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. 2018 Jun 14;9:2343. doi: 10.1038/s41467-018-04743-2

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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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Fig. 3 — Kif3b is required for metastatic HEp3 cancer cell vasculotropism and invasion into the extracellular matrix in vivo. Multicolor two-photon intravital imaging was utilized to visualize the chicken CAM structure and cancer cell behavior within metastatic cancer cell colonies. a Chicken CAM structure, 15dpf. FITC-lectin (green) was used for visualization of the vasculature, SHG (blue) for imaging of collagen fiber network and auto-fluorescence (red) for imaging of the blood. b Representative images showing similarity in the collagen matrix structure between the mouse lung (left panel) and chicken CAM (right panel). Metastatic colonies that were formed by control (c, scramble shRNA) and Kif3b shRNA2-transduced HEp3 cells (d). Right panels in (c) and (d) show higher magnification of areas in middle (SHG) panels. Red arrows point to cancer cell protrusions that are in contact with collagen fibers. Note that control cells robustly interact with the vasculature and invade into the collagen matrix while shRNA2 Kif3b cells fail to do so. Insets show only tumor cell (GFP) channel. e Quantification of the fraction of cells in contact with blood vessels for HEp3 control and Kif3b knockdown cells. f Average number of cancer cell engaged collagen fibers for HEp3 control and shRNA2 Kif3b cells. g Average cell protrusion lifetime for control and shRNA2 Kif3b cells (see also Supplementary Movie 3). Scale bars = 200 μm (a, b); 200 μm (c, d, left and middle panels) or 20 μm (c, d, right panel)