Small extracellular vesicles (sEVs) can provide information about the pathophysiology of the cells; therefore, sEVs have attracted considerable interest as possible diagnostic biomarkers. A key challenge lies in the necessity for simple and cost-effective sEV isolation methods to achieve high purity and yield suitable for research and clinical applications. We are introducing a comprehensive study on isolating sEVs using a novel cocktail strategy that integrates chemical precipitation and ultrafiltration with a two-step filtering process to ensure a highly pure and homogeneous population and further compared with PEG-based precipitation, ultra-centrifugation, and size-exclusion-chromatography columns. The isolated sEVs from each protocol are quantified for size and yield using nanoparticle tracking analysis, morphologically characterized through transmission electron microscopy, and validated by quantifying the expression profiles of sEV surface biomarkers. Furthermore, the study explores the applicability of our method for downstream multi-omics analyses. The results highlight the efficacy of the proposed protocol, demonstrating the ease and efficiency of isolating sEVs from different biofluids with minimal laboratory requirements and confirming the compatibility with multi-omics analyses. These findings position our method as particularly valuable for translational research, offering a promising avenue for advancing the study and application of sEVs in diagnostic and therapeutic research.

Cell-free RNAs (cfRNAs) can be detected in biofluids and have emerged as valuable disease biomarkers. Accurate identification of the fragmented cfRNA signals, especially those originating from pathological cells, is crucial for understanding their biological functions and clinical value. However, many challenges still need to be addressed for their application, including developing specific analysis methods and translating cfRNA fragments with biological support into clinical applications.

We present cfPeak, a novel method combining statistics and machine learning models to detect the fragmented cfRNA signals effectively. When test in real and artificial cfRNA sequencing (cfRNA-seq) data, cfPeak shows an improved performance compared with other applicable methods. We reveal that narrow cfRNA peaks preferentially overlap with protein binding sites, vesicle-sorting sites, structural sites, and novel small non-coding RNAs (sncRNAs). When applied in clinical cohorts, cfPeak identified cfRNA peaks in patients' plasma that enable cancer detection and are informative of cancer types and metastasis.

Our study fills the gap in the current small cfRNA-seq analysis at fragment-scale and builds a bridge to the scientific discovery in cfRNA fragmentomics. We demonstrate the significance of finding low abundant tissue-derived signals in small cfRNA and prove the feasibility for application in liquid biopsy.

Cancer is a leading cause of death worldwide, and despite the introduction of new therapeutic approaches for advanced cases aimed at improving patient survival, only a subset of patients benefits from a complete response. In this context, there is a growing need for new cancer biomarkers and therapeutic strategies, and the use of Extracellular Vesicles (EVs) has been widely explored in various approaches. As circulating lipid-bilayer particles carrying a variety of biological information from tumor cells, EVs can be employed as good biomarkers of diagnosis, prognosis, therapy evaluation, and as adjuvants in cancer treatment. In this review, we provide a brief overview of the different types of EVs and their biogenesis and discuss how tumor-derived EV cargo can serve as a potential biomarker in clinical settings through liquid biopsy. We also highlight recent advances in EV nanoengineering and their potential as adjuvants in cancer treatment. Finally, we discuss the key unknowns, gaps, and bottlenecks that must be addressed to fully integrate EVs into precision oncology.

Depression, particularly major depressive disorder (MDD), is a debilitating neuropsychiatric condition characterized by high disability rates, primarily driven by chronic stress and genetic predispositions. Emerging evidence highlights the critical role of microRNAs (miRNAs) in the pathogenesis of depression, with plasma-derived small extracellular vesicles (sEVs) emerging as promising biomarkers. In this study, we collected peripheral blood plasma samples from patients diagnosed with MDD, as assessed by the Hamilton Depression Rating scale, alongside healthy individuals serving as controls. Plasma-derived sEVs were isolated via ultracentrifugation, followed by high-throughput sequencing of miRNAs encapsulated within sEVs, and finally image acquisition and differential expression analysis. Our results revealed a significant elevation of miR-182-5p in plasma-derived sEVs from MDD patients compared to healthy controls, a finding further validated in chronic mild stress (CMS) models. Further analysis suggested that miRNAs encapsulated within sEVs may influence depression onset and progression by modulating hypothalamic-pituitary-adrenal (HPA) axis activity. These findings underscore the potential of miRNAs and their target genes as novel biomarkers, offering improved diagnostic accuracy and therapeutic efficacy for MDD.

Gastrointestinal (GI) cancers, which predominantly manifest in the stomach, colorectum, liver, esophagus, and pancreas, accounting for approximately 35% of global cancer-related mortality. The advent of liquid biopsy has introduced a pivotal diagnostic modality for the early identification of premalignant GI lesions and incipient cancers. This non-invasive technique not only facilitates prompt therapeutic intervention, but also serves as a critical adjunct in prognosticating the likelihood of tumor recurrence. The wealth of circulating exosomes present in body fluids is often enriched with proteins, lipids, microRNAs, and other RNAs derived from tumor cells. These specific cargo components are reflective of processes involved in GI tumorigenesis, tumor progression, and response to treatment. As such, they represent a group of promising biomarkers for aiding in the diagnosis of GI cancer. In this review, we delivered an exhaustive overview of the composition of exosomes and the pathways for cargo sorting within these vesicles. We laid out some of the clinical evidence that supported the utilization of exosomes as diagnostic biomarkers for GI cancers and discussed their potential for clinical application. Furthermore, we addressed the challenges encountered when harnessing exosomes as diagnostic and predictive instruments in the realm of GI cancers.

