formulation. Conclusion: This study identified EV LS that segregated cancerous, normal, and melanoma cell lines. We showed that LS could be used to distinguish cell populations. Moreover, we demonstrated that LS alone influences cellular uptake and that adding the segregating LS to lipid formulations in excess effects cellular uptake. These results pave the way to identify EV lipid biomarkers and better understand EV based cancer dissemination. Citation Format: Ruben R. Lopez Salazar, Prisca Bustamante, Chaymaa Zouggari, Yunxi Chen, Thupten Tsering, Ion Stiharu, Catherine Mounier, Vahe Nerguizian, Julia Burnier. Lipidomic profiling of extracellular vesicles derived from cancer cell lines: Lipid species as potential biomarkers and cellular uptake enhancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5311.