Tunneling Nanotube Conduits Facilitate The Bystander Effect After Oncolytic Viral Infection

Background: Oncolytic viruses have come to the forefront of cancer therapeutics following FDA approval for treatment of metastatic melanoma in 2015. Efficacy of viral therapy is enhanced by the bystander effect, a cellular phenomenon that amplifies the effects of the virus following activation of the prodrug ganciclovir (GCV) by virally-expressed thymidine kinase (TK) and intercellular spread of GCV via gap junctions. In the complex and stroma-rich tumor microenvironment, gap junctions may not completely account for cell-to-cell communication. Tunneling nanotubes (TNTs) are a novel and recently characterized alternative form of direct cell-to-cell communication in the tumor matrix. TNTs are fine, long, F actin-based cell extensions that serve as short and long-range conduits for efficient transfer of cellular cargo. Here, we investigate the ability of TNTs to mediate a TK-based bystander effect after oncolytic viral infection and administration of GCV. Methods: We infected 3 mesothelioma cell lines with NV1066, a mutant replication-competent strain of herpes simplex virus-1 (HSV-1) that encodes viral eGFP and viral TK. Confocal microscopy and time-lapse imaging were performed 12-36 hours later. A modified Transwell assay was used to separate infected cells from uninfected cells to assess TNT propagation of eGFP-tagged NV1066. GCV was added to infected cells in the top chamber to assess TNT propagation of TK-activated GCV. Apoptosis was measured using TUNEL assay in the bottom chamber to quantify the extent of the bystander effect. Results: Confocal microscopy demonstrated effective intercellular transfer of GFP-tagged virus between cells via TNTs prior to oncolysis. Quantification of TUNEL-positive cells at 48 hours indicated that addition of NV1066 to the top chamber resulted in 33% of the initially uninfected cells in the bottom chamber dying by 48 hours. The addition of GCV to virally infected cells in the top chamber significantly increased apoptosis in recipient cells in the bottom chamber, from 33% to 71%, producing a 2.3-fold increase in cell killing attributed to TNT transfer of viral TK-activated GCV (p = 0.007). Thus, TNTs were shown to transfer viral TK-activated GCV to non-infected cells, leading to cell death via a long-range form of the bystander effect. Conclusions: Here we demonstrate that TNTs provide a previously unknown and alternative mechanism for the bystander effect in which viral TK-activated GCV is transferred via TNTs and induces apoptosis in non-infected recipient cells. Citation Format: Emil Lou, Justin Ady, Venugopal Thayanithy, Kelly Mojica, Joshua Carson, Prassanna Rao, Yuman Fong. Tunneling nanotube conduits facilitate the bystander effect after oncolytic viral infection. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 929.