Background: Recently, exciting reports have outlined the existence and function of very thin, membranous conduits between cells that span significant distances, termed tunneling nanotubes (TnTs). TnTs have been observed in neoplastic and normal cells, and may transport soluble proteins, such as P-glycoprotein (MDR1), lipids, and whole organelles, including mitochondria and Golgi vesicles, between cells. Furthermore, TnTs mediate intercellular Ca2+ signals and transmit electrical potentials. However, their functional characterization remains incomplete.
Objectives: Our objective is to describe TnT formation and function in melanoma cell lines and primary tumor samples.
Methods: We describe the presence and composition of TnTs between cells in the melanoma cell lines SK-Mel-31, DM440, DM443, DM738, and DM598 in vitro with a combination of brightfield and fluorescent microscopy techniques. TnT formation is assessed in response to hypoxia, glucose stress, and low pH. Bulk intercellular transport of mitochondria is quantified via flow cytometry using MitotrackerTM dyes, and GFP-GRP78 fusion proteins are used for studies of protein transport. Additional flow cytometric studies include JC-1 staining to measure mitochondrial membrane potential, annexin V binding to measure apoptosis, and 7-AAD exclusion for live/dead staining. Fluorescent ATP assays are used to measure general metabolic capacity, and confocal microscopy of primary melanoma specimens is performed using melanosome-specific (P100) and dsDNA (H33342) stains.
Results All tested cell lines formed TnT-like structures with variable F-actin and tubulin composition. Mitochondria and Golgi vesicles were visible within live and fixed TnTs, which were formed constitutively and were further induced by hypoxia, glucose stress, and low pH. In an actin-dependent process, cells rapidly transferred labeled mitochondria, achieving homogeneity within 24 h as measured by flow cytometry. Transfer of healthy mitochondria from unstressed cells significantly mitigated the acute toxicity of chemotherapeutically damaged cells. GRP78 was also rapidly transferred between cells, and the transfer of GRP78 from healthy donor cells rescued cells damaged by chemotherapeutics as measured by a decreased apoptotic rate.
Conclusions: Here, we report for the first time, the characterization of TnTs between melanoma cells. Moreover, melanoma cells acquired primary and secondary chemotherapeutic resistance to alkylating agents melphalan and temozolomide and the BRAFV600E inhibitor, vemurafenib, via bulk intercellular transfer of functional mitochondria and the ER stress protein, GRP78. TnT inhibitors would represent a novel class of chemotherapeutics with implications for invasion, metastasis, and chemotherapeutic resistance.
Citation Format: Gustaaf G. de Ridder, Rupa Ray, Salvatore V. Pizzo. Tunneling nanotubes connect melanoma cells and mediate transfer of mitochondria and GRP78. [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 5248. doi:10.1158/1538-7445.AM2013-5248
Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.