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. 2019 Jan 22;9:292. doi: 10.1038/s41598-018-36646-z

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© The Author(s) 2019

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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Cancer cells adhere to fibroblasts with fibronectin (FN) deposits and become activated. (a,b) Immunofluorescent (IF) staining for FN (red) on WI-38 cells in 2D co-cultures. Arrows point Panc-1 cells (green) attaching to fibroblasts. Scale bars, 50 μm. (a) A phase-contrast image with FN signals. (b) IF image of FN fibrils (upper panel) and its merged image with GFP (Panc-1) (lower panel). Scale bars, 100 μm. (c–e) Cancer cell (green) invasion in contact with fibroblast protrusions with FN deposits (red) in 3D collagen gel: GFP-Panc-1/WI-38 (c), GFP-A549/OUS-11 (d), and GFP-A549/LuCAF (e). (f) Co-localization of integrin α5β1 (red) with FN fibrils (green) at non-labeled Panc-1/WI-38 contact sites (arrows) in 2D co-culture. Blue, DAPI stain; asterisks, Panc-1 cells; F, fibroblasts. Insets, magnified views of the indicated boxed areas. Scale bars, 10 μm. (g) IF staining of Panc-1/WI-38 co-culture for phosphorylated Erk (p-Erk, green) and F-actin (red) in the left panel and for phosphorylated Akt (p-Akt, green) and integrin β1 (red) in the right panel. Red asterisks, fibroblast-bound, activated cancer cells; white asterisks, unbound cancer cells; green arrow, positive signals in cancer cells for integrin β1 at the heterotypic contact site. When the p-Erk and p-Akt signals were quantitated using the NIH image J software, the relative intensity for the cellular p-Erk signal was 100 and 70 (85 in average) in the red asterisks and 58 and 35 (47 in average) in the white ones, while that for p-Akt was 100 (red asterisk) and 64 (white asterisk).