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. 2018 Aug 27;8:12920. doi: 10.1038/s41598-018-31332-6

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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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Comparison of the chemoresistance detected in ALI multilayered mono-cultures and 3D tumour spheroids: (A,B) Percentage (%) of live A549 cells (A) and ATP levels (B) detected in (from left to right) ALI multilayered mono-cultures and 3D tumour spheroids. Both in vitro models were grown for 14 d and then exposed to four anti-cancer drugs (docetaxel, cytarabine, vinblastine and methotrexate) at their nominal IC₅₀ concentration for 72 h. Cell cultures were exposed to drugs by direct inoculation. Data are reported as average ± standard error of the mean (n_replicates = 2; n_tests = 3). The symbols (*), (**) and (***) indicate significant differences (p values < 0.05, 0.01 and 0.001, respectively) (two-way ANOVA and Bonferroni post-test). (C) Changes in cell viability in 3D tumour spheroids cultured for 4, 7 and 14 d and analysed for their percentage of live cells (in black) and total ATP levels (in grey). Data are shown as average ± standard error of the mean (n_replicates = 2; n_tests = 3). The symbol (***) indicates a significant difference from values at t = 4 d (one-way ANOVA and Dunnett post-test). (D) Representative LSCM images of the F-actin organization (in red) in 3D tumour spheroids at different time-points. Cell nuclei were also stained with Hoechst 33342 (in blue). Z-stack images, here presented in orthogonal view, clearly demonstrate the growing thickness of the spheroids overtime. Scale bars: 10 μm (objective lens: 63×). (E) Live 3D tumour spheroids stained for cell nuclei (in blue) and hypoxia (in green) at different time-points. Z-stack images of the cultures were reconstructed and are shown as three-dimensional projections. Scale bars: 100 μm (objective lens: 10×).