Tumor cells induced ATX secretion by platelets and the formation of bioactive LPA. (A) LPA production during passive coculture of breast cancer cells and human platelets. Adherent MDA-MB-231 (left) and MDA-B02 (right) cells were incubated for up to 24 hours in the presence of human platelets from 2 different donors. Data are expressed as mean fluorescence ratio value for each concentration, normalized to the percentage of LPA 18:1 response (**P < .01; ***P < .001; vs cancer cells at 0 hours using 1-way ANOVA with a Bonferroni posttest). (B) Adherent MDA-B02 cells were incubated in the presence (+) or absence (−) of human platelets in serum-free Ham’s F-12K medium with endogenous Mg²⁺ (2.1 mM). Cell proliferation was assessed by densitometry analysis after cell staining with crystal violet (left). Data are expressed as mean density (± SD) of 6 replicates and are representative of 3 experiments (right). (***P < .001; vs MDA-BO2 (−) platelets using 2-way ANOVA with a Bonferroni posttest). (C) Platelet aggregation was stimulated by collagen (10 μg/mL) or by tumor cells (B02) (10⁶ cells) at the time indicated by the arrow, in presence or absence of peptides. RGDS or RGES (100 μM) were added prior to the addition of collagen or tumor cells. Platelet aggregation was recorded over time as the percentage of light transmission. (D) Effects of inhibitors of platelet aggregation (RGDS, ReoPro, apyrase, and clopidogrel), phospholipase A1/2 activity (palmostatin B), lysoPLD activity (RG3-39 and PF-8380), and antagonist of LPA_1/3 receptor (Ki16425) on platelet-induced MDA-BO2 cell proliferation. MDA-BO2 cells were incubated for 12 hours in the presence (+) or absence (−) of human platelets. Culture media were supplemented with the indicated compounds. Data represent proliferation as the percentage of control (MDA-B02+Plat) (± SEM) of 6 replicates from 3 independent experiments (*P < .05; **P < .01; ***P < .001; vs MDA-BO2 (+) platelets using 1-way ANOVA with a Bonferroni posttest). Plat, platelets.