Tracking Small Extracellular Vesicles Using a Minimally Invasive PicoGreen Labeling Strategy DOI Creative Commons
Sagar Rayamajhi, Benjamin K. Gibbs, Jared Sipes

et al.

ACS Applied Bio Materials, Journal Year: 2024, Volume and Issue: 7(11), P. 7770 - 7783

Published: Nov. 1, 2024

Extracellular vesicles (EVs) are cell-secreted lipid bilayer delimited particles that mediate cellular communication. These tiny sacs of information play an important role in cell communication and alter the physiological process under both normal pathological conditions. As such, tracking EVs can provide valuable regarding basic understanding communication, onset early malignancy, biomarker discovery. Most current EV-tracking strategies invasive, altering natural characteristics by modifying with lipophilic dyes or surface proteins fluorescent reporters. The invasive labeling could processes thereby have major limitations for functional studies. Here, we report alternative minimally EV strategy using PicoGreen (PG), a small molecule fluoresces at 520 nm when bound to dsDNA. We show PG binds dsDNA associated (50–200 nm), forming stable highly PG-DNA complex (PG-EVs). In 2D culture 3D organoid models, PG-EV showed efficient properties, including high signal-to-noise ratio, time- concentration-dependent uptake, ability traverse environment. further validated dual-labeled following two orthogonal strategies: (1) Bioconjugation via amine (2) donor engineering endogenously expressing mCherry-tetraspanin (CD9/CD63/CD81) reporter proteins. Our study has shown feasibility as effective be applied studying across multiple model systems.

Language: Английский

MicroRNA Profiling of Bone Marrow Plasma Extracellular Vesicles in Multiple Myeloma, Extramedullary Disease, and Plasma Cell Leukemia DOI Creative Commons
Jana Gregorová,

Monika Vlachová,

Petra Vychytilova‐Faltejskova

et al.

Hematological Oncology, Journal Year: 2025, Volume and Issue: 43(1)

Published: Jan. 1, 2025

ABSTRACT Multiple myeloma is a plasma cell malignancy characterized by an abnormal increase in monoclonal immunoglobulins. Despite significant advances treatment, some patients progress to more aggressive forms of multiple myeloma, including extramedullary disease or leukemia. Although the exact molecular mechanisms are not known, several studies have confirmed involvement small extracellular vesicle‐enriched microRNAs progression. Therefore, we performed expression profiling these molecules bone marrow disease, and leukemia using RNA sequencing identify novel involved pathogenesis. In total, 42 were significantly dysregulated among analyzed subgroups. Independent validation RT‐qPCR elevated levels miR‐140‐3p, miR‐584‐5p, miR‐191‐5p, miR‐143‐3p compared patients. Subsequent statistical analysis revealed correlations between patient clinical characteristics flow cytometry parameters microRNA expression. These results indicate that dysregulation could contribute

Language: Английский

Citations

1
Integrated Microfluidic Chip for Neutrophil Extracellular Vesicle Analysis and Gastric Cancer Diagnosis DOI Creative Commons
Dan Yu, Jianmei Gu, Jiahui Zhang

et al.

ACS Nano, Journal Year: 2025, Volume and Issue: unknown

Published: March 9, 2025

Neutrophil-derived extracellular vesicles (NEVs) are critically involved in disease progression and considered potential biomarkers. However, the tedious processes of NEV separation detection restrain their use. Herein, we presented an integrated microfluidic chip for (IMCN) analysis, which achieved immune-separation CD66b+ NEVs multiplexed contained miRNAs (termed signatures) by using 10 μL serum samples. The optimized microchannel flow rate IMCN enabled efficient capture (>90%). After recognition captured a specific CD63 aptamer, on-chip rolling circle amplification (RCA) reaction was triggered released aptamers from heat-lysed NEVs. Then, RCA products bound to molecular beacons (MBs), initiating allosteric hairpin structures amplified "turn on" fluorescence signals (RCA-MB assay). Clinical sample analysis showed that signatures had high area under curve (AUC) distinguishing between healthy control (HC) gastric cancer (GC) (0.891), benign diseases (BGD) GC (0.857). Notably, AUC reached 0.912 with combination five biomarkers (NEV signatures, CEA, CA199) differentiate HC, diagnostic accuracy further increased machine learning (ML)-based ensemble classification system. Therefore, developed is valuable platform may have use diagnosis.

