Clot formation induced by TF extracellular domain is essential for tumor cell survival. (A) Immunoblot detection of TF extracellular and cytoplasmic domains in extracts from human and murine melanoma cell lines. Representative Western blots from 3 independent experiments are shown. (B) Ex vivo assays of platelet aggregation. The 10⁴ CMFDA-stained human melanoma cells (green) were seeded on collagen type I-biocoated multichamber slides. The 30 × 10⁶ PKH26-stained platelets (red), isolated from SCID mice, were added and incubated for 2 hours. After fixation and 4,6-diamidino-2-phenylindole staining (blue), images were acquired with an epifluorescence microscope. Representative images of 1 of the 3 independent experiments performed are shown. Number and area of platelet aggregates associated with the cells were scored (15 cells per group were analyzed). (C-E) In vivo assays of clot formation (C-D) and cell survival assays (E). SCID (C and E left panels), Cx₃cr1^gfp/+ (D), or C57BL/6 (E right panel) mice were intravenously injected with 2.5 × 10⁵ CMFDA-stained human melanoma cells (green, C and E left panels), or with 5 × 10⁵ CMAC (green, D) or CMFDA (E right panel) stained murine melanoma cells and, on the opposite tail vein, with 9 × 10⁹ PKH26-stained platelets (red), isolated from SCID (C) or C57BL/6 (D) mice. At the indicated times, lungs were isolated and imaged as intact organ with an epifluorescence (C,E) or a confocal (D) microscope. Representative images from the 2-hour time point, of 1 of the 3 independent experiments performed, are shown (C-D top panels). Tumor cell association with clots (C-D middle panels), the clot area or volume at the 2-hour time point (C-D bottom panels, ≥ 16 human and ≥ 30 murine cells with clot were analyzed), and the total number of tumor cells observed from images of consecutive fields of the entire left lobe of the lungs (E) were scored. n = 3 mice for panel C middle panel (1-way ANOVA and Tukey test for 2 hours, Mann-Whitney for 24 hours). n = 3 mice for panel D (middle panel; Mann-Whitney). n = 3 mice for panel E (1-way ANOVA, left panel 2 hours; Mann-Whitney, left panel 24 hours, and right panel). A χ² test showed a significant correlation (P < 1.7 × 10⁻¹⁶) between clot formation at 2 hours (C middle panel) and cell survival at 24 hours (E left panel). (F) Cx₃cr1^gfp/+ mice were treated with hirudin (20 mg/kg), given intraperitoneally 5 minutes before and 4 hours after the intravenous injection of 5 × 10⁵ CMAC-stained B16F10-wt cells (green) and, on the opposite tail vein, 9 × 10⁹ PKH26-stained platelets (red), isolated from C57BL/6 mice. Lungs were isolated after 8 hours and imaged as intact organ with a confocal microscope. Percentage of tumor cells with clot, volume of the clots (≥ 15 cells with clot were analyzed), and the total number of tumor cells per field were scored; n = 3 mice (Mann-Whitney). (C-F) Data are mean + SD. *P < .05. **P < .01. (B-D,F) Scale bars represent 50 μm.