Profiling Of Extracellular Vesicles Research Articles

(Invited) Aiot-Integrated Digital Imaging Sensor for Molecular-Fingerprint Profiling of Extracellular Vesicles in Liquid Biopsy

The advancement of liquid biopsy techniques for non-invasive tumor analysis holds immense potential in continuously tracking tumor status—recurrence, progression, and treatment response—in real-time. To comprehensively assess tumor evolution, cellular diversity, and drug resistance mechanisms, a pivotal step involves scrutinizing proteins and nucleic acids within extracellular vesicles (EVs) present in biofluids. Our focus lies in crafting an advanced digital bead-based sensor system that seamlessly evaluates the molecular profile of EVs in glioblastoma cases. Our innovative approach enriches tumor-relevant genetic data within EVs using antibody-specific magnetic beads, subsequently amplifying it via reverse transcription recombinase polymerase amplification system across forty thousand droplets. These results are scrutinized using a compact imaging device. The core strengths of our methodology include: (a) Streamlined Sample Processing: Our system facilitates ultra-sensitive miRNA detection in EVs within a rapid ninety-minute window, owing to optimized sample preparation and a compact detection mechanism; (b) Enhanced Signal Clarity: Employing a straightforward isothermal assay significantly boosts the fluorescent signal-to-noise ratio within each 70-µm droplet, achieving over 104 turnovers of fluorescent reporters on a chip; (c) Real-Time Data Analysis: Our system seamlessly captures real-time imaging data for the edge computing, utilizing trained YOLOv5 models for classification, and presents genetic results on the smartphone. Its user-friendly design enables utilization by healthcare workers with varying skill levels. This pioneering approach has the potential to offer affordable, rapid, and informative diagnostics particularly beneficial for underserved regions and healthcare systems facing financial constraints. Furthermore, its adaptability for diverse cancers could revolutionize the pace of diagnosis and treatment decision-making through liquid biopsy, potentially establishing its widespread application in global healthcare.

MicroRNA Profile of Mouse Adipocyte-Derived Extracellular Vesicles.

The post-transcriptional control of gene expression is a complex and evolving field in adipocyte biology, with the premise that the delivery of microRNA (miRNA) species to the obese adipose tissue may facilitate weight loss. Cells shed extracellular vesicles (EVs) that may deliver miRNAs as intercellular messengers. However, we know little about the miRNA profile of EVs secreted by adipocytes during postnatal development. Here, we defined the miRNA cargo of EVs secreted by mouse adipocytes in two distinct phases of development: on postnatal day 6, when adipocytes are lipolytic and thermogenic, and on postnatal day 56, when adipocytes have active lipogenesis. EVs were collected from cell culture supernatants, and their miRNA profile was defined by small RNA sequencing. The most abundant miRNA of mouse adipocyte-derived EVs was mmu-miR-148a-3p. Adipocyte EVs on postnatal day 6 were hallmarked with mmu-miR-98-5p, and some miRNAs were specific to this developmental stage, such as mmu-miR-466i-5p and 12 novel miRNAs. Adipocytes on postnatal day 56 secreted mmu-miR-365-3p, and 16 miRNAs were specific to this developmental stage. The miRNA cargo of adipocyte EVs targeted gene networks of cell proliferation, insulin signaling, interferon response, thermogenesis, and lipogenesis. We provided here a database of miRNAs secreted by developing mouse adipocytes, which may be a tool for further studies on the regulation of gene networks that control mouse adipocyte development.

Pro-inflammatory activity of Cutibacterium acnes phylotype IA1 and extracellular vesicles: An invitro study.

