Abstract 1528: Tunneling Nanotubes: a new approach to studying intercellular communication in aggressive solid tumor malignancies.

Background: Intercellular communication is critical to malignant progression, invasion, and recurrence, yet surprisingly little is known about the mechanisms of cell-cell communication in the complex heterogeneous tumor microenvironment. Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as short and long-range conduits for transport of cellular cargo between connected cells. We have studied these unique structures in mesothelioma and lung cancer, and further confirmed that they can form and connect cells derived from cell lines and also from primary human cells from cancers of the breast, ovary, cervix, prostate, pancreas, bone (osteosarcoma), and brain (glioblastoma), indicating that nanotubes are a potential mechanism of cell communication across cancer types. We have previously demonstrated that TnTs are present in tumor specimens resected from human patients with malignant pleural mesothelioma and lung adenocarcinomas, and that they can mediate transport of mitochondria, proteins, and Golgi vesicles between connected cells. Time-lapse confocal microscopy demonstrated that TnTs can form de novo between mesothelioma cells over several hours. Exosomes - small membrane-derived nanovesicles measuring 30-100 nm in diameter - are also under extensive investigation as critical signals for communication and cell growth in cancer. We hypothesized that co-culturing exosomes with mesothelioma cells would provide a chemotactic stimulus for TnT formation. Cholesterol accumulation has been proposed to have a direct effect on TnTs; cholesterol-based microdomains known as lipid rafts have been associated with exosome signaling as well. In the current study, we also explored lipid raft expression in the context of TnTs in mesothelioma. Results: We examined whether addition of exogenous exosomes induced increased formation of tunneling nanotubes. Mesothelioma cells co-cultured in vitro with exogenous exosomes formed more TnTs than cells cultured without exosomes by 48 hours. Average number of nanotubes was higher when exosomes were co-cultured in acidic conditions as compared to non-acidic culture conditions, but this was only statistically significant within the first 24 hours. Electron microscopy revealed that exosomes were located at the base of and co-localized with TnTs, as well as being present in the extracellular environment. Mesothelioma cells connected via nanotubes were enriched with lipid rafts. Conclusions: Our findings implicate exosomes as potential chemotactic stimuli for tunneling nanotube formation. Future studies will further examine the role of lipid rafts in TnT formation and function. Citation Format: Emil Lou, Victor Babatunde, Sho Fujisawa, Afsar Barlas, Andre L. Moreira, Venugopal Thayanithy, Subbaya Subramanian, Robert Downey, Katia Manova-Todorova, Malcolm A.S. Moore. Tunneling Nanotubes: a new approach to studying intercellular communication in aggressive solid tumor malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1528. doi:10.1158/1538-7445.AM2013-1528