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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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Knockdown or antibody treatment of integrin (Itg) α5 in Panc-1 cells or fibronectin (FN) in WI-38 fibroblasts reduces fibroblast-dependent Panc-1 cell invasion in 3D collagen gel. Panc-1 cells and WI-38 cells were transfected with 10 nM control or specific siRNAs for Itg α5 or FN, respectively. These cells were harvested on the next day, mixed with non-treated counterparts, and incubated on an EZSPERE plate for 20 h. Chimeric spheroids thus produced were used in the following analyses. (a) Chimeric spheroid formation in α5-si Panc-1 (left panel) or FN-si WI-38 (right panel). Ordinate, the mean of the spheroid number per well ± SD in triplicate wells. NS, not significant (p = 0.056); ***p < 0.001. (b,c) Panc-1 cell invasion in contact with WI-38 cells in α5-si Panc-1 (left panel) or FN-si WI-38 (right panel). The above chimeric spheroids were incubated in collagen gel for 2 days, and Panc-1 cells elongated in contact with invading WI-38 cells, shown by arrows in (c), were counted for each spheroid. Ordinate, the mean of the number of elongate Panc-1 cells per spheroid ± SD in triplicate wells. The total spheroids analyzed were 14 in Ctr and α5-si Panc-1, 12 in Ctr WI-38, and 7 in FN-si WI-38. *p < 0.05, **p < 0.01. (c) Representative mages. Scale bars, 100 μm. See also Video 7. (d) Suppressive effects of anti-Itg-α5β1 and anti-FN antibodies on Panc-1 cell invasion. Invading Panc-1 cells in contact with fibroblasts were measured as above for each spheroid (total 6 to 8 spheroids per group). p = 0.066 in FN-si. (e) Representative images in (d).