Proliferative effect of platelet on ovarian cancer cells in vitro. (A) After incubation with platelets, the proliferation rate of murine (ID8 and 2C6) and human (SKOV3 and OVCAR5) ovarian cancer cells was assessed by measuring Edu incorporation. Results are normalized to Edu positivity among buffer-exposed cells. (B) Effect of manipulating platelets on proliferative response of cancer cells was investigated by either directly incubating platelets on ID8 cells (direct) or separating them using a porous membrane (indirect). Lysed platelets were prepared by repeated applications of freeze and thaw and sonication. Platelets from tumor-bearing mice were isolated from moribund C57BL/6 female mice (n = 3) by IVC venipuncture 3-4 weeks after inoculation of ID8 cells. (C) Blocking antibodies against GPIbα (5 μg/mL), P-selectin (4 μg/mL), TGF-β1 (5 μg/mL), or GPIIb-IIIa blocking agent Eptifibatide (0.5 μM) or aspirin (15 μg/mL) was added to platelets before incubation with cancer cells, and after 24 hours Edu incorporation was measured. Results were normalized to cancer cells incubated with buffer alone. (D) Increasing concentrations of TGF-β1 blocking antibody and aspirin were used for blocking platelet-induced cancer cell proliferation. All results are compared with the second bar. (E) Knockdown of TGF-βR1 gene expression after transfection of SKOV3 human ovarian cancer cells with siRNA/liposome mixture (2 μg siRNA and 3 μL lipofectamine) was investigated by measuring protein and mRNA products, using Western blotting with anti–TGF-βR1 antibody (above) and real-time RT-PCR (below), respectively. (F) ID8 cells were transfected with either TGF-βR1 siRNA or scrambled siRNA before incubation with platelets. All results are compared with the second bar. The reduction in the proliferative response induced by scrambled siRNA was not statistically significant (P > .09). Each experiment was repeated at least 3 times in triplicates. The cumulative results are summarized as bar graphs. *P < .05 (t test). **P < .01 (t test).