Language: Английский

Citations

1
Proteomic analysis of ascitic extracellular vesicles describes tumour microenvironment and predicts patient survival in ovarian cancer DOI Creative Commons
Anna Kotrbová, Kristína Gömöryová,

Antónia Mikulová

et al.

Journal of Extracellular Vesicles, Journal Year: 2024, Volume and Issue: 13(3)

Published: March 1, 2024

High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), most common type ovarian cancer, ranks among deadliest malignancies. Many HGSC patients have excess fluid in called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods analyzed them mass spectrometry. identified not only set 'core ascitic EV-associated proteins' but also defined their subset unique to Using single-cell RNA sequencing data, we mapped origin HGSC-specific different types cells present Surprisingly, did come predominantly non-malignant cell such as macrophages fibroblasts. Flow cytometry combination with analysis EV protein composition matched samples showed that type-specific markers has more substantial prognostic potential than cells. To conclude, provide evidence proteomic can define cellular TME. This finding opens numerous avenues both for better understanding EV's role promotion/prevention improved diagnostics.

Language: Английский

Citations

8
The Contribution and Perspectives of Proteomics to Epithelial Ovarian Cancer DOI Open Access
Junrui Pei,

Haifeng Qiu,

Wenjia Wang

et al.

PROTEOMICS - CLINICAL APPLICATIONS, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 26, 2025

ABSTRACT Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy which mainly consists of serous, mucinous, clear cell, and endometrioid subtypes. Due to lack classic symptoms at an early stage, EOC usually presented as advanced tumors with local and/or distant metastasis. Although a large portion was initially platinum‐sensitive, patients would acquire resistance common chemotherapeutic agents. These aforementioned issues lead challenge for clinical treatments unsatisfying outcomes. Previous studies have demonstrated genetic features are hard target alterations DNA RNA levels not fully represented protein expression profiles made it more complex. In recent years, panel attempted explore key proteins involved in development progression using high‐throughput proteomic technologies. We herein summarized them provide full view this topic. Trial Registration: ClinicalTrials.gov identifier: NCT046698990.

Language: Английский

Citations

0
Comparative evaluation of methods for isolating extracellular vesicles from ICC cell culture supernatants: Insights into proteomic and glycomic analysis DOI Creative Commons
Linlin Wu, Jiao Wei, Yueping Zhan

et al.

Cell Communication and Signaling, Journal Year: 2025, Volume and Issue: 23(1)

Published: April 29, 2025

Extracellular vesicles (EVs) are nanoscale structures involved in intercellular communication and play a key role cancer pathology. Intrahepatic cholangiocarcinoma (ICC) is highly invasive malignancy marked by abnormal sialylated glycosylation. Analyzing proteins glycans EVs provides insights into ICC molecular subtyping mechanisms. Optimizing EV isolation methods for ICC-derived enables comprehensive proteomic glycomic analysis. We systematically evaluated five methods-Ultracentrifugation (UC), exoEasy, Total Exosome Isolation (TEI), EVtrap, ÄKTA-by analyzing the biophysical properties, profiles, of EVs. Subsequently, we applied TMT-based quantitative proteome light/heavy methylamine labeling quantification N-glycan linkage isomers to investigate alterations N-glycans within secreted HuCCT1 HCCC-9810 cells with overexpressing ST6 β‑galactoside α2,6‑sialyltransferase 1 (ST6GAL1). By evaluating proteome, N-glycome extracted using different methods, UC was identified as optimal approach this study, it offered balance between operational complexity, cost-effectiveness, preservation activity. In total 1,928 high-confidence over 84 were quantified. ST6GAL1 exhibited consistent upregulation 16 proteins, downregulation 10 well 3 glycans. Quantitative analysis revealed that overexpression led significant cell adhesion glycosylation pathways, along specific changes structures. Notably, these modifications extended beyond α2,6-sialylation, suggesting interactions glycosyltransferases may drive alterations.