Acne is a chronic inflammatory skin condition that involves Cutibacterium acnes (C. acnes), which is classified into six main phylotypes (IA1, IA2, IB, IC, II and III). Acne development is associated with loss of C. acnes phylotype diversity, characterised by overgrowth of phylotype IA1 relative to other phylotypes. It was also shown that purified extracellular vesicles (EVs) secreted by C. acnes can induce an acne-like inflammatory response in skin models. We aimed to determine if the inflammatory profile of EVs secreted by C. acnes phylotype IA1 from an inflammatory acne lesion was different from C. acnes phylotype IA1 from normal skin, thus playing a direct role in the severity of inflammation. EVs were produced invitro after culture of two clinical strains of C. acnes phylotype IA1, T5 from normal human skin and A47 from an inflammatory acne lesion, and then incubated with either human immortalised keratinocytes, HaCaT cells, or skin explants obtained from abdominoplasty. Subsequently, quantitative PCR (qPCR) was performed for human β-defensin 2 (hBD2), cathelicidin (LL-37), interleukin (IL)-1β, IL-6, IL-8, IL-17α and IL-36γ, and ELISA for IL-6, IL-8 and IL-17α. We found that EVs produced invitro by C. acnes derived from inflammatory acne lesions significantly increased the pro-inflammatory cytokines and anti-microbial peptides at both transcriptional and protein levels compared with EVs derived from normal human skin. We show for the first time that C. acnes EVs from inflammatory acne play a crucial role in acne-associated inflammation invitro and that C. acnes phylotype IA1 collected from inflammatory acne lesion and normal skin produce different EVs and inflammatory profiles invitro.

Targeted proteomics of plasma extracellular vesicles uncovers MUC1 as combinatorial biomarker for the early detection of high-grade serous ovarian cancer

BackgroundThe five-year prognosis for patients with late-stage high-grade serous carcinoma (HGSC) remains dismal, underscoring the critical need for identifying early-stage biomarkers. This study explores the potential of extracellular vesicles (EVs) circulating in blood, which are believed to harbor proteomic cargo reflective of the HGSC microenvironment, as a source for biomarker discovery.ResultsWe conducted a comprehensive proteomic profiling of EVs isolated from blood plasma, ascites, and cell lines of patients, employing both data-dependent (DDA) and data-independent acquisition (DIA) methods to construct a spectral library tailored for targeted proteomics. Our investigation aimed at uncovering novel biomarkers for the early detection of HGSC by comparing the proteomic signatures of EVs from women with HGSC to those with benign gynecological conditions. The initial cohort, comprising 19 donors, utilized DDA proteomics for spectral library development. The subsequent cohort, involving 30 HGSC patients and 30 control subjects, employed DIA proteomics for a similar purpose. Support vector machine (SVM) classification was applied in both cohorts to identify combinatorial biomarkers with high specificity and sensitivity (ROC-AUC > 0.90). Notably, MUC1 emerged as a significant biomarker in both cohorts when used in combination with additional biomarkers. Validation through an ELISA assay on a subset of benign (n = 18), Stage I (n = 9), and stage II (n = 9) plasma samples corroborated the diagnostic utility of MUC1 in the early-stage detection of HGSC.ConclusionsThis study highlights the value of EV-based proteomic analysis in the discovery of combinatorial biomarkers for early ovarian cancer detection.

Do extracellular vesicles have specific target cells?; Extracellular vesicle mediated embryo maternal communication.

Extracellular vesicles (EVs) serve as messengers for intercellular communication, yet the precise mechanisms by which recipient cells interpret EV messages remain incompletely understood. In this study, we explored how the origin of EVs, their protein cargo, and the recipient cell type influence the cellular response to EVs within an embryo implantation model. We treated two types of EVs to 6 different recipient cell types and expression of zinc finger protein 81 (ZNF81) gene expression in the recipient cells were quantified using quantitative polymerase chain reaction (qPCR). The proteomic contents of the EV cargos were also analyzed. The results showed that downregulation of the ZNF81 gene was a specific cellular response of receptive endometrial epithelial cells to trophoblast derived EVs. Protein cargo analysis revealed that the proteomic profile of EVs depends on their cell of origin and therefore may affect the recipient cell response to EVs. Furthermore, trophoblastic EVs were found to be specifically enriched with transcription factors such as CTNNB1 (catenin beta-1), HDAC2 (histone deacetylase 2), and NOTCH1 (neurogenic locus notch homolog protein 1), which are known regulators of ZNF81 gene expression. The current study provided compelling evidence supporting the existence of EV specificity, where the characteristics of both the EVs and the recipient cell type collectively contribute to regulating EV target specificity. Additionally, EV protein cargo analysis suggested a potential association between transcription factors and the specific functionality of trophoblastic EVs. This in vitro embryo implantation model and ZNF81 read-out provides a unique platform to study EV specific functionality in natural cell-cell communication.

