Coagulation is required for the establishment of the premetastatic niche. (A) Experimental design to study the role of TF in the premetastatic niche. C57BL/6 mice were subcutaneously implanted with B16F10-wt cells and tumors allowed to grow for 21 days. Hirudin was administered intraperitoneally (20 mg/kg) 5 minutes before and 4 hours after intravenous injection of B16F10-wt cells. Lungs were isolated after either 24 hours or 14 days after intravenous injection to score for either cell survival or number of metastatic lung nodules, respectively. (B) Immunohistochemistry against CD11b (AlexaFluor-488, green) in lung sections 14 days after the subcutaneous implantation of 5 × 103 B16F10-wt tumor cells, before either intravenous injection or hirudin treatment. CD11b+ cells per field were scored from tiled images, acquired with a confocal microscope, from naive and primary tumor-bearing mice; n = 3 mice, ≥ 15 single fields analyzed per mouse, extracted from ≥ 8 tiled images (Mann-Whitney). Scale bars represent 400 μm. (C) At 21 days after subcutaneous implantation of 5 × 103 B16F10-wt cells with Matrigel (growth factor-reduced), mice were treated with hirudin as described in panel A and intravenously injected with 2.5 × 105 CMFDA-stained B16F10-wt cells. Lungs isolated after 24 hours were sectioned, and the number of tumor cells per section was scored under an epifluorescence microscope; n = 3 mice, 9 nonconsecutive sections analyzed per mouse (Mann-Whitney). (D) At 21 days after subcutaneous implantation of 5 × 103 B16F10-wt cells, mice were treated with hirudin as described in panel A and intravenously injected with 2.5 × 105 unstained B16F10-wt cells. Lungs were isolated 14 days after and the number of metastatic lung nodules was scored; n = 3 mice (+s.c.; Mann-Whitney), n ≥ 9 mice (−s.c.; Mann-Whitney). (E-G) C57BL/6 mice were subcutaneously implanted with 5 × 103 B16F10-wt cells and pretreated with hirudin (20 mg/kg, intraperitoneally) on days 5 and 9 after palpable tumor growth (average of 12.6 days after implantation). On day 11, 2.5 × 105 B16F10-wt cells were intravenously injected, and lungs isolated 14 days later to score for metastatic lung nodules (F, n ≥ 5 mice, 1-way ANOVA and Tukey test). (G) The recruitment of CD11b cells to the premetastatic niche was analyzed by immunohistochemistry of lung sections from mice just before intravenous injection; n = 3 mice, ≥ 30 single fields analyzed per mouse (1-way ANOVA and Tukey test). (B-D,F-G) Data are mean + SD. *P < .05. **P < .01.
