Fig. 5
AK4 overexpression reduces hypoxic necrosis and promotes liver metastasis in vivo. a NOD scid Gamma (NSG) mice were injected subcutaneously with CL1-0 Vec and CL1-0 AK4 cells (1 × 106 cells/100 μL) in the left and right flanks, respectively. Volumes of CL1-0 Vec and CL1-0 AK4 tumors were measured weekly as indicated. b Left, pimonidazole staining and IHC images of AK4 expression in CL1-0 Vec and CL1-0 AK4 subcutaneous xenograft tumors. Scale bar represents 2 mm. Right, pimonidazole-positive tumor area was detected and quantified by Definiens imaging analysis algorithm. **P ≤ 0.01. c Representative IHC staining for AK4, HIF-1α, and E-cadherin expression in subcutaneous xenograft tumors from CL1-0 vector or CL1-0 AK4 cells. Scale bar represents 100 μm. d NSG mice were injected orthotopically in the left lung with CL1-0 Vec or CL1-0 AK4 cells at a concentration of 1 × 105 cells in 10 μL of PBS/Matrigel mixture. Gross view (formalin-fixed) and H&E staining images of lungs and livers from mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The white and black arrows indicate tumor nodules in the gross view and H&E staining images, respectively. e Quantification of liver nodule number in mice orthotopically injected with CL1-0 Vec or CL1-0 AK4 cells at day 30. The results are presented as the mean ± SD of at least three separate experiments. Two-tailed, unpaired Student’s t tests were used for all pairwise comparisons. *P ≤ 0.05; **P ≤ 0.01