Integrated miRNA Profiling of Extracellular Vesicles from Uterine Aspirates, Malignant Ascites and Primary-Cultured Ascites Cells for Ovarian Cancer Screening.

Extracellular vesicles (EVs) are of growing interest in the context of screening for highly informative cancer markers. We have previously shown that uterine aspirate EVs (UA EVs) are a promising source of ovarian cancer (OC) diagnostic markers. In this study, we first conducted an integrative analysis of EV-miRNA profiles from UA, malignant ascitic fluid (AF), and a conditioned medium of cultured ascites cells (ACs). Using three software packages, we identified 79 differentially expressed miRNAs (DE-miRNAs) in UA EVs from OC patients and healthy individuals. To narrow down this panel and select miRNAs most involved in OC pathogenesis, we aligned these molecules with the DE-miRNA sets obtained by comparing the EV-miRNA profiles from OC-related biofluids with the same control. We found that 76% of the DE-miRNAs from the identified panel are similarly altered (differentially co-expressed) in AF EVs, as are 58% in AC EVs. Interestingly, the set of miRNAs differentially co-expressed in AF and AC EVs strongly overlaps (40 out of 44 miRNAs). Finally, the application of more rigorous criteria for DE assessment, combined with the selection of miRNAs that are differentially co-expressed in all biofluids, resulted in the identification of a panel of 29 miRNAs for ovarian cancer screening.

Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity

BackgroundObesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity’s impact on EV secretion from human AT remains limited.MethodsWe investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography–mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry.ResultsEVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation.ConclusionsWe are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

P-704 Distinctive proteomic profiling and pathway analysis of plasma extracellular vesicles provided clues for their regulatory roles in the pathogenesis of polycystic ovarian syndrome

