Failure to detect functional transfer of active K-Ras protein from extracellular vesicles into recipient cells in culture

Exosomes, extracellular nanovesicles that carry nucleic acids, lipids, and proteins, have been the subject of several studies to assess their ability to transfer functional cargoes to cells. We recently characterized extracellular nanovesicles released from glioblastoma cells that carry active Ras in complex with proteins regulating exosome biogenesis. Here, we investigated whether a functional transfer of Ras from exosomes to other cells can initiate intercellular signaling. We observed that treatment of serum-starved, cultured glioblastoma cells with exogenous glioblastoma exosomes caused a significant increase in cellular viability over time. Moreover, we detected fluorescent signal transfer from lipophilic dye-labeled exogenous glioblastoma exosomes into cultured glioblastoma cells. To probe possible signaling from cell-to-cell, we utilized bimolecular luciferase complementation to examine the ability of K-Ras in exosomes to interact with the Raf-Ras Binding domain (Raf-RBD) expressed in a recipient cell line. Although the K-Ras/Raf-RBD interaction was readily detectable upon co-expression in a single cell line, or following lysis of co-cultured cell lines separately expressing K-Ras and RBD, bearing in mind the limitations of our assay, we were unable to detect the interaction in the intact, co-cultured cell lines or upon treatment of the Raf-RBD-expressing cells with exosomes containing K-Ras. Furthermore, HA-Tag-BFP fused to the K-Ras hypervariable region and CAAX sequence failed to be transferred at significant levels from extracellular vesicles into recipient cells, but remained detectable in the cell supernatants even after 96 hours of culture of naive cells with extracellular vesicles. We conclude that if transfer of functional K-Ras from extracellular vesicles into the cytoplasm of recipient cells occurs, it must do so at an extremely low efficiency and therefore is unlikely to initiate Ras-ERK MAP kinase pathway signaling. These results suggest that studies claiming functional transfer of protein cargoes from exosomes should be interpreted with caution.