Accurate diagnosis of early gastric cancer is valuable for asymptomatic populations, while current endoscopic examination combined with pathological tissue biopsy often encounters bottlenecks for early-stage cancer and causes pain to patients. Liquid biopsy shows promise for noninvasive diagnosis of early gastric cancer; however, it remains a challenge to achieve accurate diagnosis due to the lack of highly sensitive and specific biomarkers. Herein, we propose a protocol combining metabolomics profiling from plasma extracellular vesicles (EVs) and machine learning to identify the metabolomics discrepancies of early gastric cancer individuals from other populations. Efficient metabolomics profiling is achieved by efficient, high-purity, and damage-free plasma EVs separation using elaborately designed nanotrap-structured microparticles (NanoFisher) by taking advantage of stereoscopic interaction and affinity interaction. Significant metabolomics discrepancies are obtained from 150 early gastric cancer (50), benign gastric disease (50), and non-disease control (50) plasma samples. Machine learning enables ideal distinction between early gastric cancer and non-disease control samples with an area under the curve (AUC) of 1.000, achieves an AUC of 0.875-0.975 for differentiating early gastric cancer from benign gastric diseases, and demonstrates an overall accuracy of 92% in directly classifying these three categories. The plasma EV metabolomics profiling enabled by NanoFisher materials, integrated with machine learning, holds considerable promise for broad clinical acceptance, enhancing gastric cancer screening outcomes.

Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer and one of the leading causes of cancer death worldwide. Despite diagnostic and therapeutic advances, CRC mortality remains high, especially in industrialized countries. Numerous studies have highlighted the pathogenetic role of altered microRNA (miRNA) expression among the various factors contributing to the development and progression of colorectal cancer (CRC). However, the data regarding specific miRNAs involved in CRC pathogenesis remain inconsistent, and no miRNAs have been recognized so far as reliable or effective biomarkers for the diagnosis of this tumor type. To identify novel miRNA biomarkers in CRC, this study validated the expression levels of a four-miRNA signature predicted to be involved in CRC by analyzing both tissue and liquid biopsy samples. Our experimental and bioinformatics results highlighted the diagnostic potential of hsa-miR-21-5p, hsa-miR-503-5p, and hsa-miR-375, as well as the potential prognostic value of hsa-miR-497-5p overexpression and hsa-miR-375-3p downregulation. Overall, the results obtained suggest the diagnostic and prognostic significance of this four-miRNA signature in CRC.

Extracellular vesicles (EVs) and their cargoes are increasingly being recognized as noninvasive diagnostic markers, necessitating the isolation of EVs from complex biological samples. Herein, a distearoyl phospholipid ethanolamine-functionalized single-crystal ordered macroporous three-dimensional zeolitic imidazolate framework (SOM-ZIF-8-DSPE) was developed, which combines the surface charge interaction of ZIF-8 with the synergistic effect of DSPE insertion into the phospholipid membrane of EVs to improve the isolating selectivity of EV capture. The materials have porous structures larger than 300 nm in diameter, providing enough space and active sites to trap EVs. Benefiting from this feature, the entire isolation process takes only 10 min and is well compatible with the subsequent analysis of RNA in EVs. Consequently, 10 upregulated miRNA of plasma EVs in the primary colorectal cancer (pCRC) patients is found over the healthy donors, and 6 upregulated miRNA of plasma EVs in the metastatic colorectal cancer (mCRC) patients over pCRC patients. These findings suggest that the isolation of EV-based SOM-ZIF-8-DSPE is a promising strategy to identify biomarkers for disease diagnosis, such as miRNAs in plasma EVs for the early detection of CRC.

The challenges associated with liquid biopsy of colorectal cancer (CRC) are closely linked to the substantial variations observed in gene expression profiles among patients. This variability complicates the selection of an ideal biomarker for accurate diagnosis. In this report, we propose that employing a combination of miRNAs offers a better change for enhancing the accuracy of CRC diagnosis compared to solely relying on single miRNAs. As an illustrative example, we measured 9 miRNAs from 45 patient samples (comprising 31 CRC cases and 14 healthy controls) via RT-qPCR. We then utilized two methods: (1) LASSO regression for marker ranking and (2) linear discriminant analysis (LDA) to identify the optimal weighted combination of multiple markers. Our data indicates that combination of triple markers, selected based on their ranking, exhibited the highest diagnostic performance, including a sensitivity of 93.6% (95% confidence interval, CI 79.3-98.9%), specificity of 100% (CI 78.5-100.0%), positive predictive value (PPV) of 100%, negative predictive value (NPV) of 87.5%, and an overall accuracy of 95.6%. In contrast, the diagnostic performance of each individual miRNA used in the triple marker combination ranged from 53.3 to 80.0% in accuracy. While we acknowledge the need for further extensive studies involving larger patient cohorts and the consideration of additional miRNA candidates, our research undeniably highlights the potential of combining multiple markers as a robust methodology for identifying biomarkers among heterogeneous patient profiles.

A diverse range of gastrointestinal tract disorders are called gastrointestinal (GI) malignancies. The transformation of normal cells into precursor cells, precursor cells into premalignant cells, and premalignant cells into cancerous cells is facilitated by the interaction of many modifiable and non-modifiable risk factors. Developing relevant therapy alternatives based on a better knowledge of the illness's aetiology is essential to enhance patient outcomes. The exosome is crucial in regulating intercellular interaction because it may send molecular signals to nearby or distant cells. Exosomes produced from cancer can introduce a variety of chemicals and vast concentrations of microRNA (miRNA) into the tumour microenvironment. These miRNAs significantly impact immunological evasion, metastasis, apoptosis resistance, and cell growth. Exosomal miRNAs, or exosomal miRNAs, are essential for controlling cancer resistance to apoptosis, according to mounting data. Exosomal miRNAs function as an interaction hub between cancerous cells and the milieu around them, regulating gene expression and various signalling pathways. Our research examines the regulatory function of exosomal miRNAs in mediating interactions between cancer cells and the stromal and immunological cells that make up the surrounding milieu.

