polyP on EV derived from cancer cells mediates ligand binding. (A) The amount of polyP associated with cancer cell–derived EV was estimated by comparison with a standard curve (supplemental Figure 2A). (B) Detection of polyP on L3.6-derived EVs following incubation with buffer (HBS), DNase I and RNase A (D/R), or CIP. polyP of different chain lengths was used to estimate the size of EV-associated polyP. L3.6-derived EVs were preincubated with buffer or E coli PPX, and the binding of PPX-Δ12–AF488 (C), anti-CD81–AF488 (D), and or FXII-AF488 (E) was measured. The number of total EV was determined in scatter mode, and the number of ligand-binding EV was determined in fluorescence mode using a 488-nm excitation laser. The differences in fluorescent particle numbers were compared by estimating the area under each curve (AUC) using GraphPad Prism 8. Bars represent means ± SEM. ***P < .001, 1-way ANOVA with multiple comparisons. L, long; M, medium; S, short.