Redox-signaling transmitted in trans to neighboring cells by melanoma-derived TNF-containing exosomes.

Hydrogen peroxide is known to be involved in redox signaling pathways that regulate normal processes and disease progression, including cytokine signaling, oxidative stress, and cancer. In studies on immune surveillance against cancer, hydrogen peroxide was found to disrupt cytotoxic T-cell function, thus contributing to tumor escape. In this study, secretion of TNF-containing vesicles of rab9+ endosomal origin, termed exosomes, was investigated using GFP-TNF constructs. We observed a polarized intracellular trafficking and apical secretion of TNF-positive nanovesicles. Cell-to-cell transfer of TNF was observed in exosomes in real-time microscopy, occurring separate from the melanin/melanosome compartment. Exosomes were prepared by ultracentrifugation or immunoisolation on anti-beta2-microglobulin magnetic beads. TNF as well as TNF receptors 1 and 2 were present in the exosomes as determined by Western blot, flow cytometry, and deconvolution microscopy. The functional significance of melanoma-derived exosomes was established by their signaling competence with ability to generate significantly higher ROS levels in T cells compared with sham exosomes (P=0.0006). In conclusion, we report here, for the first time, that TNF is found in tumor cell-derived exosomes and that these exosomes transmit redox signaling in trans to neighboring cells. The results are of importance for a better understanding of tumor escape mechanisms.