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. 2017 Oct 26;7:14167. doi: 10.1038/s41598-017-14326-8

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© The Author(s) 2017

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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Confocal Microscopy to Evaluate Cellular Morphology and Population Dynamics. (a) Multiphoton confocal images (3D renderings) of ER+ tumor surrogates at days 0, 3, & 7, showing the GFP positive MCF-7 cells alone (green, left panels), the mCherry positive CAF-hTERT (red, middle panels), and both cell types together (merge, right panels) (250x magnification, 3D reconstructions are between 150 and 400 μm in thickness). (b) Epithelial to fibroblast ratio (green: red) was calculated from confocal maximum projections using CellProfiler and showed no significant change over time (Kruskal-Wallis test). The percentage of each cell population seeded (initial E:F = 2:1) is indicated by the dashed lines. (c) Total cell number (epithelial cells and CAF) was calculated from confocal maximum projections using CellProfiler and demonstrated an increase over time (Kruskal-Wallis test, p < 0.001, n = 4–7 FOV). (d) FormFactor, a measure of cellular circularity was calculated from confocal images using CellProfiler, with changes in CAF over time indicating cellular elongation (Kruskal-Wallis test, p < 0.0001, n = 4–7 FOV). Data in (b–d) represent mean ± SEM.