Abstract Study question To elucidate the distinctive proteomic profiling of plasma extracellular vesicles (EVs) and the pathogenic contributions in polycystic ovarian syndrome (PCOS). Summary answer In PCOS, differentially expressed proteins in the circulatory exosomes are associated with the several characteristic signaling pathways related to cardiovascular and metabolism risks. What is known already EVs from various tissues or body fluids have been shown to be involved in the pathogenesis of PCOS and might serve as potential biomarkers for the diagnosis and prognostic prediction. Most of previous studies focus on the miRNAs expression of EVs isolated from blood, endometrium and follicular fluid. However, a comprehensive quantitative protein analysis of circulatory EVs has seldom been conducted in PCOS. Whether and how the proteomic profiling of circulatory EVs is related to the characteristic phenotypic expression is also unclear. Study design, size, duration This is a case-control study conducted at the reproductive endocrinologic department of the tertiary medical center from 2019 to 2022. Blood samples were collected from sixty PCOS and thirty non-PCOS control subjects. Participants/materials, setting, methods Plasma EVs were isolated by sucrose gradient-based ultracentrifugation. Quantitative proteomic analysis was performed by tandem mass tag labeling and liquid chromatography tandem mass spectrometry (TMT) to identify differentially expressed proteins. The functions and pathways of identified proteins were analyzed with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The correlation between the identified proteins and the clinical, hormonal, and biochemical parameters were also analyzed. Main results and the role of chance Nanoparticle tracking analysis suggested the majority of EVs were 70-190 nm in diameter, which were corresponsive to the size of exosome. Differential expressed protein (DEP) analysis revealed distinctive upregulated proteins in the circulatory exosomes of PCOS patients, includes IGF-1, HIST1H1E, PF4, HBA1, HBB, SNC73, which were enriched in nitric oxide transport and negative regulation of extrinsic apoptotic signaling pathway. Proteins which were downregulated in PCOS patients, including RAA1, RARRES2, CFHR1, LBP, IGFBP3, TPI1, SERPINA5, SNC73, were enriched in lipid transport and complement and coagulation cascades. There is significant correlation between the differentially expressed exosome proteins and several clinical phenotypes, including hypertension, hyperandrogenism and iron metabolism. The enriched pathways and characterized phenotypes have been shown to be closely related to cardiovascular and metabolic aberrations of PCOS, which could provide pathogenic clues for how the circulatory EVs proteins contribute to the disease development and progression. Limitations, reasons for caution PCOS is highly heterogeneous and the case number of our study might not be large enough to differentiate distinctive patterns among different subtypes of PCOS. The enrolled population is limited to reproductive-age Asian patients and might not be generalized to other age or ethnic groups. Wider implications of the findings Our results could be apply to further studies for biomarker identification and mechanistic researches, and to elucidate whether and how the proteomic biology of circulatory EVs is related to the pathogenesis of PCOS. Trial registration number not applicable

P-040 The characteristic of human seminal plasma extracellular vesicle (SPEVs) profiles with different sperm features in a large cohort of 309 Taiwanese men

Abstract Study question To investigate human seminal plasma extracellular vesicle profiles in different sperm characteristics and their potential clinical application. Summary answer Normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic samples. Moreover, SPEV concentration was highly correlated with total motile sperm count. What is known already Seminal plasma extracellular vesicles (SPEVs) are cell-derived membrane structures that carry proteins, nucleic acid, and lipids, which can modify the constitution and biological function of sperm. However, the characteristics of SPEVs in different sperm features remained unclear. Study design, size, duration From January 2021 to September 2023, 309 men who visited the NCKUH andrology clinic agreed to collect semen samples for this study, which the Institutional Review Board has approved. Participants/materials, setting, methods Semen parameters were measured by the X1 CASA system (Bonraybio, Taiwan). SPEVs were isolated by size exclusion chromatography column and ExoQuick™. Next, the SPEVs’ concentration and size distribution were performed with nanoparticle tracking analysis. The Kruskal-Wallis test calculated comparison in multiple groups, and Dunn’s multiple comparison tests accessed group-group comparison. The Pearson correlation coefficient r of exosome concentration with individual sperm parameters was calculated. A p-value less than 0.05 was set as statistical significance. Main results and the role of chance Among them, 59 semen samples were presented with normozoospermia, 36 were oligozoospermia, 73 were asthenozoospermia, and 141 samples had no sperm in semen. Among these 141 azoospermic men, 89 men are characterized by non-obstructive azoospermia (NOA), while the other 52 men had a vasectomy. Our data showed that normozoospermic semen has significantly higher SPEV concentration than asthenozoospermic or oligozoospermic semen samples (26 x109 [IQR 17.25 x109-56.25 x109] versus 12 x109 [IQR 5.6 x109-19.5 x109] versus 15.5 x109 [IQR 8.575 x109-26.75 x109], p = 0.0001). Nevertheless, azoospermic semen has the lowest SPEV concentrations. The concentration of SPEVs from infertile men diagnosed with NOA is slightly lower than vasectomized men (3.9 x109 [IQR 1.875 x109-7.675 x109] versus 5.4 x109 [IQR 2.9 x109-8 x109], p = 0.0001). The Pearson r coefficient of SPEV concentration with sperm concentration, progressive motility, and total motile sperm count was 0.238, 0.303, and 0.451, respectively. (P < 0.0001) Limitations, reasons for caution Because our data were collected from Taiwanese men, it is not yet known whether SPEV profiles differ among races. Wider implications of the findings Human SPEV concentration and its bio-content may be a potential non-invasive biomarker to predict the male reproductive outcome. Trial registration number not applicable

Optimized AF4 combined with density cushion ultracentrifugation enables profiling of high-purity human blood extracellular vesicles.