Tumor-derived small extracellular vesicle (sEV) microRNAs (miRNAs) are emerging biomarkers for cancer diagnostics. Conventional sEV miRNA detection methods necessitate the lysis of sEVs, rendering them laborious and time-consuming and potentially leading to damage or loss of miRNAs. Membrane fusion-based in situ detection of sEV miRNAs involves the preparation of probe-loaded vesicles (e.g., liposomes or cellular vesicles), which are typically sophisticated and require specialist equipment. Membrane perforation methods employ chemical treatments that can induce severe miRNA degradation or leaks. Inspired by previous studies that loaded nucleic acids into EVs or cells using hydrophobic tethers for therapeutic applications, herein, we repurposed this strategy by conjugating a hydrophobic tether onto molecular beacons to aid their transportation into sEVs, allowing for in situ detection of miRNAs in a fusion-free and multiplexing manner. This method enables simultaneous detection of multiple miRNA species within serum-derived sEVs for the diagnosis of prostate cancer, breast cancer, and gastric cancer with an accuracy of 83.3%, 81.8%, and 100%, respectively, in a cohort of 66 individuals, indicating that it holds a high application potential in clinical diagnostics.

The potential of serum extracellular vesicles (EVs) as non-invasive biomarkers for diagnosing colorectal cancer (CRC) remains elusive. We employed an in-depth 4D-DIA proteomics and machine learning (ML) pipeline to identify key proteins, PF4 and AACT, for CRC diagnosis in serum EV samples from a discovery cohort of 37 cases. PF4 and AACT outperform traditional biomarkers, CEA and CA19-9, detected by ELISA in 912 individuals. Furthermore, we developed an EV-related random forest (RF) model with the highest diagnostic efficiency, achieving AUC values of 0.960 and 0.963 in the train and test sets, respectively. Notably, this model demonstrated reliable diagnostic performance for early-stage CRC and distinguishing CRC from benign colorectal diseases. Additionally, multi-omics approaches were employed to predict the functions and potential sources of serum EV-derived proteins. Collectively, our study identified the crucial proteomic signatures in serum EVs and established a promising EV-related RF model for CRC diagnosis in the clinic.

It takes more than 20 years for normal colorectal mucosa to develop into metastatic carcinoma. The long time window provides a golden opportunity for early detection to terminate the malignant progression. Here, we aim to enable liquid biopsy of T1a stage colorectal cancer (CRC) and precancerous advanced adenoma (AA) by profiling circulating small extracellular vesicle (sEV)-derived RNAs. We exhibited a full RNA landscape for the circulating sEVs isolated from 60 participants. A total of 58,333 annotated RNAs were detected from plasma sEVs, among which 1,615 and 888 sEV-RNAs were found differentially expressed in plasma from T1a stage CRC and AA compared to normal controls (NC). Then we further categorized these sEV-RNAs into six modules by a weighted gene coexpression network analysis and constructed a 60-gene t-SNE model consisting of the top 10 RNAs of each module that could well distinguish T1a stage CRC/AA from NC samples. Some sEV-RNAs were also identified as indicators of specific endoscopic and morphological features of different colorectal lesions. The top-ranked biomarkers were further verified by RT-qPCR, proving that these candidate sEV-RNAs successfully identified T1a stage CRC/AA from NC in another cohort of 124 participants. Finally, we adopted different algorithms to improve the performance of RT-qPCR-based models and successfully constructed an optimized classifier with 79.3% specificity and 99.0% sensitivity. In conclusion, circulating sEVs of T1a stage CRC and AA patients have distinct RNA profiles, which successfully enable the detection of both T1a stage CRC and AA via liquid biopsy.

Exosomes play a crucial role in various biological processes, such as human development, immune responses, and disease occurrence. The membrane proteins on exosomes are pivotal factors for their biological functionality. Currently, numerous membrane proteins have been identified on exosome membranes, participating in intercellular communication, mediating target cell recognition, and regulating immune processes. Furthermore, membrane proteins from exosomes derived from cancer cells can serve as relevant biomarkers for early cancer diagnosis. This article provides a comprehensive review of the composition of exosome membrane proteins and their diverse functions in the organism's biological processes. Through in-depth exploration of exosome membrane proteins, it is expected to offer essential foundations for the future development of novel biomedical diagnostics and therapies.

Extracellular vesicles (EVs) have emerged as promising biomaterials for the treatment of different disease. However, only handful types of EVs with clinical transformation potential have been reported to date, and their preparation on a large scale under biosafety-controlled conditions is limited. In this study, we characterize a novel type of EV with promising clinical application potential: dehydration-induced extracellular vesicles (DIMVs). DIMV is a type of micron-diameter cell vesicle that contains more bioactive molecules, such as proteins and RNA, but not DNA, than previously reported cell vesicles. The preparation of DIMV is extraordinarily straightforward, which possesses a high level of biosafety, and the protein utilization ratio is roughly 600 times greater than that of naturally secreted EVs. Additional experiments demonstrate the viability of pre- or post-isolation DIMV modification, including gene editing, nucleic acid encapsulation or surface anchoring, size adjustment. Finally, on animal models, we directly show the biosafety and immunogenicity of DIMV, and investigate its potential application as tumour vaccine or drug carrier in cancer treatment.

