En bloc release of MVB-like small extracellular vesicle clusters by colorectal carcinoma cells

Gábor Valcz; Edit I Buzás; Ágnes Kittel; Tibor Krenács; Tamás Visnovitz; Sándor Spisák; György Török; László Homolya; Sára Zsigrai; Gábor Kiszler; Géza Antalffy; Krisztina Pálóczi; Zoltán Szállási; Vanessza Szabó; Anna Sebestyén; Norbert Solymosi; Alexandra Kalmár; Kristóf Dede; Péter Lőrincz; Zsolt Tulassay; Péter Igaz; Béla Molnár

doi:10.1080/20013078.2019.1596668

En bloc release of MVB-like small extracellular vesicle clusters by colorectal carcinoma cells

J Extracell Vesicles. 2019 Apr 8;8(1):1596668. doi: 10.1080/20013078.2019.1596668. eCollection 2019.

Authors

Gábor Valcz^{1

2}, Edit I Buzás^{3

4}, Ágnes Kittel⁵, Tibor Krenács⁶, Tamás Visnovitz^{3

4}, Sándor Spisák⁷, György Török⁸, László Homolya⁸, Sára Zsigrai², Gábor Kiszler⁹, Géza Antalffy⁹, Krisztina Pálóczi³, Zoltán Szállási¹⁰, Vanessza Szabó⁹, Anna Sebestyén⁶, Norbert Solymosi¹¹, Alexandra Kalmár^{1

2}, Kristóf Dede¹², Péter Lőrincz¹³, Zsolt Tulassay^{1

2}, Péter Igaz^{1

2}, Béla Molnár^{1

2}

Affiliations

¹ Molecular Medicine Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.
² 2nd Department of Medicine, Semmelweis University, Budapest, Hungary.
³ Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
⁴ MTA-SE Immune-Proteogenomics Extracellular Vesicle Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
⁵ Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
⁶ 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
⁷ Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, USA.
⁸ Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
⁹ Department of Image Analysis, 3DHISTECH Ltd, Budapest, Hungary.
¹⁰ Computational Health Informatics Program (CHIP), Boston Children's Hospital and Harvard Medical School, Boston, USA.
¹¹ Centre for Bioinformatics, University of Veterinary Medicine, Budapest, Hungary.
¹² Department of General Surgery and Surgical Oncology, Uzsoki Teaching Hospital, Budapest, Hungary.
¹³ Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, Hungary.

Abstract

Small extracellular vesicles (EVs) are membrane enclosed structures that are usually released from cells upon exocytosis of multivesicular bodies (MVBs) as a collection of separate, free EVs. In this study, we analysed paraffin embedded sections of archived human colorectal cancer samples. We studied 3D reconstructions of confocal microscopic images complemented by HyVolution and STED imaging. Unexpectedly, we found evidence that large, MVB-like aggregates of ALIX/CD63 positive EV clusters were released en bloc by migrating tumour cells. These structures were often captured with partial or complete extra-cytoplasmic localization at the interface of the plasma membrane of the tumour cell and the stroma. Their diameter ranged between 0.62 and 1.94 μm (mean±S.D.: 1.17 ± 0.34 μm). High-resolution 3D reconstruction showed that these extracellular MVB-like EV clusters were composed of distinguishable internal particles of small EV size (mean±S.D.: 128.96 ± 16.73 nm). In vitro, HT29 colorectal cancer cells also showed the release of similar structures as confirmed by immunohistochemistry and immune electron microscopy. Our results provide evidence for an en bloc transmission of MVB-like EV clusters through the plasma membrane. Immunofluorescent-based detection of the MVB like small EV clusters in archived pathological samples may represent a novel and unique opportunity which enables analysis of EV release in situ in human tissues.

Keywords: EV; MVB; colorectal carcinoma; migrating cancer cells.

Grants and funding

This work was supported by National Research, Development and Innovation Office NKFIH, Hungary KMR-12-1-2012-0216, NVKP_16-1-2016-0004, NVKP_16-1-2016-0017, OTKA11958, OTKA120237, K115398, European Regional Development Fund and the Hungarian Government TRAIN-EV H2020-MSCA-ITN-722148 by the European Commission, VEKOP-2.3.2-16-2016-00002 and VEKOP-2.3.3-15-2016-00016, as well as STIA_17. The study that served as a basis of this publication was also supported by the Institutional Higher Education Excellence Program of the Ministry of Human Capacities within the framework of the “Molecular biology” and the “Therapeutic development” thematic programs. The study also supported by Bolyai Fellowship (590/2015), MTA Premium postdoctoral research grant PPD-222/2018, Momentum Program of the Hungarian Academy of Sciences (LP2012-025), Breast Cancer Research Foundation (BCRF-17-156) and Research and Technology Innovation Fund (KTIA_NAP_13-2014-0021).