Extracellular vesicles (EVs) have emerged as a promising tool for clinical liquid biopsy. However, the identification of EVs derived from blood samples is hindered by the presence of abundant plasma proteins, which impairs the downstream biochemical analysis of EV-associated proteins and nucleic acids. Here, we employed optimized asymmetric flow field-flow fractionation (AF4) combined with density cushion ultracentrifugation (UC) to obtain high-purity and intact EVs with very low lipoprotein contamination from human plasma and serum. Further proteomic analysis revealed more than 1000 EV-associated proteins, a large proportion of which has not been previously reported. Specifically, we found that cell-line-derived EV markers are incompatible with the identification of plasma-EVs and proposed that the proteins MYCT1, TSPAN14, MPIG6B and MYADM, as well as the traditional EV markers CD63 and CD147, are plasma-EV markers. Benefiting from the high-purity of EVs, we conducted comprehensive miRNA profiling of plasma EVs and nanosized particles (NPs), as well as compared plasma- and serum-derived EVs, which provides a valuable resource for the EV research community. Overall, our findings provide a comprehensive assessment of human blood EVs as a basis for clinical biopsy applications.

Lipidome profiling of neutrophil-derived extracellular vesicles unveils their contribution to the ensemble of synovial fluid-derived extracellular vesicles during joint inflammation

The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies.

Differential extracellular vesicle concentration and their biomarker expression of integrin αv/β5, EpCAM, and glypican-1 in pancreatic cancer models

Tumor-derived extracellular vesicles (EVs) show great potential as biomarkers for several diseases, including pancreatic cancer, due to their roles in cancer development and progression. However, the challenge of utilizing EVs as biomarkers lies in their inherent heterogeneity in terms of size and concentration, making accurate quantification difficult, which is highly dependent on the isolation and quantification methods used. In our study, we compared three EV isolation techniques and two EV quantification methods. We observed variations in EV concentration, with approximately 1.5-fold differences depending on the quantification method used. Interestingly, all EV isolation techniques consistently yielded similar EV quantities, overall size distribution, and modal sizes. In contrast, we found a notable increase in total EV amounts in samples from pancreatic cancer cell lines, mouse models, and patient plasma, compared to non-cancerous conditions. Moreover, individual tumor-derived EVs exhibited at least a 3-fold increase in several EV biomarkers. Our data, obtained from EVs isolated using various techniques and quantified through different methods, as well as originating from various pancreatic cancer models, suggests that EV profiling holds promise for the identification of unique and cancer-specific biomarkers in pancreatic cancer.

Comparing the Proteomic Profiles of Extracellular Vesicles Isolated using Different Methods from Long-term Stored Plasma Samples