Inflammation is one of the key injury factors for spinal cord injury (SCI). Exosomes (Exos) derived from M2 macrophages have been shown to inhibit inflammation and be beneficial in SCI animal models. However, lacking targetability restricts their application prospects. Considering that chemokine receptors increase dramatically after SCI, viral macrophage inflammatory protein II (vMIP-II) is a broad-spectrum chemokine receptor binding peptide, and lysosomal associated membrane protein 2b (Lamp2b) is the key membrane component of Exos, we speculated that vMIP-II-Lamp2b gene-modified M2 macrophage-derived Exos (vMIP-II-Lamp2b-M2-Exo) not only have anti-inflammatory properties, but also can target the injured area by vMIP-II. In this study, using a murine contusive SCI model, we revealed that vMIP-II-Lamp2b-M2-Exo could target the chemokine receptors which highly expressed in the injured spinal cords, inhibit some key chemokine receptor signaling pathways (such as MAPK and Akt), further inhibit proinflammatory factors (such as IL-1β, IL-6, IL-17, IL-18, TNF-α, and iNOS), and promote anti-inflammatory factors (such as IL-4 and Arg1) productions, and the transformation of microglia/macrophages from M1 into M2. Moreover, the improved histological and functional recoveries were also found. Collectively, our results suggest that vMIP-II-Lamp2b-M2-Exo may provide neuroprotection by targeting the injured spinal cord, inhibiting some chemokine signals, reducing proinflammatory factor production and modulating microglia/macrophage polarization.

Gastric cancer (GC) poses a significant global health challenge because of its high mortality rate attributed to the late-stage diagnosis and lack of early symptoms. Early cancer diagnostics is crucial for improving the survival rates in GC patients, which emphasizes the importance of identifying GC markers for liquid biopsy. The review discusses a potential use of extracellular vesicle microRNAs (EV miRNAs) as biomarkers for the diagnostics and prognostics of GC. Methods. Original articles on the identification of EV miRNA as GC markers published in the Web of Science and Scopus indexed issues were selected from the PubMed and Google Scholar databases. We focused on the methodological aspects of EV analysis, including the choice of body fluid, methods for EV isolation and validation, and approaches for EV miRNA analysis. Conclusions. Out of 33 found articles, the majority of authors investigated blood-derived extracellular vesicles (EVs); only a few utilized EVs from other body fluids, including tissue-specific local biofluids (washing the tumor growth areas), which may be a promising source of EVs in the context of cancer diagnostics. GC-associated miRNAs identified in different studies using different methods of EV isolation and analysis varied considerably. However, three miRNAs (miR-10b, miR-21, and miR-92a) have been found in several independent studies and shown to be associated with GC in experimental models. Further studies are needed to determine the optimal miRNA marker panel. Another essential step necessary to improve the reliability and reproducibility of EV-based diagnostics is standardization of methodologies for EV handling and analysis of EV miRNA.

Cell-free long RNAs in human plasma and extracellular vesicles (EVs) have shown promise as biomarkers in liquid biopsy, despite their fragmented nature.

To investigate these fragmented cell-free RNAs (cfRNAs), we developed a cost-effective cfRNA sequencing method called DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing). DETECTOR-seq utilised a meticulously tailored set of customised guide RNAs to remove large amounts of unwanted RNAs (i.e., fragmented ribosomal and mitochondrial RNAs) in human plasma. Early barcoding strategy was implemented to reduce costs and minimise plasma requirements.

Using DETECTOR-seq, we conducted a comprehensive analysis of cell-free transcriptomes in both whole human plasma and EVs. Our analysis revealed discernible distributions of RNA types in plasma and EVs. Plasma exhibited pronounced enrichment in structured circular RNAs, tRNAs, Y RNAs and viral RNAs, while EVs showed enrichment in messenger RNAs (mRNAs) and signal recognition particle RNAs (srpRNAs). Functional pathway analysis highlighted RNA splicing-related ribonucleoproteins (RNPs) and antimicrobial humoral response genes in plasma, while EVs demonstrated enrichment in transcriptional activity, cell migration and antigen receptor-mediated immune signals. Our study indicates the comparable potential of cfRNAs from whole plasma and EVs in distinguishing cancer patients (i.e., colorectal and lung cancer) from healthy donors. And microbial cfRNAs in plasma showed potential in classifying specific cancer types.

Our comprehensive analysis of total and EV cfRNAs in paired plasma samples provides valuable insights for determining the need for EV purification in cfRNA-based studies. We envision the cost effectiveness and efficiency of DETECTOR-seq will empower transcriptome-wide investigations in the fields of cfRNAs and liquid biopsy.

DETECTOR-seq (depletion-assisted multiplexed cell-free total RNA sequencing) enabled efficient and specific depletion of sequences derived from fragmented ribosomal and mitochondrial RNAs in plasma. Distinct human and microbial cell-free RNA (cfRNA) signatures in whole Plasma versus extracellular vesicles (EVs) were revealed. Both Plasma and EV cfRNAs were capable of distinguishing cancer patients from normal individuals, while microbial RNAs in Plasma cfRNAs enabled better classification of cancer types than EV cfRNAs.

We provided an overview which evaluated the diagnostic performance of circulation EV biomarkers for CRC from PubMed, Medline, and Web of Science until 21 August 2022.Weidentified 48 studies that involved 7727 participants and evaluated 162 plasma/serum individual EV biomarkers including 117 RNAs and 45 proteins, as well as 45 EV biomarker panels for CRC detection. 12 studies evaluated the diagnostic performance of EV biomarkers for early CRC. The summarized sensitivity, specificity, and AUC value of individual EV RNAs and EV RNA panels were 76%, 75%, 0.87 and 82%, 79% and 0.90, respectively. Meanwhile, those of individual EV proteins and EV protein panels were 85%, 84%, 0.92 and 87%, 83%, 0.92, respectively. These results indicated that EV biomarker panels revealed superior diagnostic performance than the corresponding individual biomarkers. In early CRC, EV biomarkers showed available diagnostic value with the sensitivity, specificity, and AUC value of 80%, 75%, and 0.89.In subgroup analyses, EV miRNAs and LncRNAs held similar diagnostic value with the sensitivity, specificity and AUC value of 75%, 78%, 0.90 and 79%, 72%, 0.83, which was highly consistent with the whole EV RNAs. Significantly, the diagnostic values of EV miRNAs in plasma were marginally higher than those based on serum. In detail, the sensitivity, specificity, and AUC values were 79%, 81%, and 0.92 in plasma, as well as 74%, 77%, and 0.88 in serum, respectively. Therefore, circulation EV biomarkers could be considered as a promising biomarker for the early detection of CRC.

