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. 2018 Dec 4;8:17608. doi: 10.1038/s41598-018-35710-y

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© The Author(s) 2018

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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BST-2 expressing cells resist hemodynamic shear stress in vivo, survive in circulation, and colonize the lungs: (A) Cartoon of experimental set up. (B) Representative 10x images of cell mixture used for injections. Beads are DAPI⁻, which auto-fluoresce red (red arrows), while shCTL cells are DAPI⁺ and CMFDA⁺ (Green, green arrows), and shBST-2 cells are DAPI⁺ and CMTPX⁺ (Red, purple arrows). (C) Percent of CTCs present in blood at indicated times (0–6 h) after intravenous injection. Percentages were determined using flow cytometry where shCTL cells are EPCAM⁺ and CMFDA⁺ and shBST-2 cells are EPCAM⁺ and CMTPX⁺. (D,E) Percent of live and dead shCTL and shBST-2 cells at 0 and 4 h post injection. Percentages were determined using flow cytometry and staining for the early apoptotic marker Annexin V and the late apoptotic marker 7-AAD. (F) 10x images of lung sections from mice carrying a mixture of beads (red arrows), shCTL (green arrows), and shBST-2 cells (purple arrows) and euthanized at the indicated times (0, 2, 4, and 6 h). Merge column show all cells and beads. Numbers in parenthesis next to sample name to the right depict the number of shCTL cells, shBST-2 cells, and beads within the representative images. (G) Number of shCTL and shBST-2 cells per field found in the lungs at 0, 2, 4, and 6 hours. 100 beads were counted for each time point. Error bars represent SEM and significance was taken at P < 0.05*, P < 0.01**, and P < 0.001***. ns = not significant. CTCs = circulating tumor cells.