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. 2020 Nov 7;18:162. doi: 10.1186/s12951-020-00722-2

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — Characterization of extracellular vesicles (EVs) derived from regular (Reg) and brain metastases (Br) breast cancer cells. The conditioned media of regular human MDA-MB-231 breast cancer cells and a brain metastases variant of MDA-MB-231 cells (MDA-MB-231-BrM2-831) were processed by tangential flow filtration to obtain Reg-EVs and Br-EVs, respectively. a Schematic of in vivo selection cycles to obtain a brain metastases cell line: (1) intracardiac injection of cells, (2) development of brain metastases, (3) excision of brain metastases and in vitro culture, and (4) intracardiac injection of cultured cells. b Size distribution profiles (10 nmincrements) measured by nanoparticle tracking analysis (NTA). Data are shown as biological triplicates. c Particles isolated from 100 mL of conditioned cell culture media. d Protein markers of EVs (annexin V, cluster of differentiation (CD)9, and CD81) and intracellular contaminants (calnexin) obtained by Western blot. Arrows represent the expected protein band. e Zeta potential measured by laser Doppler micro-electrophoresis. Data are presented as mean ± SD of three technical replicates. Dashed line represents the mean value of three biological replicates. f Representative cryogenic transmission electron microscopy images. Scale bar, 100 nm (upper panels) and 50 nm (lower panels). Ann V annexin V, Caln calnexin, H cell homogenate; L protein ladder