PDPN is released from glioma cells as cargo of EVs. (A) Detection of PDPN immunoreactivity in glioma cells and EVs; highly positive U373P cells release ample PDPN⁺ EVs relative to PDPN-downregulated U373vIII and U87vIII cell lines. Lower levels of PDPN expression in U87P cells result in the absence of PDPN signal in EVs (n = 3). (B) Immunogold staining and electron microscopy of U373P EVs for PDPN (10-nm gold particles, black arrows) and exosomal marker CD63 (5-nm gold particles, white arrowheads). Multiple small EVs (≤100 nm) stain for both PDPN and CD63 (n = 2). (C) Glioma EVs float at exosomal density in the iodixanol gradient (F4-F5) and commonly express multiple EV markers (flotillin-1, CD81, syntenin, and CD9). EVs released from EGFRvIII⁻ (U373PT) cells carry PDPN, but not TF, whereas their isogenic counterparts from EGFRvIII⁺ cells (U373vIII) carry TF, but not PDPN (n = 3). (D) Single EV nanoflow cytometry of EV populations from glioma cells expressing PDPN alone (U373PT), PDPN and TF (U373TF-G11-PT), or TF alone (U373vIII-PT). EVs are heterogenous, but their subsets coexpress CD81, PDPN, and TF (n = 5) (supplemental Methods).