Fig. 8 | Nature Communications

Fig. 8

From: Fate-mapping post-hypoxic tumor cells reveals a ROS-resistant phenotype that promotes metastasis

Fig. 8

Post-hypoxic CTCs have ROS-resistant phenotype. a Schematic representation of the proposed mechanism. Post-hypoxic cells (GFP+) are resistant to ROS and have a survival advantage over DsRed+ cells in the bloodstream. b Mitochondrial ROS levels were measured by using MitoROS by flow cytometry in matched tumor and blood samples freshly harvested from mice (N = 2, n = 8); ****P < 0.0001 GFP versus DsRed and ####P < 0.0001 tumor versus blood (Two-way ANOVA with Bonferroni posttest). The box extends from the 25th to 75th percentiles, the median is the vertical line inside the box, and the whiskers represent the minimum and maximum. c Freshly isolated CTCs were treated with the ROS inhibitor N-acetylcysteine (NAC) for 1 h, and ROS levels were measured using MitoROS by flow cytometry (N = 1, n = 7); ****P < 0.0001 GFP versus DsRed and ####P < 0.0001 NAC– versus NAC+ (Two-way ANOVA with Bonferroni posttest). d Cell viability was measured by using Sytox Blue by flow cytometry (N = 1, n = 9); ****P < 0.0001 GFP versus DsRed (two-tailed t-test). e, f Tumor-derived cells sorted for DsRed+ or GFP+ expression were treated ex vivo with H2O2 for 1 h and f were quantified 48 h later by image analysis by using nuclei segmentation to determine cell confluence (e) (N = 4, n = 16); ****P < 0.0001 GFP versus DsRed (Two-way ANOVA with Bonferroni posttest). g Tumor-derived hypoxia fate-mapping cells (1 × 105 cells) were injected directly into the tail vein of NSG mice. Organs were harvested either 48 h or 25 days after injection. h, i Cryo-sections of the lung were stained with DAPI and imaged for DsRed and GFP to detect micrometastasis after 48 h (h) or macrometastasis after 25 days (i). Scale bar: 50 μm (h), 1 mm (i). j Quantification of the number of metastatic events by image analysis of mounted lung sections after 48 h or 25 days (N = 1, n = 9–10); ****P < 0.0001 GFP versus DsRed (Two-way ANOVA with Bonferroni posttest). k Quantification of metastatic burden by flow cytometry analysis of lungs after 48 h or 25 days (N = 1, n = 10); ****P < 0.0001 GFP versus DsRed (Two-way ANOVA with Bonferroni posttest)

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