Endogenous APC protects against cancer cell–induced vascular leakage in the lung through S1P1. Vascular leakage in the lungs was assessed in C56Bl/6 mice treated intraperitoneally with 200 μg of an antibody that both blocks anticoagulant and signaling properties (MPC1609; ■) and a control antibody (MCO1716; □) at 30 minutes before the administration of 1.75 × 105 B16F10 into the lateral tail vein in the absence or presence of the S1P1 agonist SEW2871 (10 mg/kg), using the Evans Blue Dye assay. Antibody administration was repeated at 48 and 96 hours after cancer cell inoculation. Mice were killed at 5 days after cancer cell inoculation. Evans Blue Dye was injected intravenously (20 mg/kg) at 3 hours before mice were killed and lungs were harvested, homogenized, and incubated at 60°C in formamide for 16 hours to extract the Evans Blue dye. Vascular leakage was subsequently assessed spectrophotometrically at a wavelength of 620 nm and is indicated as OD620 per gram of lung tissue. Error bars represent means ± SEMs (n = 7-8), ***P < .001; **P < .01.
