Characterization of Extracellular Vesicles from Bronchoalveolar Lavage Fluid and Plasma of Patients with Lung Lesions Using Fluorescence Nanoparticle Tracking Analysis

Dlugolecka, Magdalena; Szymański, Jacek; Zaręba, Łukasz; Homoncik, Zuzanna; Domagała‐Kulawik, Joanna; Polubiec-Kownacka, Małgorzata; Czystowska-Kuźmicz, Małgorzata

doi:10.3390/cells10123473

Cited by 11 publications

(7 citation statements)

References 46 publications

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“…At present, there are no EV extraction methods that could purify biological fluid samples from all cell debris and proteins ( 35 ) and, in our study, TEM, CLSM, and WB analyses showed potential sample contamination. EV surface proteins are attached to the EV membrane with a smooth diffuse manner that forms a protein corona for the EV.…”

Section: Discussionmentioning

confidence: 59%

“…The lack of a universal EV isolation protocol is a key challenge for EV research. Earlier studies have shown successful isolation of EVs from human and mouse BALF with ultrafiltration and ultracentrifugation (15,(35)(36)(37). Recently, SEC has become a popular method for isolating EVs because of the relatively pure and intact EVs that it can produce.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, SEC has become a popular method for isolating EVs because of the relatively pure and intact EVs that it can produce. However, the total separation of non-vesicular material from EVs remains impossible with currently available methods ( 35 , 38 , 39 ). Dilute samples are another potential downside of SEC, caused by the separation of the EVs into several fractions, but this can be avoided by pooling and by performing an additional concentration step.…”

Section: Discussionmentioning

confidence: 99%

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Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses

et al. 2022

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Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell–cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, n = 35) and healthy horses (n = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.

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Section: Discussionmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses

et al. 2022

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“…The content of EVs in peripheral blood is higher than that in BALF, but the source is complex, and the heterogeneity is strong [29]; plasma also contains high concentrations of proteins and lipoproteins, making the puri cation of EVs di cult [30,31]. However, BALF does not have high lipoprotein and protein contents.…”

Section: Discussionmentioning

confidence: 99%

MiR-1246b, a novel miRNA molecule of extracellular vesicles in bronchoalveolar lavage fluid promotes the nodules growth through FGF14 in patients with lung cancers

Huang

Ding

Yuan

et al. 2023

Preprint

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With the widespread development of chest computed tomography (CT), the detection rate of pulmonary nodules has been increasing, therefore, the judgment of benign and malignant nodules has become a common problem in clinic. MicroRNA, a potential tool, is expected to become a good choice for diagnosing and studying the occurrence and development of diseases through the vector of bronchoalveolar lavage fluid extracellular vesicles (BALF-EVs). In this study, radial endobronchial ultrasound (R-EBUS) was used to locate pulmonary nodules in patients, BALF was obtained and EVs were isolated, and small RNA sequencing was performed to screen differential miRNAs between benign and malignant pulmonary nodules groups. The binding targets and underlying mechanisms of the differential miRNAs were verified by in vitro and in vivo experiments. EVs were successfully isolated and identified after obtaining BALF by R-EBUS localization. Differential miRNAs in BALF-EVs of patients with benign or malignant pulmonary nodules were screened by high-throughput small RNA sequencing. A new sequence, miR-1246b, was noticed. We found that FGF14 was the binding target of miR-1246b by luciferase Assay. Subsequent mechanistic studies showed that miR-1246b inhibited the expression of FGF14 in lung cancer cells, further led to ERK phosphorylation and epithelial-to-mesenchymal transition (EMT), which ultimately contributed to lung cancer cell proliferation, migration and invasion. In summary, our study demonstrates that the detection of miRNAs in BALF-EVs, a means of liquid biopsy, could assist to distinguish malignant nodules from benign nodules. miR-1246b, extracted from BALF-EVs, targets FGF14 to promote lung cancer cell proliferation, migration and invasion.

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“…Except for the apoptotic bodies, all EVs are released into circulation by living cells, either by direct budding of the plasma membrane (oncosomes and microvesicles) or via the endosomal pathway (exosomes) [ 200 , 202 ]. EVs can be found in many biological fluids, including blood [ 203 ], amniotic fluid [ 204 ], urine [ 205 ], CSF [ 206 ], breast milk [ 207 ], saliva [ 208 ], ascites [ 209 ], tears [ 210 ], semen [ 211 ], bronchoalveolar lavage fluid [ 212 ], and in the conditioned media of cultured cells [ 213 ].…”

Section: Ddpcr-based Liquid Biopsies For Skin Cancer Monitoring and P...mentioning

confidence: 99%

Skin Cancer Research Goes Digital: Looking for Biomarkers within the Droplets

Dobre

Constantin

Neagu

2022

JPM

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Skin cancer, which includes the most frequent malignant non-melanoma carcinomas (basal cell carcinoma, BCC, and squamous cell carcinoma, SCC), along with the difficult to treat cutaneous melanoma (CM), pose important worldwide issues for the health care system. Despite the improved anti-cancer armamentarium and the latest scientific achievements, many skin cancer patients fail to respond to therapies, due to the remarkable heterogeneity of cutaneous tumors, calling for even more sophisticated biomarker discovery and patient monitoring approaches. Droplet digital polymerase chain reaction (ddPCR), a robust method for detecting and quantifying low-abundance nucleic acids, has recently emerged as a powerful technology for skin cancer analysis in tissue and liquid biopsies (LBs). The ddPCR method, being capable of analyzing various biological samples, has proved to be efficient in studying variations in gene sequences, including copy number variations (CNVs) and point mutations, DNA methylation, circulatory miRNome, and transcriptome dynamics. Moreover, ddPCR can be designed as a dynamic platform for individualized cancer detection and monitoring therapy efficacy. Here, we present the latest scientific studies applying ddPCR in dermato-oncology, highlighting the potential of this technology for skin cancer biomarker discovery and validation in the context of personalized medicine. The benefits and challenges associated with ddPCR implementation in the clinical setting, mainly when analyzing LBs, are also discussed.

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Characterization of Extracellular Vesicles from Bronchoalveolar Lavage Fluid and Plasma of Patients with Lung Lesions Using Fluorescence Nanoparticle Tracking Analysis

Cited by 11 publications

References 46 publications

Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses

Isolation of Extracellular Vesicles From the Bronchoalveolar Lavage Fluid of Healthy and Asthmatic Horses

MiR-1246b, a novel miRNA molecule of extracellular vesicles in bronchoalveolar lavage fluid promotes the nodules growth through FGF14 in patients with lung cancers

Skin Cancer Research Goes Digital: Looking for Biomarkers within the Droplets

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