Fig. 4

Implementing a hypoxia fate-mapping system in breast cancer models. a Fluorescent images of full cross sections of orthotopic mammary tumors derived from MDA-MB-231 hypoxia fate-mapping cells. Tumors were excised at various times over a 45-day period and sectioned for imaging. The white dashed line outlines the necrotic core. b Fluorescent images of DsRed and GFP expression, and TUNEL (blue) and Hypoxyprobe™ (purple) labeling. Normalized intensity plots for each fluorophore (top) (mean ± SEM, n = 8), or O₂ measurements (bottom) are displayed as a function of distance from the tumor core. O₂ measurements were performed by using a fixed-needle microprobe (mean ± SEM, N = 8; ****P < 0.0001 vs. 0 (one-way ANOVA with Bonferroni posttest). c Fluorescent images of GFP expression and Hypoxyprobe™ (purple) and HIF-1α (yellow) immunofluorescent labeling. d Fluorescent images of DsRed and GFP expression, and CD31 (yellow) immunofluorescent labeling to detect endothelial cells lining blood vessels in a tumor region far from the necrotic core. e Full tumor cross sections were imaged in tiles, linearly stitched, binarized by ImageJ, and used to determine the ratio of DsRed-, double- (both DsRed and GFP), and GFP-positive areas of MDA-MB-231 orthotopic tumors (see Supplementary Fig. 5b for an illustration of the calculation method). Ratios (%) are displayed in the graph at different time points of tumor progression (mean ± SEM, N = 3, n = 48); ****P < 0.0001 versus day 15 (Two-way ANOVA with Bonferroni posttest). The box extends from the 25th to 75th percentiles, the median is marked by the vertical line inside the box, and the whiskers represent the minimum and maximum points. f MCF7 hypoxia fate-mapping cells were injected into the nipple and delivered to a single ductal tree of multiparous NSG mice. A whole mount of the mammary fat pad was imaged by fluorescent microscopy 60 days after injection. g Tissue sections were stained with DAPI to detect cell nuclei and imaged for DsRed and GFP. h Tissue sections were stained with DAPI to detect cell nuclei and labeled with a HIF-1α antibody. Scale bar: 50 μm