Effects of tumor-expressed FVII on tumor characteristics and metastasis. (A) Primary tumor growth after orthotopic injection of MDA^pcDNA and MDA^FVII cells in immunodeficient mice (n = 6). In the extracted tumors, KI67 positivity of the outer proliferative zone (B), CD31⁺ vessel density (C), CD11b⁺ (D), F4/80⁺ immune cells (E), and active β-catenin (F) were determined. (G) In the same experiment, metastasis was determined in the indicated organs using qPCR of human GAPDH corrected for mouse β-actin. (H) Tumor growth and metastasis were also determined after orthotopic grafting in the presence of the FVII-blocking antibody 3G12 (n = 6). (I) Tumor growth and metastasis at the end of the experiment at week 11 were also determined in the presence of downstream coagulation inhibitors dabigatran (thrombin) or rivaroxaban (FXa) (n = 6). (J) Experimental liver metastasis after tail vein injection of MDA^pcDNA and MDA^FVII cells (n = 9). (K) Metastasis was also determined using qPCR at equal tumor burden (pcDNA at 11 weeks, FVII at 10 weeks; n = 8). (L) SOX9 expression in isolated lung, liver, and bone metastatic FVII cells (n = 4). Statistically significant differences were tested using t tests. Graphs show the mean and SD. Scale bars, 40 μm. DAPI, 4′,6-diamidino-2-phenylindole.