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. 2019 Nov 27;9:17674. doi: 10.1038/s41598-019-54117-x

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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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Histological sections of HCT116 spheroids fixed at three different time points after seeding, stained with pimonidazole (green, hypoxia), Ki-67 (red, proliferation), and DAPI (blue, all cell nuclei). No significant hypoxia is observed on treatment day (day four). A hypoxic core that is surrounded by a rim of cells many of which are proliferating (red) develops over time. Necrosis in the spheroid core may be indicated by a decreased cellular density at the centre in the section of a spheroid fixed on day 25.