Fig. 5

HNSCC EVs promote platelet aggregation through activation of the coagulation cascade mediated by Tissue Factor (CD142). (A) Flow cytometric analysis of TF (CD142) expression on EVs isolated from SAS (n = 3), UD5 (n = 4) and Raji cells (n = 3). The left panels display representative histograms of CD142-PE staining (colored peaks) compared to the matched isotype controls (black lines) from SAS EVs, UD5 EVs and Raji EVs. The data are normalized to mode for comparison. The right panels summarize the MFI of TF expression normalized to the isotype control for each EV-type. (B) Western blot analysis of TF (CD142) protein expression in SAS cells, SAS EVs, UD5 cells and UD5 EVs. The molecular weight markers (in kDa) indicate bands corresponding to TF (~ 47 kDa) and GAPDH (~ 36 kDa, loading control). (C) The effect of Raji-derived EVs on PLT aggregation was evaluated using aggregometry. PLTs were suspended in Tyrode’s buffer containing 2 mM Ca²⁺ (+) or without Ca²⁺ (-), and aggregation was monitored following the addition of Raji-derived EVs (60 µg/mL) at 300 s. TRAP was used as positive control to induce PLT aggregation, while PBS was considered as negative control (n = 3). (D) Inhibition of SAS- and UD5-derived EV-induced PLT aggregation following mAb-mediated blockade of TF (CD142). SAS (n = 3–9) and UD5 EVs (n = 3–4) were first pre-incubated with CD142 mAb (1 µg/mL and 10 µg/mL), and IgG1 as a matched isotype was used as a negative control. The pre-incubated EVs (60 µg/mL) were subsequently added to PLTs in the aggregometer at the 300 s time point. (E) Representative images of TF (CD142) internalization analyzed by Imaging flow cytometry. PLTs were incubated with SAS-derived EVs (60 µg/mL) for 5 min and analyzed across three channels: bright field (Ch01), TF-associated SAS-derived EVs labeled with CD142-PE (yellow, Ch03), and PLTs labeled with CD41-BV421 (purple, Ch07). The interaction between PLTs and EVs was compared to an unstained control. The scale bar (dashed line) is 7 μm. Data are presented as mean + SD. Statistical significance is as follows: ns-p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001. Mann-Whitney test for (A, first and third graph) and unpaired Student’s t-test for (A, second graph). One-way ANOVA followed by Tukey’s post-hoc test for (C) and (D, second graph). Kruskal-Wallis test followed by Dunn’s post-hoc test for (D, first graph)