Fig 1: Tumors with a high hypoxia signature and low expression of DNA repair genes have a poor prognosisKaplan Meier curves show the survival differences between the subgroups identified in Figure 1c, based on median expression values of the HIF2a signature and the indicated repair proteins. (a) HIF2α-BRCA1. (b) HIF2α-RAD51. (c) HIF2α-RIF1. (d) HIF2α-Ku70. (e) Tumors belonging to all 4 HIF2α-high/repair-low and to all 4 HIF2α-low/repair-high quadrants in a-d were identified by GeneVenn (www.genevenn.sourceforge.net) and analyzed separately. Tumors with high expression of the HIF2α signature and low levels of all 4 repair proteins had a very poor prognosis (green), when compared to tumors with low expression of the HIF2α signature and high expression of all 4 repair proteins (magenta).
Fig 2: Hypoxic areas in primary tumors and liver metastases are characterized by DNA damage and low expression of the DNA repair proteins RAD51 and RIF1(a) Immunohistochemistry (IHC) for CAIX, HIF1α and γH2AX in human primary colorectal tumours. Robust expression of all three markers was observed surrounding necrotic lesions. (b) Quantification of CAIX, HIF1α and γH2AX expression in peri-necrotic areas and reference zones in primary human colon tumors by IHC (n=20). (c) Analysis of CAIX and γH2AX expression in human liver metastases by IHC. A 20x magnification of the area depicted in the square in lower panel. (d) Quantification of CAIX and γH2AX staining (IHC) in peri-necrotic and reference tissue in liver metastases (n=30). (e) Scatterplot showing the correlation between the expression of (randomly chosen microscopic field areas of) CAIX and γH2AX, assessed with the Spearman test (rho) (n=60). (f) Patient-derived colonospheres were injected into the liver parenchyma of immune-deficient mice. Following tumor initiation, the tumor-bearing liver lobes were subjected to a vascular clamping or sham protocol [24] to induce hypoxia. After 24 hours, the livers were excised and expression of CAIX and γH2AX was examined by IHC. (g) IHC on peri-necrotic areas of clamped livers (experiment as in (f)) for CAIX, γH2AX, RIF1, RAD51, and KU70. * p<0.001, N=necrosis, T=tumor, PN=peri-necrotic, RZ=Reference Zone.
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