BackgroundThe lack of standardized protocols for isolating extracellular vesicles (EVs), especially from biobank-stored blood plasma, translates to limitations for the study of new biomarkers. This study examines whether a combination of current isolation methods could enhance the specificity and purity of isolated EVs for diagnosis and personalized medicine purposes.ResultsEVs were isolated from healthy human plasma stored for one year by ultracentrifugation (UC), size exclusion chromatography (SEC), or SEC and UC combined (SEC + UC). The EV isolates were then characterized by transmission electron microscopy imaging, nanoparticle tracking analysis (NTA) and western blotting. Proteomic procedures were used to analyze protein contents. The presence of EV markers in all isolates was confirmed by western blotting yet this analysis revealed higher albumin expression in EVs-UC, suggesting plasma protein contamination. Proteomic analysis identified 542 proteins, SEC + UC yielding the most complex proteome at 364 proteins. Through gene ontology enrichment, we observed differences in the cellular components of EVs and plasma in that SEC + UC isolates featured higher proportions of EV proteins than those derived from the other two methods. Analysis of proteins unique to each isolation method served to identify 181 unique proteins for the combined approach, including those normally appearing in low concentrations in plasma. This indicates that with this combined method, it is possible to detect less abundant plasma proteins by proteomics in the resultant isolates.ConclusionsOur findings reveal that the SEC + UC approach yields highly pure and diverse EVs suitable for comprehensive proteomic analysis with applications for the detection of new biomarkers in biobank-stored plasma samples.

MiRNome Profiling of Extracellular Vesicles in Patients With Severe COVID-19 and Identification of Predictors of Mortality.

Extracellular vesicles (EVs), containing microRNAs (miRNAs) and other molecules, play a central role in intercellular communication, especially in viral infections caused by SARS-CoV-2. This study explores the miRNA profiles in plasma-derived EVs from patients with severe COVID-19 vs controls, identifying potential mortality predictors. This prospective study included 36 patients with severe COVID-19 and 33 controls without COVID-19. EV-derived miRNAs were sequenced, and bioinformatics and differential expression analysis between groups were performed. The plasma miRNA profile of an additional cohort of patients with severe COVID-19 (n = 32) and controls (n = 12) was used to compare with our data. Survival analysis identified potential mortality predictors among the significantly differentially expressed (SDE) miRNAs in EVs. Patients with severe COVID-19 showed 50 SDE miRNAs in plasma-derived EVs. These miRNAs were associated with pathways related to inflammation and cell adhesion. Fifteen of these plasma-derived EV miRNAs were SDE in the plasma of severe cases vs controls. Two miRNAs, hsa-miR-1469 and hsa-miR-6124, were identified as strong mortality predictors with an area under the receiver operating characteristic curve of 0.938. This research provides insights into the role of miRNAs within EVs in severe COVID-19 and their potential as clinical biomarkers for mortality.

Proteomic profiling of extracellular vesicles derived from human serum for the discovery of biomarkers in Avascular necrosis

BackgroundAvascular necrosis (AVN) is a medical condition characterized by the destruction of bone tissue due to a diminished blood supply. When the rate of tissue destruction surpasses the rate of regeneration, effective treatment becomes challenging, leading to escalating pain, arthritis, and bone fragility as the disease advances. A timely diagnosis is imperative to prevent and initiate proactive treatment for osteonecrosis. We explored the potential of differentially expressed proteins in serum-derived extracellular vesicles (EVs) as biomarkers for AVN of the femoral head in humans. We analyzed the genetic material contained in serum-derived exosomes from patients for early diagnosis, treatment, and prognosis of avascular necrosis.MethodsEVs were isolated from the serum of both patients with AVN and a control group of healthy individuals. Proteomic analyses were conducted to compare the expression patterns of these proteins by proteomic analysis using LC-MS/MS.ResultsOur results show that the levels of IGHV3-23, FN1, VWF, FGB, PRG4, FCGBP, and ZSWIM9 were upregulated in the EVs of patients with AVN compared with those of healthy controls. ELISA results showed that VWF and PRG4 were significantly upregulated in the patients with AVN.ConclusionsThese findings suggest that these EV proteins could serve as promising biomarkers for the early detection and diagnosis of AVN. Early diagnosis is paramount for effective treatment, and the identification of new osteonecrosis biomarkers is essential to facilitate swift diagnosis and proactive intervention. Our study provides novel insights into the identification of AVN-related biomarkers that can enhance clinical management and treatment outcomes.

Proteomic Analysis of Butyrate-Resistant Colorectal Cancer-Derived Exosomes Reveals Potential Resistance to Anti-Cancer Drugs.