Distant metastases are the primary cause of therapy failure and mortality in patients with papillary thyroid carcinomas (PTCs). However, the underlying mechanism responsible for the initiation of tumor cell dissemination and metastasis in PTCs has rarely been investigated.

The aim of this study was to investigate effects and underlying molecular mechanisms of circulating exosomal microRNAs (miRNAs) in distant metastatic PTCs.

The most relevant circulating exosomal miRNA to distant metastatic PTCs were verified between distant metastatic PTCs and nondistant metastatic PTCs by miRNA microarray, quantitative real-time polymerase chain reaction (qRT-PCR) assays and receiver operating characteristic (ROC) curves. The parental and recipient cells of that circulating exosomal miRNA were then explored. In vitro and in vivo experiments were further performed to elucidate the function and potential mechanisms of circulating exosomal miRNAs that contribute to the development of distant metastases.

We determined that PTC-derived exosomal miR-519e-5p was significantly upregulated in the circulatory system in distant metastatic PTCs. Further tests demonstrated that PTC cells can acquire a more malignant phenotype via hnRNPA2B1-mediated sorting of tumor suppressor miR-519e-5p into exosomes to activate Wnt signaling pathway via upregulating PLAGL2. Furthermore, miR-519e-5p included in PTC-derived exosomes can be transferred to recipient CD8+ T cells and aid in tumor immune escape in distant organs through inhibiting Notch signaling pathway by downregulating NOTCH2.

Our findings highlight the dual role of PTC-derived exosomal miR-519e-5p in distant metastasis, which may improve our understanding of exosome-mediated distant metastatic mechanisms.

Extracellular vesicles (EVs) were promising circulating biomarkers for multiple diseases, but whether serum EVs-derived proteins could be used as a reliable tumor biomarker for colorectal cancer (CRC) remained inconclusive. In this study, we identified CXCL4 by a 4D data-independent acquisition-based quantitative proteomics assay of serum EVs-derived proteins in 40 individuals and subsequently analyzed serum EVs-derived CXCL4 levels by ELISA in 2 cohorts of 749 individuals. The results revealed that EVs-derived CXCL4 levels were dramatically elevated in CRC patients than in benign colorectal polyp patients or healthy controls (HC). Furthermore, receiver operating characteristic curves revealed that EVs-derived CXCL4 exhibited superior diagnostic performance with area under the curve of 0.948 in the training cohort. Additionally, CXCL4 could effectively distinguish CRC in stage I/II from HC. Notably, CRC patients with high levels of EVs-derived CXCL4 have shorter 2-year progression-free survival than those with low levels. Overall, our findings demonstrated that serum EVs-derived CXCL4 was a candidate diagnostic and prognostic biomarker for CRC.

Analysis of miRNA (18-23nt) encapsulated in small extracellular vesicles (sEVs) (diameter ~30-200 nm) is critical in understanding the diagnostic and therapeutic value of sEV miRNA. However, various sEV enrichment techniques yield different quantities and qualities of sEV miRNA. Here, we compare the efficacy of three sEV isolation techniques in four combinations for miRNA next-generation sequencing.

Blood plasma from four Holstein-Friesian dairy cows (Bos taurus) (n = 4) with similar genetic traits and physical characteristics were pooled to isolate sEV. Ultracentrifugation (UC) (100,000 × g, 2 h at 4 °C), size-exclusion chromatography (SEC) and ultrafiltration (UF) were used to design four groups of sEV isolations (UC+SEC, SEC+UC, SEC+UF and UC+SEC+UF). sEV miRNAs were isolated using a combination of TRIzol, Chloroform and miRNeasy mini kit (n = 4/each), later sequenced utilizing Novaseq S1 platform (single-end 100 bp sequencing).

All four sEV methods yielded > 1,700 miRNAs and sEV miRNAs demonstrated a clear separation from control blood plasma circulating miRNA (PCA analysis). MiR-381-3p, miR-23-3p, and miR-18b-3p are among the 25 miRNAs unique to sEV, indicating potential sEV-specific miRNA markers. Further, those 25 miRNAs mostly regulate immune-related functions, indicating the value of sEV miRNA cargo in immunology.

The four sEV miRNA isolation methods employed in this study are valid techniques. The choice of method depends on the research question and study design. If purity is of concern, the UC+SEC method resulted in the best particles/µg protein ratio, which is often used as an indication of sample purity. These results could eventually establish sEV miRNAs as effective diagnostic and therapeutic tools of immunology.

Extracellular microRNAs (miRNAs) can be detected in body fluids and hold great potential as cancer biomarkers. Extracellular miRNAs are protected from degradation by binding various proteins and through their packaging into extracellular vesicles (EVs). There is evidence that the diagnostic performance of cancer-associated extracellular miRNAs can be improved by assaying EV-miRNA instead of total cell-free miRNA, but several challenges have hampered the advancement of EV-miRNA in liquid biopsy. Because almost all types of cells release EVs, cancer cell-derived EVs might constitute only a minor fraction of EVs in body fluids of cancer patients with low volume disease. Furthermore, a given cell type can release several subpopulations of EVs that vary in their cargo, and there is evidence that the majority of EVs contain low copy numbers of miRNAs. In this mini-review, we discuss the potential of several candidate EV membrane proteins such as CD147 to define cancer cell-derived EVs, and approaches by which subpopulations of miRNA-rich EVs in body fluids might be identified. By integrating these insights, we discuss strategies by which EVs that are both cancer cell-derived and miRNA-rich could be isolated to enhance the diagnostic performance of extracellular miRNAs.

