Aspirin treatment of platelets inhibits tumor cell IL-8 and invasion. Platelets were pretreated with 100 μM aspirin (ASA) or vehicle control for 1 hour, washed to remove residual aspirin, and then activated with TRAP (5 μM) or breast tumor cells (3 × 10⁶/mL). (A) Platelet activation status was determined by p-selectin staining, using flow cytometry. (B) MDA-MB-231 tumor cells were treated with aspirin-treated platelets, lysed, run on an Akt signaling array, and compared with array results from Figure 4A. (C) MDA-MB-231 or MCF-7 cells were exposed to releasates from aspirin-treated or control platelets, and IL-8 release from tumor cells was measured by ELISA at 24 hours. The effect of aspirin pretreatment on the metastatic potential of APR was tested using transwell invasion assays. In addition, 1 ng/mL rhIL-8 was added to TRAP-activated releasates from aspirin-treated platelets. Representative 10× images are shown in panel D, and quantification of MDA-MB-231 invasion is shown in panel E. One value for TRAP-APR was considered an outlier (17.2-fold, with standard deviation from the mean >2.5) and removed from graphical representation and statistical analysis. P values were determined by unpaired Student t tests. n = 3 to 7 independent replicates per treatment group, with the exception of the Akt signaling array, which represents a single experiment. Scale bars represent 100 μm. The dashed black line represents invasion on incubation with resting platelet releasate.