Optimal isolation of extracellular vesicles from pleural fluid and profiling of their microRNA cargo

Our plain-English summary of Optimal isolation of extracellular vesicles from pleural fluid and profiling of their microRNA cargo, by T.M. Chee, H.E. O'Farrell, L.G. Lima, et al (Journal of Extracellular Biology, 2023).

The study

• Authors: T.M. Chee, H.E. O'Farrell, L.G. Lima, A. Möller, K.M. Fong, I.A. Yang, R.V. Bowman
• Journal: Journal of Extracellular Biology
• Published: 2023;2(10):e119
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Pleural effusion occurs in both benign and malignant pleural disease. In malignant pleural effusions, the diagnostic accuracy and sensitivity of pleural fluid cytology is less than perfect, particularly for the diagnosis of malignant pleural mesothelioma, but also in some cases for the diagnosis of metastatic pleural malignancy with primary cancer in the lung, breast or other sites. Extracellular vesicles (EVs) carry an enriched cargo of microRNAs (miRNAs) which are selectively packaged and differentially expressed in pleural disease states. To investigate the diagnostic potential of miRNA cargo in pleural fluid extracellular vesicles (PFEVs), we evaluated methods for isolating the extracellular vesicle (EV) fraction including combinations of ultracentrifugation, size-exclusion chromatography (SEC) and ultrafiltration (10 kDa filter unit). PFEVs were characterized by total and EV-associated protein, nanoparticle tracking analysis and visualisation by transmission electron microscopy. miRNA expression was analyzed by Nanostring nCounter® in separate EV fractions isolated from pleural fluid with or without additional RNA purification by ultrafiltration (3 kDa filter unit). Optimal PFEV yield, purity and miRNA expression were observed when PFEV were isolated from a larger volume of pleural fluid processed through combined ultracentrifugation and SEC techniques. Purification of total RNA by ultrafiltration further enhanced the detectability of PFEV miRNAs. This study demonstrates the feasibility of isolating PFEVs, and the potential to examine PFEV miRNA cargo using Nanostring technology to discover disease biomarkers.

© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.

In plain English

This short laboratory study, from The Prince Charles Hospital in Queensland, Australia, compared ways to isolate extracellular vesicles (tiny particles released by cells) from pleural fluid and measure their microRNA. The authors used stored fluid from 11 patients: three with malignant pleural mesothelioma, three with lung cancer that had spread to the pleura, one with metastatic melanoma and four with benign effusions. The article does not mention asbestos, and did not test diagnostic accuracy. Total protein yield was not associated with markers of vesicles. Adding a step called size-exclusion chromatography lowered albumin, which the authors read as a sign of purer samples. In one sample, this step plus an extra RNA purification step improved microRNA detection. In that same sample, doubling the starting volume gave higher raw counts for 143 microRNAs (1.1- to 3.5-fold) and lower counts for four, after low-count microRNAs were excluded. The authors concluded that isolating vesicles from pleural fluid is feasible. They noted that samples were archival, stored for up to ten years, and limited in number and volume, so findings need validation in freshly collected fluid.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory methods (proof-of-principle) study comparing techniques for isolating extracellular vesicles from pleural fluid and profiling their microRNA; published as a short communication
• Who was studied: Archival pleural fluid from 11 patients who underwent thoracentesis at The Prince Charles Hospital, Queensland, Australia: malignant pleural mesothelioma (n = 3), metastatic melanoma (n = 1), lung cancer metastatic to the pleura (n = 3) and benign effusions (n = 4). Fluid had been stored for up to 10 years. Not every sample was used in every experiment.
• Exposure: No asbestos or other environmental exposure was studied, and asbestos is not mentioned. The study compared laboratory isolation methods: ultracentrifugation, size-exclusion chromatography and ultrafiltration, used alone or in combination, with different starting volumes of fluid.
• Main results:
  • Across preparation methods, total protein yield was not associated with vesicle abundance as judged by vesicle-specific protein markers (FLOT1 and CD9).
  • Exosome-enriched fractions isolated with size-exclusion chromatography (SEC) showed lower albumin than preparations without SEC, which the authors said indicates SEC may improve purity.
  • Fractions that kept microvesicles showed more vesicle markers. They also showed more albumin, meaning more non-vesicle protein was co-isolated.
  • In sample PF6129, nanoparticle tracking found vesicles despite weak markers. Ultracentrifugation alone gave 5.94 × 10^8 plus or minus 1.14 × 10^7 particles per mL, with a size peak at 114 nm. Ultracentrifugation plus SEC gave fewer particles: 1.21 × 10^8 plus or minus 2.93 × 10^6 particles per mL.
  • Electron microscopy of two samples showed cup-shaped vesicles with either method. Preparations using ultracentrifugation only had high background.
  • In sample PF7278, adding SEC and RNA purification improved microRNA detectability compared with ultracentrifugation alone.
  • Also in sample PF7278 alone, raw counts were compared between a preparation from 5 mL and one from 2.5 mL of fluid. MicroRNAs with raw counts under 50 in both preparations were excluded. Of those remaining, 143 had higher counts in the 5 mL preparation (1.1- to 3.5-fold increase) and four had lower counts (0.8- to 0.9-fold decrease).
  • In four further samples, Method 2 (ultracentrifugation only, 16 mL of fluid) gave overall greater microRNA expression than Method 1 (with SEC and RNA purification, 5 mL). The authors said a direct comparison was not justified because the methods and volumes differed, and that both methods can be considered.
• The authors' conclusion: The authors concluded that isolating the vesicle fraction of pleural fluid is feasible. They said the study clarifies the advantages and disadvantages of different methods for analysing vesicle cargo in pleural effusions, as groundwork for future searches for diagnostic biomarkers.
• Limitations the authors state:
  • The fluids were archival and stored for up to ten years. Long-term storage and freeze-thaw cycles may alter microRNA content, so results need validation in freshly collected fluid.
  • Samples were limited in number and volume, so full particle analysis could not be done on all samples.
  • Some experiments used very few samples, which limited how many replications were possible.
  • Vesicles concentrated by 10 kDa ultrafiltration were tested only by western blotting, not by nanoparticle tracking or electron microscopy.

Topics

Medical terms in this study, explained in our Lexicon: Pleural Effusion, Fluid Cytology, Pleural Mesothelioma, Transmission Electron Microscopy (TEM), Biomarker.

Related studies

• Potential utility of miRNAs derived from pleural fluid extracellular vesicles to differentiate between benign and malignant pleural effusions — T.M. Chee, C.J. Zahra, et al. (2025)
• Exploring RNA cargo in extracellular vesicles for pleural mesothelioma detection — A. Kraft, M.B. Kirschner, et al. (2025)
• Plasmatic extracellular vesicle microRNAs in malignant pleural mesothelioma and asbestos-exposed subjects suggest a 2-miRNA signature as potential biomarker of disease — T. Cavalleri, L. Angelici, et al. (2017)
• Serum Extracellular Vesicle-Derived microRNAs as Potential Biomarkers for Pleural Mesothelioma in a European Prospective Study — E. Casalone, G. Birolo, et al. (2022)
• Selection of microRNAs in extracellular vesicles for diagnosis of malignant pleural mesothelioma by in vitro analysis — T. Jotatsu, H. Izumi, et al. (2020)
• CARGO: A Cytometry Analysis framework via Regularized Graph Optimal-transport — A.S. Shemonti, G.B. Gmyrek, et al. (2026)

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