Fig. 5.

Hypoxia fate-mapping system facilitates profiling of intratumoral hypoxia. a Tumors derived from hypoxia fate-mapping MDA-MB-231 cells were harvested 2 weeks after implantation and sorted into DsRed+/GFP− or GFP+ populations directly into Trizol (N = 2). Venn diagram displaying the overlap of the number of genes with differential expression (−1.5 ≥ FC ≥ 1.5) in GFP+ versus DsRed+ tumor cells (green circle) and MDA-MB-231 cells exposed to 1% O₂ versus 20% O₂ (pink circle) (Supplementary Table 6). b–d Relative expression of mRNA measured by qPCR in tumor-derived cells (TG or TR) or MDA-MB-231 cells exposed to 20% or 1% O₂ in vitro, b CRE, c genes co-regulated by intratumoral and in vitro hypoxia (CA9, DNAH11, EGLN3, and LOX), and d genes exclusive to upregulation upon intratumoral hypoxia (ITGA10 and CP) (mean ± SEM, N = 3, n = 3); ****P < 0.0001 TG versus TR and 1% versus 20% (two-tailed t-test). 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. e Heat map of the 41-gene signature derived from the overlap of intratumoral and in vitro hypoxia. The distribution of the relative fold change of each gene in the 41-gene signature is displayed for GFP+ versus DsRed+ sorted tumor cells (T) or cells (C) exposed to 1% versus 20% O₂ conditions. Genes with fold change higher than 75% of the fold change of genes in the set are red and genes with fold change lower than 25% of the fold change of genes in the set are blue (Pearson correlation factor r = 0.84 and P = 9.6 × 10⁻⁷). f–h Microarray expression data from 664 breast cancer patients were used to perform multigene survival analysis (n = 664; HGU133 plus 2.0 arrays, KMplotter). Kaplan–Meier analysis of distant metastasis-free survival (DMFS) of breast cancer patients stratified by high or low expression by using f the 40 most induced genes by intratumoral hypoxia, g the 41-gene signature derived from the overlap of intratumoral and in vitro hypoxia, or h the 40 most induced genes by in vitro hypoxia