Butyrate-resistant (BR) cells play an important role in acquiring chemoresistance in colorectal cancer (CRC). Our previous study demonstrated that BR CRC cells showed cross-resistance to chemotherapy drugs, including 5-fluorouracil and oxaliplatin, in both monolayer and spheroid cultures. The mechanisms underlying drug resistance were also elucidated. However, the link between parental (PT) and BR cells remains unclear. Extracellular vesicles (EVs) are key cell-cell communications that transport various molecules, including DNA, RNA, and proteins, between the donor and target cells. EVs contribute to drug resistance in cancers, such as melanoma and lung cancer. Recently, we focused on the correlation of proteomic profiles of EVs from different cell types. In this study, we analyzed the proteomic profiles of EVs derived from PT and BR cells to investigate the mechanisms underlying the butyrate- and chemo-resistant phenotypes. EVs were isolated from PT and BR cells using ultracentrifugation. The characteristics of the EVs were evaluated using western blot and transmission electron microscopy. The EV proteomic data were further analyzed using liquid chromatography-mass spectrometry. We identified a unique protein expressed in BR cells related to the chemoresistant phenotype. Functional enrichment analysis showed that BR cells had higher protein catalytic activity, binding, and transcription activity. The STITCH database showed a greater correlation between protein-drug interactions in BR cells than in PT cells. Moreover, our findings support the hypothesis that EVs promote tumor progression and metastasis and affect the tumor microenvironment. Proteomic analysis of EVs from BR CRC cells reveals insights into drug resistance mechanisms, including protein-mediated carcinogenesis and reduced drug uptake, offering potential strategies to overcome resistance in clinical practice.

Extracellular vesicles originating from steatotic hepatocytes promote hepatic stellate cell senescence via AKT/mTOR signaling.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing rapidly due to the obesity epidemic. In the inflammatory stages of MASLD (MASH), activation of hepatic stellate cells (HSCs) leads to initiation and progression of liver fibrosis. Extracellular vesicles (EVs) are released from all cell types and play an important role in intercellular communication. However, the role of EVs released from hepatocytes in the context of MASLD is largely unknown. Therefore, the present study aimed to investigate the role of EVs derived from both normal and steatotic (free fatty acid-treated) hepatocytes on the phenotype of HSCs via the senescence pathway. Primary rat hepatocytes were treated with free fatty acids (FFAs: oleic acid and palmitic acid). EVs were collected by ultracentrifugation. EVs markers and HSCs activation and senescence markers were assessed by Western blot analysis, qPCR and cytochemistry. Reactive oxygen species (ROS) production was assessed by fluorescence assay. RNA profiles of EVs were evaluated by sequencing. We found that EVs from hepatocytes treated with FFAs (FFA-EVs) inhibit collagen type 1 and α-smooth muscle actin expression, increase the production of ROS and the expression of senescence markers (IL-6, IL-1β, p21 and senescence-associated β-galactosidase activity) in early activating HSCs via the AKT-mTOR pathway. Sequencing showed differentially enriched RNA species between the EVs groups. In conclusion, EVs from FFA-treated hepatocytes inhibit HSC activation by inducing senescence via the AKT-mTOR signaling pathway. Determining the components in EVs from steatotic hepatocytes that induce HSC senescence may lead to the identification of novel targets for intervention in the treatment of MASLD in the future.

Buoyant Metal-Organic Framework Corona-Driven Fast Isolation and Ultrasensitive Profiling of Circulating Extracellular Vesicles.