Colorectal cancer (CRC) is a major cause of mortality and morbidity. Gambogic acid (GA) is a promising antitumor drug for treating CRC. We aimed to elucidate its mechanism in CRC invasion/metastasis via tumor cell-derived extracellular vesicle (EV)-carried miR-21. Nude mice peritoneal carcinomatosis (PC) model was subjected to GA treatment liver collection, followed by observation/counting of metastatic liver tissues/liver metastatic nodules by hematoxylin and eosin staining. miR-21 expression in metastatic liver tissues/CD68 + CD86, CD68 + CD206 cell percentages and M2 macrophage marker CD206 level in tumor tissues/interleukin (IL)-12 and IL-10 levels were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR)/flow cytometry/enzyme-linked immunosorbent assay. HT-29 cells were treated with GA/miR-21 mimics/negative control for 48 h. miR-21 expression/cell proliferation/migration/invasion/apoptosis were assessed by RT-qPCR/cell counting kit-8/scratch assay/transwell assay/flow cytometry. EVs were extracted from HT-29 cells and identified by transmission electron microscope/nanoparticle tracking analysis/Western blot. IL-4/IL-13-induced macrophages/PC nude mice were treated with GA and EVs, with the internalization of EVs by macrophages assessed through the uptake test. After intraperitoneal injection of GA, PC nude mice exhibited decreased tumor cell density/irregular cell number/liver metastatic nodule number/miR-21 expression, and CRC cells manifested reduced CD68 + CD206 cells/IL-10/miR-21/proliferation/migration/invasion and increased CD68 + CD86 cells/IL-12/apoptosis, while these trends were opposite after miR-21 overexpression, implying that GA curbed CRC/cell invasion/metastasis and macrophage polarization by diminishing miR-21 levels. miR-21 was encapsulated in HT-29 cell-derived EVs. M2 polarization elevated CD206 cells/IL-10, which were decreased by simultaneous GA treatment. EVs could be uptaken by macrophages. CRC cell-EV-miR-21 annulled the suppression effects of GA on macrophage M2 polarization. GA suppressed macrophage M2 polarization by lessening tumor cell derived-EV-shuttled miR-21, thereby weakening CRC invasion/metastasis.

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction secondary to body infection without overt central nervous system infection. Dysregulation of miRNA expression in the transcriptome can spread through RNA transfer in exosomes, providing an early signal of impending neuropathological changes in the brain. Here, we comprehensively analyzed brain-derived exosomal miRNA profiles in SAE rats (n = 3) and controls (n = 3). We further verified the differential expression and correlation of brain tissue, cerebrospinal fluid, and plasma exosomal miRNAs in SAE rats. High-throughput sequencing of brain-derived exosomal miRNAs identified 101 differentially expressed miRNAs, of which 16 were downregulated and 85 were upregulated. Four exosomal miRNAs (miR-127-3p, miR-423-3p, mR-378b, and miR-106-3p) were differentially expressed and correlated in the brain tissue, cerebrospinal fluid, and plasma, revealing the potential use of miRNAs as SAE liquid brain biopsies. Understanding exosomal miRNA profiles in SAE brain tissue and exploring the correlation with peripheral exosomal miRNA can contribute to a comprehensive understanding of miRNA changes in the SAE pathological process and provide the possibility of establishing early diagnostic assays.

Gastrointestinal (GI) cancers, including colorectal, gastric, esophageal, pancreatic, and liver, are associated with high mortality and morbidity rates worldwide. One of the underlying reasons for the poor survival outcomes in patients with these malignancies is late disease detection, typically when the tumor has already advanced and potentially spread to distant organs. Increasing evidence indicates that earlier detection of these cancers is associated with improved survival outcomes and, in some cases, allows curative treatments. Consequently, there is a growing interest in the development of molecular biomarkers that offer promise for screening, diagnosis, treatment selection, response assessment, and predicting the prognosis of these cancers. Extracellular vesicles (EVs) are membranous vesicles released from cells containing a repertoire of biological molecules, including nucleic acids, proteins, lipids, and carbohydrates. MicroRNAs (miRNAs) are the most extensively studied non-coding RNAs, and the deregulation of miRNA levels is a feature of cancer cells. EVs miRNAs can serve as messengers for facilitating interactions between tumor cells and the cellular milieu, including immune cells, endothelial cells, and other tumor cells. Furthermore, recent years have witnessed considerable technological advances that have permitted in-depth sequence profiling of these small non-coding RNAs within EVs for their development as promising cancer biomarkers -particularly non-invasive, liquid biopsy markers in various cancers, including GI cancers. Herein, we summarize and discuss the roles of EV-associated miRNAs as they play a seminal role in GI cancer progression, as well as their promising translational and clinical potential as cancer biomarkers as we usher into the area of precision oncology.

