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

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
• migrating cancer cells
• MVB
• colorectal carcinoma

Acknowledgements

We are grateful to Gabriella Kónyáné Farkas, Zita Bratu, Anna Tamási, Éva Balogh Mátrainé, Tibor Horváth, Mónika Paulusz, Titanilla Dankó, Ádám Oszvald and Györgyné Vidra for their support. The authors thank Gábor Juhász (Eötvös Loránd University, Budapest and Biological Research Centre, Szeged) for the access to transmission electron microscope at Eötvös Loránd University and his financial support for the operation of the TEM core facility.

Competing financial interest

The authors declare no competing financial interests.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental materials data can be accessed here.

Additional information

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).