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. 2019 Aug 19;7:133. doi: 10.1186/s40478-019-0788-1

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© The Author(s). 2019

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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.

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Fig. 4 — Blebbing of metastatic and endothelial cells of cerebral vessels. a: Representative two-photon micrograph (z-projection) of a blebbing breast cancer cell (arrow) migrating from the lumen of the capillary to the brain parenchyma. Dotted arrow indicates vasoconstriction. b: 3D confocal image representation of a blebbing metastatic cell projecting out the blebs through the vessel wall during initial phase of transendothelial migration. c: Confocal microscopy z-projection indicating blebs detaching from the tumour cell (red) resulting in large oncosome-like vesicles (arrows). d: Endothelial apoptotic blebbing during diapedesis of a metastatic cell (arrow) through the capillary wall, as represented in two-photon microscopy z-sections (i-iii). e: Basolateral blebs (arrows) in endothelial cells of a cerebral capillary hosting a breast cancer cell, as represented in confocal microscopy z-sections (i-ii). f: Claudin-5-positivity (grey) of endothelial blebs (arrows). STED (claudin-5) and confocal (blue, green and red channels) z-projection images are shown. g: Two-photon micrograph (z-projection) presenting intensive blebbing of cerebral microvessels during transmigration of cancer cells. Arrow indicates blebbing, dashed arrow shows extravasating tumour cell. 36 h later, the tumour cell reached an extravascular position (arrowhead), while intensive blebbing is almost completely resolved. Numbers indicate matching vessels in the two images. h: Transmission electron micrograph of a cerebral capillary and an intravascular tumour cell. Insets show mitochondrial damage (1) and blebbing (2) of the endothelium. EC = endothelial cell. i: Vesicle-like cleaved caspase-3 positivity (grey) in endothelial cells close to metastatic cells, as shown in a confocal z-projection. Arrows indicate cleaved caspase-3-positive endothelial cells, dashed arrows mark tumour cells. j: Cleaved caspase-3 positivity (grey) of endothelial cells and metastatic cells, as shown in a confocal z-stack. Arrow indicates cleaved caspase-3 positivity in endothelial cells, dashed arrows mark cleaved caspase-3-positive tumour cells. Red = tumour cells (tdTomato), green = endothelium (YFP), blue = nuclei (Hoechst)