Abstract 4814: Tunneling nanotubes facilitate intercellular communication and trafficking between mesothelioma cells

Abstract

Abstract Introduction: Mesothelioma is an aggressive malignancy which spreads via local invasion. We have observed fine, long, non-adherent cable-like structures connecting mesothelioma cells in vitro which are consistent with tunneling nanotubes, an entity recently described in malignant as well as neuronal and immune cells. We hypothesized that tunneling nanotubes facilitate intercellular communication between distant and neighboring cells via transfer of intracellular components. Materials and Methods: Biphasic and sarcomatoid mesothelioma cells, as well as primary mesothelioma and lung cancer cells, were grown using medium of variable concentrations of serum, glucose, and cytokines at low or normal pH. Fluorescent lipophilic dyes were used to stain cytoplasmic membranes and mitochondria. Red and green fluorescent protein-expressing cells were examined following mixing of the two populations. Brightfield and fluorescent microscopy were performed, as was time-lapse imaging to determine time frames for transport via the nanotubes. To determine composition, nanotubes and cells were fixed and immunofluorescent staining was performed. Electron microscopy was also performed to elucidate nanotube structure and cell membrane insertions. Results: Nanotubes connected cells in close and distant proximity. The proportion of cells growing one or more nanotubes was highest in medium with low pH and/or low serum, high glucose, and cytokines. We have reproduced this finding in primary tumor cells from resected mesothelioma cells following surgery, as well as mesothelioma and lung cancer cells from pleural effusions. Brightfield and fluorescent microscopy revealed transfer of material (including mitochondria and proteins) between cells following nanotube formation, confirming the tubes are tunneling. Cells positive for both GFP and RFP and connected by nanotubes were observed, indicating protein transfer was bidirectional. Fluorescent immunostaining elucidated the presence of actin, vimentin, Zo-1, and fascin in nanotubes, supporting actin as a key component. Electron microscopy demonstrated the presence of early nanotubes and insertion of mature nanotubes into receptive cells. Conclusions: Tunneling nanotubes facilitate intercellular communication and trafficking between cells from cell lines and primary tumors. For the first time, we demonstrate that specific medium conditions can reproducibly promote increased growth of nanotubes to accelerate cell-to-cell communication via these structures, as well the ability to fix these sensitive structures for immunostaining. We also demonstrate that proteins and mitochondria are among cell components transported bidirectionally via nanotubes. Tunneling nanotubes provide a significant alternative for cell-to-cell communication between invasive cancer cells, as well as a plausible mechanism for tumoral resistance to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4814.