Accurately assaying tumor-derived circulating extracellular vesicles (EVs) is fundamental in noninvasive cancer diagnosis and therapeutic monitoring but limited by challenges in efficient EV isolation and profiling. Here, we report a bioinspired buoyancy-driven metal-organic framework (MOF) corona that leverages on-bubble coordination and dual-encoded surface-enhanced Raman scattering (SERS) nanotags to streamline rapid isolation and ultrasensitive profiling of plasma EVs in a single assay for cancer diagnostics. This integrated bubble-MOF-SERS EV assay (IBMsv) allows barnacle-like high-density adhesion of MOFs on a self-floating bubble surface to enable fast isolation (2 min, near 90% capture efficiency) of tumor EVs via enhanced EV-MOF binding. Also, IBMsv harnesses four-plexed SERS nanotags to profile the captured EV surface protein markers at a single-particle level. Such a sensitive assay allows multiplexed profiling of EVs across five cancer types, revealing heterogeneous EV surface expression patterns. Furthermore, the IBMsv assay enables cancer diagnosis in a pilot clinical cohort (n = 55) with accuracies >95%, improves discrimination between cancer and noncancer patients via an algorithm, and monitors the surgical treatment response from hepatocellular carcinoma patients. This assay provides a fast, sensitive, streamlined, multiplexed, and portable blood test tool to enable cancer diagnosis and response monitoring in clinical settings.

Effect of 3D and 2D cell culture systems on trophoblast extracellular vesicle physico-chemical characteristics and potency.

The growing understanding of the role of extracellular vesicles (EVs) in embryo-maternal communication has sparked considerable interest in their therapeutic potential within assisted reproductive technology, particularly in enhancing implantation success. However, the major obstacle remains the large-scale production of EVs, and there is still a gap in understanding how different culture systems affect the characteristics of the EVs. In the current study, trophoblast analogue human chorionic carcinoma cell line was cultivated in both conventional monolayer culture (2D) and as spheroids in suspension culture (3D) and how the cell growth environment affects the physical, biochemical and cellular signalling properties of EVs produced by them was studied. Interestingly, the 3D system was more active in secreting EVs compared to the 2D system, while no significant differences were observed in terms of morphology, size, and classical EV protein marker expression between EVs derived from the two culture systems. There were substantial differences in the proteomic cargo profile and cellular signalling potency of EVs derived from the two culture systems. Notably, 2D EVs were more potent in inducing a cellular response in endometrial epithelial cells (EECs) compared to 3D EVs. Therefore, it is essential to recognize that the biological activity of EVs depends not only on the cell of origin but also on the cellular microenvironment of the parent cell. In conclusion, caution is warranted when selecting an EV production platform, especially for assessing the functional and therapeutic potential of EVs through in vitro studies.

Comparative Proteome Profiling of Extracellular Vesicles from Three Growth Phases of Haematococcus pluvialis under High Light and Sodium Acetate Stresses.

Extracellular vesicles (EVs) are nano-sized particles involved in intercellular communications that intrinsically possess many attributes as a modern drug delivery platform. Haematococcus pluvialis-derived EVs (HpEVs) can be potentially exploited as a high-value-added bioproduct during astaxanthin production. The encapsulation of HpEV cargo is a crucial key for the determination of their biological functions and therapeutic potentials. However, little is known about the composition of HpEVs, limiting insights into their biological properties and application characteristics. This study examined the protein composition of HpEVs from three growth phases of H. pluvialis grown under high light (350 µmol·m-2·s-1) and sodium acetate (45 mM) stresses. A total of 2038 proteins were identified, the majority of which were associated with biological processes including signal transduction, cell proliferation, cell metabolism, and the cell response to stress. Comparative analysis indicated that H. pluvialis cells sort variant proteins into HpEVs at different physiological states. It was revealed that HpEVs from the early growth stage of H. pluvialis contain more proteins associated with cellular functions involved in primary metabolite, cell division, and cellular energy metabolism, while HpEVs from the late growth stage of H. pluvialis were enriched in proteins involved in cell wall synthesis and secondary metabolism. This is the first study to report and compare the protein composition of HpEVs from different growth stages of H. pluvialis, providing important information on the development and production of functional microalgal-derived EVs.