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. 2019 Oct 24;10:4862. doi: 10.1038/s41467-019-12412-1

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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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Fig. 6 — Post-hypoxic cells have enhanced metastatic potential. a Orthotopic tumors derived from MDA-MB-231 hypoxia fate-mapping cells. Blood, lungs, and tumor were harvested at 2 weeks for half of the mouse cohort. Surgical tumor resection was performed on the second half of the cohort, and lungs were harvested 2 weeks after tumor removal to assess late-stage metastasis. Pie charts represent the ratio of DsRed+ to GFP+ cells in each site. b The probability of a GFP+ (or DsRed+) CTC in the blood was obtained by dividing the percentage of GFP+ (or DsRed+) CTCs detected in the bloodstream with the percentage of GFP+ (or DsRed+) cells in the matched primary tumor (N = 3, n = 17) by using flow cytometry (FC); ****P < 0.0001 GFP versus DsRed (two-tailed t-test). c Cross-sectional imaging of DsRed and GFP expression and DAPI labeling of lung micrometastasis at week 2. The inset shows magnified GFP+ micrometastasis. d, e The probability of a GFP+ metastatic event in the lung is obtained by dividing the percentage of GFP+ micrometastases by the percentage of GFP+ cells in the matched primary tumor by using d image analysis or e flow cytometry (N = 3, IA: n = 23; FC: n = 22); ****P < 0.0001 GFP versus DsRed (two-tailed t-test). The box extends from the 25th to 75th percentiles, median is marked by the line inside the box, and whiskers represent the minimum and maximum. f Cross-sectional imaging of DsRed and GFP expression and DAPI labeling of lung macrometastasis 2 weeks after tumor resection. g, h The probability of a GFP+ metastatic event in the lung is obtained by dividing the percentage of individual GFP+ macrometastatic colonies by the percentage of GFP+ cells in the matched primary tumor by using g image analysis or h flow cytometry (N = 3, IA: n = 16; FC: n = 22); ****P < 0.0001 GFP versus DsRed (two-tailed t-test). i, j Tumor and lungs from triple-transgenic mice were harvested at 4 months of age. Probability of lung metastatic events was obtained as described in g (mean ± SEM, N = 8); ****P < 0.0001 GFP versus tdTom (two-tailed t-test). k MDA-MB-231 hypoxia fate-mapping cells were injected into the tail vein of NSG mice and harvested 5 weeks later