Figure 5 from HuR-Regulated Extracellular Vesicles Promote Endothelial Cell Remodeling in Pancreatic Cancer

Tumor-intrinsic HuR directly alters endothelial cell function via EV signaling. A, Schematic of the PalmGRET reporter labeling the inner leaflet of all membranes with GFP-nLuc via integration of a palmitoylation sequence. The construct for the PalmGRET EV reporter labels the inner leaflet of all cellular membranes and secreted membranous particles. B, PalmGRET-expressing cells produce GFP⁺ EVs detected by fluorescent nanoparticle tracking analysis (n = 3). C, Cell growth of PalmGRET-expressing KPC cells over 6 days. D, Immunoblot of cell lysates utilized for mouse studies probed for HuR, nLuc, and loading control vinculin. E, PalmGRET-expressing cells were orthotopically implanted into C57BL6 mice, and tumors were harvested at 14 days for flow cytometry. Levels of EV import (GFP MFI) in endothelial cells (CD45⁻CD31⁺) were equal in WT and HuR-KO tumors (n = 7). F, Total endothelial cell (CD45⁻CD31⁺) presence as quantified by cells per gram of tumor in WT vs. HuR-KO tumors (n = 7). G, ICAM-1 geometric mean surface expression of ICAM-1 in endothelial cells (CD45⁻CD31⁺) of WT vs. HuR-KO tumors (n = 7). H, ICAM-1 geometric mean surface expression (MFI) of ICAM-1 in endothelial cells of WT vs. HuR-KO tumors that have (GFP⁺) or have not (GFP⁻) imported PDAC EVs (n = 7). P values were calculated using an unpaired two-tailed Student t test or an ordinary one-way ANOVA (H only). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. MFI, mean fluorescence intensity.