One of the biggest challenges in the fight against cancer is early detection. Early diagnosis is vital, but there are some barriers such as economic, cultural, and personal factors. Considering the disadvantages of radiological imaging techniques or serological analysis methods used in cancer diagnosis, such as being expensive, requiring expertise, and being time-consuming, there is a need to develop faster, more reliable, and cost-effective diagnostic methods for use in cancer diagnosis. Exosomes, which are responsible for intercellular communication with sizes ranging from 30-120 nm, are naturally produced biological nanoparticles. Thanks to the cargo contents they carry, they are a potential biomarker to be used in the diagnosis of cancer. Exosomes, defined as extracellular vesicles of endosomal origin, are effective in cancer growth, progression, metastasis, and drug resistance, and changes in microenvironmental conditions during tumor development change exosome secretion. Due to their high cellular activity, tumor cells produce much higher exosomes than healthy cells. Therefore, it is known that the number of exosomes in body fluids is significantly rich compared to other cells and can act as a stand-alone diagnostic biomarker. Cancer- derived exosomes have received great attention in recent years for the early detection of cancer and the evaluation of therapeutic response. In this article, the content, properties, and differences of exosomes detected in common types of cancer (lung, liver, pancreas, ovaries, breast, colorectal), which are the leading causes of cancer-related deaths, are reviewed. We also discuss the potential utility of exosome contents as a biomarker for early detection, which is known to be important in targeted cancer therapy.

Fulminant myocarditis (FM) is the most serious type of myocarditis. However, the molecular mechanism underlying the pathogenesis of FM has not been fully elucidated. Small extracellular vesicles (sEVs) play important roles in many diseases, but any potential role in paediatric FM has not been reported. Here, the differential signatures of lncRNAs in plasma sEVs were studied in FM children and healthy children using transcriptome sequencing followed by functional analysis. Then immune-related lncRNAs were screened to study their role in immune mechanisms, the levels and clinical relevance of core immune-related lncRNAs were verified by qRT-PCR in a large sample size. Sixty-eight lncRNAs had increased levels of plasma sEVs in children with FM and 11 had decreased levels. Functional analysis showed that the sEVs-lncRNAs with different levels were mainly related to immunity, apoptosis and protein efflux. Seventeen core immune-related sEVs-lncRNAs were screened, functional enrichment analysis showed that these lncRNAs were closely related to immune activation, immune cell migration and cytokine pathway signal transduction. The results of the study show that sEVs-lncRNAs may play an important role in the pathogenesis of fulminant myocarditis in children, especially in the mechanism of immune regulation.

The potential of urinary-derived extracellular vesicle (uEV) microRNAs (miRNAs) as noninvasive molecular biomarkers for identifying early-stage renal cell carcinoma (RCC) patients is rarely explored. The present study aims to explore the possibility of uEV miRNAs as novel molecular biomarkers for distinguishing early-stage RCC.

uEVs were extracted by ExoQuick-TC™ kit and miRNA concentrations were measured by RT-qPCR. ROC curves and bioinformatics analysis were employed to predict the diagnostic efficacy and regulatory mechanisms of dysregulated miRNAs.

Through a multiphase case-control study on uEV miRNAs screening, training, and validation in RCC cells (ACHN, Caki-1) and control cells (HK-2) and in uEVs of 125 RCC patients and 128 age- and sex-matched controls, we successfully identified four uEVs miRNAs (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) were significantly and stably upregulated in RCC in vitro and in vivo. When adjusted with estimated glomerular filtration rate (eGFR), the AUC of the three-uEV miRNA panel (miR-135b-5p, miR-200c-3p, and miR-203a-3p) was 0.785 (95 % CI = 0.729-0.842, P < 0.0001) for discriminating RCC patients from controls. Notably, this panel exhibited similar performance in distinguishing early-stage (stage Ⅰ) RCC patients, with an AUC of 0.786 (95 %CI = 0.727-0.844, P < 0.0001). Bioinformatics analysis predicted that candidate miRNAs were involved in cancer progressing.

Our study identified a four uEV miRNAs panel (miR-135b-5p, miR-196b-5p, miR-200c-3p, and miR-203a-3p) may serve as an auxiliary noninvasive indication of early-stage RCC.

No residual disease (R0 resection) after debulking surgery is the most critical independent prognostic factor for advanced ovarian cancer (AOC). There is an unmet clinical need for selecting primary or interval debulking surgery in AOC patients using existing prediction models.

RNA sequencing of circulating small extracellular vesicles (sEVs) was used to discover the differential expression microRNAs (DEMs) profile between any residual disease (R0, n = 17) and no residual disease (non-R0, n = 20) in AOC patients. We further analyzed plasma samples of AOC patients collected before surgery or neoadjuvant chemotherapy via TaqMan qRT-PCR. The combined risk model of residual disease was developed by logistic regression analysis based on the discovery-validation sets.

Using a comprehensive plasma small extracellular vesicles (sEVs) microRNAs (miRNAs) profile in AOC, we identified and optimized a risk prediction model consisting of plasma sEVs-derived 4-miRNA and CA-125 with better performance in predicting R0 resection. Based on 360 clinical human samples, this model was constructed using least absolute shrinkage and selection operator (LASSO) and logistic regression analysis, and it has favorable calibration and discrimination ability (AUC:0.903; sensitivity:0.897; specificity:0.910; PPV:0.926; NPV:0.871). The quantitative evaluation of Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) suggested that the additional predictive power of the combined model was significantly improved contrasted with CA-125 or 4-miRNA alone (NRI = 0.471, IDI = 0.538, p < 0.001; NRI = 0.122, IDI = 0.185, p < 0.01).

Overall, we established a reliable, non-invasive, and objective detection method composed of circulating tumor-derived sEVs 4-miRNA plus CA-125 to preoperatively anticipate the high-risk AOC patients of residual disease to optimize clinical therapy.

The aim of this study was to analyze the existence of miRNAs derived from serum extracellular vesicles (EVs) in familial Mediterranean fever (FMF) patients. Our group has previously shown the association of certain miRNAs with FMF.

Serum samples of adult and pediatric FMF patients and their age matched controls were used in the study. Serum EVs were characterized by transmission electron microscopy (TEM) and flow cytometry. RNAs were isolated from EVs and levels of miR-197-3p and miR-20a-5p were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).

