Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours

ABSTRACT

Cells release heterogeneous nano-sized vesicles either as exosomes, being derived from endosomal compartments, or through budding from the plasma membrane as so-called microvesicles, commonly referred to as extracellular vesicles (EVs). EVs are known for their important roles in mammalian physiology and disease pathogenesis and provide a potential biomarker source in cancer patients. EVs are generally often analysed in bulk using Western blotting or by bead-based flow-cytometry or, with limited parameters, through nanoparticle tracking analysis. Due to their small size, single EV analysis is technically highly challenging. Here we demonstrate imaging flow cytometry (IFCM) to be a robust, multiparametric technique that allows analysis of single EVs and the discrimination of distinct EV subpopulations. We used IFCM to analyse the tetraspanin (CD9, CD63, CD81) surface profiles on EVs from human and murine cell cultures as well as plasma samples. The presence of EV subpopulations with specific tetraspanin profiles suggests that EV-mediated cellular responses are tightly regulated and dependent on cell environment. We further demonstrate that EVs with double positive tetraspanin expression (CD63⁺/CD81⁺) are enriched in cancer cell lines and patient plasma samples. In addition, we used IFCM to detect tumour-specific GFP-labelled EVs in the blood of mice bearing syngeneic intracerebral gliomas, indicating that this technique allows unprecedented disease modelling. In summary, our study highlights the heterogeneous and adaptable nature of EVs according to their marker profile and demonstrates that IFCM facilitates multiparametric phenotyping of EVs not only in vitro but also in patient plasma at a single EV level, with the potential for future functional studies and clinically relevant applications.

Abbreviation: EDTA = ethylenediamine tetraacetic acid

KEYWORDS:

• Imaging flow cytometry
• extracellular vesicle
• biomarker
• glioma
• tetraspanin

Acknowledgments

The authors thank Keith Ligon for providing the BT112 glioblastoma cell line and Christel Herold-Mende for the NCH644 cell line. We further thank the FACS core facility and the Mouse Pathology Core Facility at the University Medical Center Hamburg-Eppendorf for help with cell sorting and tissue embedding, respectively. This work was funded by the Deutsche Forschungsgemeinschaft (scholarship to FLR RI2616/2-1), by the Anni Hofmann Stiftung (KL) and by ERANET GlioEX (MG).

Author contribution

The manuscript was written through contributions of all authors. All authors approved of the final version of the manuscript. F.L.R. and C.L.M. conceived the idea, designed the experiments and wrote the manuscript. F.L.R, C.L.M., K.K. and M.H performed the experiments. R.R. designed and performed CLEM experiments. L.D. performed and interpreted MRI images and helped with the in vivo work, E.S, A.R., C.H. and A.B. performed in vitro cell isolation and culture, D.H.H. provided astrocytes and additional glioblastoma cell lines and assisted in writing the manuscript, J.F., T.M., N.O.S and S.P. acquired patient specimens and assisted in data analysis. K.P, S.G, S.K., M.G., X.O.B., S.L., E.A.C., B.G., A.G., B.F. and M.W. assisted with writing the manuscript, analysis of data and contributed relevant material and/or expertise and discussed the results. F.L.R. designed and assembled all figures. K.L designed the study, analysed data and wrote the paper.

Disclosure

A.G. received three travel awards (each 1000 USD) from Merck Millipore/Amnis Corporation. A.G. is a consultant for and has equity interests in Evox Therapeutics Ltd., Oxford, UK. B.G. is SAB member of Evox Therapeutics and Innovex Therapeutics. All other authors declare that no potential conflict of interest exists.

Supplementary materials

Supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the Deutsche Forschungsgemeinschaft [RI2616/2-1];Anni Hofmann Stiftung [];ERANET GlioEX