Fig 2: Platelet-derived mEVs generated during the coculture were internalized by HT29 cells. A–E: HT29 cells (1 × 106) were incubated with CFSE-loaded platelets (1 × 108) up to 20 h. At the end of the incubation, cancer cells were washed, harvested by trypsin, and resuspended in 100 µl of PBS and analyzed for the CFSE fluorescence signal by flow-cytometry (A, D) or Amnis imaging flow cytometry (B, C). A: Flow cytometry dot plots represent CFSE negative (red) and positive (purple) HT29 cells cultured alone or with CFSE-loaded platelets for 20 h, respectively. B and C: HT29 cell suspension was analyzed by Amnis imaging flow cytometry for CFSE fluorescence; the representative images of cells, which internalized CFSE + mEVs, are reported (B), and the percentage of cells with internalized CFSE + mEVs to total CFSE+ cells are shown (C), all data are shown as scatter dot plots with mean + SEM, n = 4; **P < 0.01 versus HT alone. D: HT29 cells were cocultured with CFSE-loaded platelets in the presence of vehicle (DMSO) or dm-amiloride (250 µM) for 20 h; at the end of the incubation, cancer cells were harvested and analyzed for CFSE signal by flow-cytometry after trypan blue quenching; data are reported as % of cells with internalized CFSE + mEVs versus control (HT + PLT, vehicle), all data are shown as scatter dot plots with mean + SEM, n = 4, **P < 0.01 versus vehicle. E and F: Western blot analysis of 12-LOX in HT29 cells cultured with platelets in the presence of vehicle (DMSO) or dm-amiloride (250 µM) for 20 h; the optical density of 12-LOX bands was normalized with those of ß-actin, and results are reported as % of control (vehicle), all data are shown as scatter dot plots with mean + SEM, n = 3, *P < 0.05 versus vehicle. C: Two-way ANOVA with Tukey's multiple comparisons test and (D and F) unpaired t test (two-tailed) were used.

Fig 3: Characterization of mEVs generated during the interaction between platelets and cancer cells. mEVs were isolated from the conditioned medium of HT29 cells cultured alone (HT) or with platelets (HT + PLT) and assessed by flow cytometry for the positivity to CD41, CD66, and Annexin V. A: The percentage of EVs positive to CD41 and CD66 is reported; all data are shown as scatter dot plots with mean + SEM, n = 3; **P < 0.01 versus CD41+ mEVs. B: The count of CD41+ mEVs in the medium of coculture at 2 and 20 h was assessed; results are reported as CD41+ mEV/µl, all data are shown as scatter dot plots with mean + SEM, n = 5–9; **P < 0.01 versus HT alone. C: The count of CD41+ AnnexinV+ MPs in the medium of coculture at 2 and 20 h was assessed; results are reported as CD41+ AnnexinV+ mEVs/µl, all data are shown as scatter dot plots with mean + SEM, n = 6–13; **P < 0.01 versus HT alone. D and E: Western Blot analysis of 12-LOX and ß-actin in mEVs isolated from the conditioned medium of HT + PLT (D) or from the releasate of thrombin-stimulated platelets(E); platelets (PLT) were loaded into the gels as a positive control for the 12-LOX. F: mEVs isolated from the releasate of thrombin-stimulated platelets (250,000 mEVs in 250 µl of HEPES buffer) were incubated with and without AA (30 µM) for 30 min 37°C, and the levels of 12-HETE were assessed in the medium by LC-MS/MS; results are reported as the total amount (ng), all data are shown as scatter dot plots with mean + SEM, n = 7–9; **P < 0.01 versus mEVs. A and F: unpaired t test (two-tailed) was used; (B and C) Two-way ANOVA with Tukey's multiple comparisons test was used.

Fig 4: Effects of 12-LOX inhibition on the expression of mesenchymal genes in HT29 cells cocultured with platelets. HT29 cells (HT, 1 × 106) were cultured alone or cocultured with platelets (PLT) (1 × 108) for 20 h. Twenty min before the addition of platelets, vehicle (DMSO), CDC (3–30 µM), baicalein (3 µM), and esculetin (3 µM) were added to cancer cells. The gene expression of CDH1, VIM, TWIST1, ZEB1, FIN1, and RHOA was assessed by qRT-PCR and normalized to GAPDH mRNA levels; values are reported as fold-change versus HT29 cells cultured alone, all data are shown as scatter dot plots with mean + SEM (n = 4–7). A: Concentration-dependent effects of CDC on gene expression of CDH1, VIM, TWIST1, ZEB1, FIN1, and RHOA in HT29 cells cultured with platelets: **P < 0.01 versus HT29 cells alone (multiple unpaired t test), *P < 0.05, #P < 0.01 versus HT + PLT (vehicle) (mixed-effects analysis followed by Tukey test for multiple comparisons). B: Heat map of % Control (vehicle) (mean) values of 12-HETE biosynthesis and gene expression versus increasing CDC concentrations. C: Reduction of 12-HETE biosynthesis in HT29 cell-platelet cocultures by baicalein and esculetin (3 µM); **P < 0.01 versus vehicle (n = 3–4) using one-way ANOVA and Dunnett's multiple comparisons test. D: Effects of baicalein or esculetin (3 µM) on gene expression of CDH1, VIM, TWIST1, ZEB1, FIN1, and RHOA in HT29 cells cultured with platelets; **P < 0.01 versus HT29 cells alone (multiple unpaired t test), #P < 0.01, *P < 0.05 versus vehicle (n = 4–7) (mixed-effects analysis followed by Tukey test for multiple comparisons).