Clot formation contributes to endothelial activation in pulmonary metastasis. (A) C57BL/6-wt or Mac1 KO mice were intravenously injected with 5 × 105 CMFDA-stained B16F10 tumor cells (green). Lungs were harvested 8 hours later and assessed for VCAM-1 expression (red, Alexa Fluor 633; imaged with a confocal microscope). Percentage of tumor cells associated with VCAM-1 expression is shown; n = 3 mice (Mann-Whitney). (B) BALB/c or SCID mice were treated with hirudin and intravenously injected 5 × 105 4T1-GFP or 1205Lu-GFP tumor cells, respectively. Eight hours after the introduction of tumor cells, lungs were harvested and assessed for VCAM-1 expression (red, Alexa Fluor 633). Representative images, acquired with a confocal microscope, are shown. Percentage of tumor cells associated with VCAM-1 expression was analyzed; n ≥ 4 mice (Mann-Whitney for each cell line). (C) C57BL/6 mice were intravenously injected 5 × 105 CMFDA-stained-B16F10-wt, -B16F10-TFPI, or -B16F10-vector tumor cells (green). Lungs were harvested 8 hours after and assessed for VCAM-1 expression (red, Alexa Fluor 633). Representative images, acquired with a confocal microscope, are shown. Percentage of tumor cells associated with VCAM-1 expression was analyzed; n = 3 mice (one-way analysis of variance and the Tukey test). In (A-C), data represent mean + SD and *P < .05; **P < .01. Scale bars represent 50 µm.
