Aims: Chronic kidney disease (CKD) is an independent risk factor for the development of coronary artery disease (CAD). For both, CKD and CAD, the intercellular transfer of microRNAs (miRs) through extracellular vesicles (EVs) is an important factor of disease development. Whether the combination of CAD and CKD affects endothelial function through cellular crosstalk of EV-incorporated miRs is still unknown. Methods and results: Out of 172 screened CAD patients, 31 patients with CAD + CKD were identified and matched with 31 CAD patients without CKD. Additionally, 13 controls without CAD and CKD were included. Large EVs from CAD + CKD patients contained significantly lower levels of the vasculo-protective miR-130a-3p and miR-126-3p compared to CAD patients and controls. Flow cytometric analysis of plasma-derived EVs revealed significantly higher numbers of endothelial cell-derived EVs in CAD and CAD + CKD patients compared to controls. EVs from CAD + CKD patients impaired target human coronary artery endothelial cell (HCAEC) proliferation upon incubation in vitro. Consistent with the clinical data, treatment with the uraemia toxin indoxyl sulfate (IS)-reduced miR-130a-3p levels in HCAEC-derived EVs. EVs from IS-treated donor HCAECs-reduced proliferation and re-endothelialization in EV-recipient cells and induced an anti-angiogenic gene expression profile. In a mouse-experiment, intravenous treatment with EVs from IS-treated endothelial cells significantly impaired endothelial regeneration. On the molecular level, we found that IS leads to an up-regulation of the heterogenous nuclear ribonucleoprotein U (hnRNPU), which retains miR-130a-3p in the cell leading to reduced vesicular miR-130a-3p export and impaired EV-recipient cell proliferation. Conclusion: Our findings suggest that EV-miR-mediated vascular intercellular communication is altered in patients with CAD and CKD, promoting CKD-induced endothelial dysfunction.

Glioblastoma is a devastating disease, for which biomarkers allowing a prediction of prognosis are urgently needed. microRNAs have been described as potentially valuable biomarkers in cancer. Here, we studied a panel of microRNAs in extracellular vesicles (EVs) from the serum of glioblastoma patients and evaluated their correlation with the prognosis of these patients. The levels of 15 microRNAs in EVs that were separated by size-exclusion chromatography were studied by quantitative real-time PCR, followed by CD44 immunoprecipitation (SEC + CD44), and compared with those from the total serum of glioblastoma patients (n = 55) and healthy volunteers (n = 10). Compared to total serum, we found evidence for the enrichment of miR-21-3p and miR-106a-5p and, conversely, lower levels of miR-15b-3p, in SEC + CD44 EVs. miR-15b-3p and miR-21-3p were upregulated in glioblastoma patients compared to healthy subjects. A significant correlation with survival of the patients was found for levels of miR-15b-3p in total serum and miR-15b-3p, miR-21-3p, miR-106a-5p, and miR-328-3p in SEC + CD44 EVs. Combining miR-15b-3p in serum or miR-106a-5p in SEC + CD44 EVs with any one of the other three microRNAs in SEC + CD44 EVs allowed for a prognostic stratification of glioblastoma patients. We have thus identified four microRNAs in glioblastoma patients whose levels, in combination, can predict the prognosis for these patients.

Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.

Detection of tumor progression in patients with glioblastoma remains a major challenge. Extracellular vesicles (EVs) are potential biomarkers and can be detected in the blood of patients with glioblastoma. In our study, we evaluated the potential of serum‐derived EVs from glioblastoma patients to serve as biomarker for tumor progression. EVs from serum of glioblastoma patients and healthy volunteers were separated by size exclusion chromatography and ultracentrifugation. EV markers were defined by using a proximity‐extension assay and bead‐based flow cytometry. Tumor progression was defined according to modified RANO criteria. EVs from the serum of glioblastoma patients (n = 67) showed an upregulation of CD29, CD44, CD81, CD146, C1QA and histone H3 as compared to serum EVs from healthy volunteers (P value range: <.0001 to .08). For two independent cohorts of glioblastoma patients, we noted upregulation of C1QA, CD44 and histone H3 upon tumor progression, but not in patients with stable disease. In a multivariable logistic regression analysis, a combination of CD29, CD44, CD81, C1QA and histone H3 correlated with RANO‐defined tumor progression with an AUC of 0.76. Measurement of CD29, CD44, CD81, C1QA and histone H3 in serum‐derived EVs of glioblastoma patients, along with standard MRI assessment, has the potential to improve detection of true tumor progression and thus could be a useful biomarker for clinical decision making.

Background/Objectives: Despite the significant advancements made in the diagnosis of lung cancer, the traditional diagnostic methods remain limited because they are often invasive, expensive, and not suitable for regular screening, creating a need for more accessible and non-invasive alternatives. In this context, the analysis of miRNAs in EVs and free circulating microRNA may be used as liquid biopsies in lung cancer to identify individuals at risk. This study aimed to compare miRNA profiles in the serum and EVs derived from lung cancer patients by focusing on Let-7a-5p and miR-21-3p. Materials and Methods: Serum and EVs were isolated from lung cancer patients and healthy controls. EVs were characterized using nanoparticle tracking analysis, electron microscopy, and Western blotting for surface markers (CD63, CD81, TSG101). Total miRNA levels were quantified in the serum and EVs, and specific miRNAs (hsa-let-7a-5p and hsa-miR-21-3p) were analyzed using RT-qPCR. Statistical analysis evaluated miRNA expression across clinicopathological features, including age, gender, smoking status, tumor stage, cancer type, and EGFR mutation status. Results: Total miRNA levels were significantly enriched in EVs compared to the serum. Let-7a-5p was downregulated in EVs from patients with advanced-stage lung cancer (Stage III–IV) compared to those with early-stage cancer and controls (p < 0.05), while no differences were observed in the serum. Conversely, miR-21-3p was significantly upregulated in EVs and serum from advanced-stage patients (p < 0.01) and in adenocarcinoma compared to squamous cell carcinoma (p < 0.05). No significant differences were observed for age, gender, or smoking status. Conclusions: Our findings highlight the differential expression of miRNAs in EVs and the serum, emphasizing the diagnostic potential of EV-associated Let-7a-5p and miR-21-3p in lung cancer. These results suggest that EVs are a more robust source for miRNA biomarkers compared to the serum.

The epidermal growth factor receptor signaling pathway is essential for cellular processes such as proliferation, survival, and migration. Dysregulation of EGFR signaling is frequently observed in non-small cell lung cancer and is associated with a poor prognosis. Aim: This study aims to isolate and characterize extracellular vesicles released by mutant-EGFR lung cancer cell line PC9 and compare them with wild type EGFR lung cancer cell line A549. Methodology: The two lung cancer cell lines were cultured with 2% EV-free serum, and EVs were subsequently isolated by differential (ultra)centrifugation. EVs were analyzed by nanoparticle tracking analysis for quantification and size determination. The presence and characterization of the isolated EVs was conducted with immunoblotting. Dysregulation of hsa-miR-128-3p in both cell lines and EVs was assessed by qRT-PCR. Results showed that EGFR mutation significantly increased EV release and altered their size, compared to EVs released by wild type EGFR cell line. In addition to classical EV marker such as CD9, CD81, Flotillin-1 and TSG101, Western blot analysis also detected phosphorylated EGFR. Furthermore, EGFR mutation led to a significant downregulation of hsa-miR-128-3p in EVs. In conclusion, this study highlights the significant influence of EGFR activation on EV release and cargo, providing insights into tumor behavior and potential biomarkers for lung cancer.