EV characterization using TEM demonstrated fraction of 30-120 nm-sized particles with cup-shaped morphology. Flow cytometry results revealed the CD63 and CD81 positive populations as 53.3% in serum EVs. We showed that miR-197-3p and miR-20a-5p were "circulating miRNAs" and carried in EVs of FMF patients and controls. In FMF patients, level of miR-197-3p was significantly decreased. There was no significant alteration in the level for miR-20a-5p between patients and controls.

We showed the differential level of miR-197-3p in serum EVs of the FMF patients. miR-197-3p's potential as a biomarker and therapeutic target in FMF pathogenesis warrants further investigation.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly form of cancer, with limited progress in 5-year survival rates despite significant research efforts. The main challenges in treating PDAC include difficulties in early detection, and resistance to current therapeutic approaches due to aggressive molecular and microenvironment features. These challenges emphasize the importance of identifying clinically validated biomarkers for early detection and clinical management. Extracellular vesicles (EVs), particularly exosomes, have emerged as crucial mediators of intercellular communication by transporting molecular cargo. Recent research has unveiled their role in initiation, metastasis, and chemoresistance of PDAC. Consequently, utilizing EVs in liquid biopsies holds promise for the identification of biomarkers for early detection, prognosis, and monitoring of drug efficacy. However, numerous limitations, including challenges in isolation and characterization of homogeneous EVs populations, as well as the absence of standardized protocols, can affect the reliability of studies involving EVs as biomarkers, underscoring the necessity for a prudent approach. EVs have also garnered considerable attention as a promising drug delivery system and novel therapy for tumors. The loading of biomolecules or chemical drugs into exosomes and their subsequent delivery to target cells can effectively impede tumor progression. Nevertheless, there are obstacles that must be overcome to ensure the accuracy and efficacy of therapies relying on EVs for the treatment of tumors. In this review, we examine both recent advancements and remaining obstacles, exploring the potential of utilizing EVs in biomarker discovery as well as for the development of drug delivery vehicles.

Extracellular vesicle-derived microRNAs (EV-miRNAs) are promising biomarkers for early cancer diagnosis. However, existing EV-miRNA extraction technologies have a complex two-step process that results in low extraction efficiency and inconsistent results. This study aimed to develop and evaluate a new single-step extraction method, called miRQuick, for efficient and high-recovery extraction of EV-miRNAs from samples. The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. A membrane lysate is then added to extract only miRNA. The entire process can be completed within an hour using standard laboratory equipment. We validated the miRQuick method using various analytical techniques and compared its performance to other methods for plasma, urine and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected nine biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick. Our results demonstrate that miRQuick is a rapid and efficient method for EV-miRNA extraction with excellent repeatability, making it suitable for various applications including cancer diagnosis.

Parkinson's disease (PD) is an increasingly common neurodegenerative condition, which causes movement dysfunction and a broad range of non-motor symptoms. There is no molecular or biochemical diagnosis test for PD. The miRNAs are a class of small non-coding RNAs and are extensively studied owing to their altered expression in pathological states and facile harvesting and analysis techniques.

A total of 48 samples (16 each of PD, aged-matched, and young controls) were recruited. The small extracellular vesicles (sEVs) were isolated and validated using Western blot, transmission electron microscope, and nanoparticle tracking analysis. Small RNA isolation, library preparation, and small RNA sequencing followed by differential expression and targeted prediction of miRNA were performed. The real-time PCR was performed with the targeted miRNA on PD, age-matched, and young healthy control of plasma and plasma-derived sEVs to demonstrate their potential as a diagnostic biomarker.

In RNA sequencing, we identified 14.89% upregulated (fold change 1.11 to 11.04, p < 0.05) and 16.54% downregulated (fold change -1.04 to -7.28, p < 0.05) miRNAs in PD and controls. Four differentially expressed miRNAs (miR-23b-3p, miR-29a-3p, miR-19b-3p, and miR-150-3p) were selected. The expression of miR-23b-3p was "upregulated" (p = 0.002) in plasma, whereas "downregulated" (p = 0.0284) in plasma-derived sEVs in PD than age-matched controls. The ROC analysis of miR-23b-3p revealed better AUC values in plasma (AUC = 0.8086, p = 0.0029) and plasma-derived sEVs (AUC = 0.7278, p = 0.0483) of PD and age-matched controls.

We observed an opposite expression profile of miR-23b-3p in PD and age-matched healthy control in plasma and plasma-derived sEV fractions, where the expression of miR-23b-3p is increased in PD plasma while decreased in plasma-derived sEV fractions. We further observed the different miR-23b-3p expression profiles in young and age-matched healthy control.

Human bone marrow mesenchymal stem cells (BMSCs) are efficient mass producers of exosomes that can potentially be utilized for delivery of miRNAs in cancer therapy. The current study aimed to assess the role of MSC-exosomal miR-99b-5p during the development of colorectal cancer (CRC). The potential value of using plasma levels of exosomal miR-99b-5p for predicting the liver metastasis of colorectal cancer was also assessed. In this study, we found that overexpression of fibroblast growth factor receptor 3 (FGFR3) was associated with tumor progression in CRC and FGFR3 was the target gene of miR-99b-5p, which was down-regulated in CRC tissues. Furthermore, we observed that elevated miR-99b-5p inhibited CRC cell proliferation, invasion and migration, while reduced levels had the opposite effect on CRC cells. Moreover, exosomal miR-99b-5p delivered by BMSCs was able to limit the proliferation, invasion and migration of CRC cells in vitro, as well as suppressing tumor growth in vivo. Collectively, these findings revealed that MSC-derived exosomal miR-99b-5p can be transferred into CRC cells and which can suppress tumor progression by targeting FGFR3. This highlights the potential of using exosomal miR-99b-5p as a novel diagnostic marker for CRC, while providing a therapeutic target to combat CRC.