Language: Английский

Citations

0
Ovarian cancer ascites proteomic profile reflects metabolic changes during disease progression DOI Creative Commons
Diana Luísa Almeida-Nunes, Mariana Nunes, Hugo Osório

et al.

Biochemistry and Biophysics Reports, Journal Year: 2024, Volume and Issue: 39, P. 101755 - 101755

Published: June 13, 2024

Ovarian cancer (OC) patients develop ascites, an accumulation of ascitic fluid in the peritoneal cavity anda sign tumour dissemination within cavity. This body is under-researched, mainly regarding ascites formed during progression that have no diagnostic value and, therefore, are discarded. We performed a discovery proteomics study to identify new biomarkers supernatant OC patients. In this preliminary study, we analyzed small amount highlight importance not discarding such biological material treatment, which could be valuable for management. Our findings reveal malignant (MAF) displays proliferative environment promotes growth cells shift metabolic pathway using alternative sources nutrients, as cholesterol pathway. Also, drained from treatment showed immunosuppressive environment, with up-regulation proteins signaling pathways IL-4 and IL-13 down-regulation MHC-II. pinpointed protein (Transmembrane Protein 132A) context deserves better explored more extensive cohort patients' samples. The proteomic profile MAF provides unique insight into kinetics disease progression, information can used effective strategies.

Language: Английский

Citations

1
Vesiclemania: ILGD INSTAND 2024 - A German extravaganza of tiny packages! DOI

Sophia H. Montigel,

Luise Meder,

Kendr K.a Maaß

et al.

Trillium Extracellular Vesicles, Journal Year: 2024, Volume and Issue: 6(1), P. 12 - 17

Published: Sept. 25, 2024

The 2024 IGLD INSTAND Symposium brought together leading experts in EV research. Key themes included the importance of methodological rigor and standardization, emphasized through latest MISEV 2023 guidelines, advances isolation technologies. Several presentations explored clinical applications EVs, including their use stem cell treatments, liquid biopsies, as gene therapy delivery systems using EV-mediated CRISPR/Cas9. Discussions also focused on interplay between EVs immune system, such role modulating viral infections therapeutic potential pathological situations cancer. In conclusion, event need for inter-laboratory reproducibility collaborative research to ensure technologies are primed translation, underscoring efforts drive innovations field.

Language: Английский

Citations

0
Heterogeneity in extracellular vesicles: Same same - but different? DOI
Kerstin Menck, Christian Preußer

Trillium Extracellular Vesicles, Journal Year: 2024, Volume and Issue: 6(1), P. 18 - 21

Published: Sept. 25, 2024

Language: Английский

Citations

0
Tracking Small Extracellular Vesicles Using a Minimally Invasive PicoGreen Labeling Strategy DOI Creative Commons
Sagar Rayamajhi, Benjamin K. Gibbs, Jared Sipes

et al.

ACS Applied Bio Materials, Journal Year: 2024, Volume and Issue: 7(11), P. 7770 - 7783

Published: Nov. 1, 2024

Extracellular vesicles (EVs) are cell-secreted lipid bilayer delimited particles that mediate cellular communication. These tiny sacs of information play an important role in cell communication and alter the physiological process under both normal pathological conditions. As such, tracking EVs can provide valuable regarding basic understanding communication, onset early malignancy, biomarker discovery. Most current EV-tracking strategies invasive, altering natural characteristics by modifying with lipophilic dyes or surface proteins fluorescent reporters. The invasive labeling could processes thereby have major limitations for functional studies. Here, we report alternative minimally EV strategy using PicoGreen (PG), a small molecule fluoresces at 520 nm when bound to dsDNA. We show PG binds dsDNA associated (50–200 nm), forming stable highly PG-DNA complex (PG-EVs). In 2D culture 3D organoid models, PG-EV showed efficient properties, including high signal-to-noise ratio, time- concentration-dependent uptake, ability traverse environment. further validated dual-labeled following two orthogonal strategies: (1) Bioconjugation via amine (2) donor engineering endogenously expressing mCherry-tetraspanin (CD9/CD63/CD81) reporter proteins. Our study has shown feasibility as effective be applied studying across multiple model systems.

Language: Английский

Citations

0