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. 2019 Nov 28;9:17782. doi: 10.1038/s41598-019-54001-8

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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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Monitoring doxorubicin (dox) accumulation and spheroid size change during treatment under in vitro normoxia (20% O₂), chronic severe hypoxia (0% O₂), and cycling hypoxia (cycling gas profile with 4 h 0%, 4 h 20% O₂). (a) Example transmitted light images used for spheroid segmentation. (b) Doxorubicin fluorescence at the beginning of the experiment, prior to starting the doxorubicin flow. Almost no fluorescence is visible. (c–e) Two-photon doxorubicin fluorescence images after 24, 48, and 72 h of doxorubicin treatment. (f) Quantified average doxorubicin fluorescence intensity in the spheroid region (the total summed pixel intensity in the segmented region divided by its pixel area). (g) Quantified segmented spheroid area change during the 72 h treatment, for spheroids in each of the tested oxygen conditions. Doxorubicin accumulation is higher under cycling hypoxia than under chronic severe hypoxia and normoxia. (h–o) Doxorubicin penetration, visualized as average doxorubicin fluorescence intensity vs. depth into the spheroid, for different oxygen conditions, at varying times in the experiment. Imaging the complete set of spheroids in all three conditions took ~30 minutes, so the average time for each set of images of each condition is reported. Issues with the microscope stage automation reduced temporal resolution over the first ~24 h of the experiment. Shaded regions depict standard error of the mean for N = 5 (cycling), 6 (20%), 4 (0%) spheroids.