Tunneling nanotubes: an alternate route for propagation of the bystander effect following oncolytic viral infection

Our plain-English summary of Tunneling nanotubes: an alternate route for propagation of the bystander effect following oncolytic viral infection, by J. Ady, V. Thayanithy, K. Mojica, et al (Molecular Therapy Oncolytics, 2016).

The study

• Authors: J. Ady, V. Thayanithy, K. Mojica, P. Wong, J. Carson, P. Rao, Y. Fong, E. Lou
• Journal: Molecular Therapy Oncolytics
• Published: 2016;3:16029
• Cited by: 39 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Tunneling nanotubes (TNTs) are ultrafine, filamentous actin-based cytoplasmic extensions which form spontaneously to connect cells at short and long-range distances. We have previously described long-range intercellular communication via TNTs connecting mesothelioma cells in vitro and demonstrated TNTs in intact tumors from patients with mesothelioma. Here, we investigate the ability of TNTs to mediate a viral thymidine kinase based bystander effect after oncolytic viral infection and administration of the nucleoside analog ganciclovir. Using confocal microscopy we assessed the ability of TNTs to propagate enhanced green fluorescent protein (eGFP), which is encoded by the herpes simplex virus NV1066, from infected to uninfected recipient cells. Using time-lapse imaging, we observed eGFP expressed in infected cells being transferred via TNTs to noninfected cells; additionally, increasing fluorescent activity in recipient cells indicated cell-to-cell transmission of the eGFP-expressing NV1066 virus had also occurred. TNTs mediated cell death as a form of direct cell-to-cell transfer following viral thymidine kinase mediated activation of ganciclovir, inducing a unique long-range form of the bystander effect through transmission of activated ganciclovir to nonvirus-infected cells. Thus, we provide proof-of-principle demonstration of a previously unknown and alternative mechanism for inducing apoptosis in noninfected recipient cells. The conceptual advance of this work is that TNTs can be harnessed for delivery of oncolytic viruses and of viral thymidine kinase activated drugs to amplify the bystander effect between cancer cells over long distances in stroma-rich tumor microenvironments.

In plain English

This was a laboratory (in vitro) study of tunneling nanotubes (TNTs) — fine actin-based extensions connecting cells — using three human mesothelioma cell lines (MSTO-211H, VAMT and JMN). Cells were infected with NV1066, a replication-competent attenuated herpes simplex virus-1 carrying an eGFP transgene, and examined by confocal and time-lapse microscopy. The authors reported that infected cells still formed TNTs; in MSTO-211H cells specifically, there were significantly more TNTs per cell at 48 hours after infection than in controls. eGFP moved along TNTs from infected to noninfected cells for as long as 16 hours after infection, with fluorescence rising in recipient cells. In a modified transwell assay with a 0.4 micrometre filter separating cell populations, adding NV1066 to the top chamber gave 33% cell death in the bottom chamber at 48 hours; adding ganciclovir as well raised this to 71% (a 2.3-fold increase; P = 0.007). Adding cytochalasin B, which blocks TNT formation, lowered killing from 30% to 11% with NV1066 alone (P = 0.0016) and from 71% to 18% with NV1066 plus ganciclovir (P = 0.002). The authors described this as proof-of-principle.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory study of human mesothelioma cell lines using oncolytic virus infection, confocal and time-lapse microscopy, and a modified transwell (Boyden chamber) assay
• Who was studied: Three human malignant mesothelioma cell lines of differing histologic subtypes: MSTO-211H (biphasic), JMN, and VAMT (sarcomatoid); TNT quantification and the transwell apoptosis assay used MSTO-211H cells; work done at Memorial Sloan-Kettering Cancer Center and the University of Minnesota
• Exposure: Infection with NV1066, a replication-competent attenuated eGFP-expressing herpes simplex virus-1, given at a multiplicity of infection of 0.1, with or without the nucleoside analog ganciclovir; cytochalasin B 400 nmol/l used to block TNT formation
• Main results:
  • Infection did not prevent TNT formation; in MSTO-211H cells, at 48 hours after infection there were significantly more TNTs per cell than in controls
  • eGFP was transferred along TNTs from infected to noninfected cells for up to 16 hours after infection
  • Fluorescence per area fell in the initially infected donor cell and rose steadily in the recipient cell
  • In the transwell assay, NV1066 in the top chamber produced 33% death of initially uninfected cells in the bottom chamber at 48 hours
  • Adding ganciclovir to the virally infected cells in the top chamber raised apoptosis in the bottom-chamber recipient cells from 33% to 71%, a 2.3-fold increase (P = 0.007)
  • Positive control with virus added directly to the bottom well gave 80% apoptotic cells
  • Positive control with ganciclovir plus NV1066 added directly to the bottom well gave 95% apoptosis
  • Blocking TNT formation with cytochalasin B lowered cell killing in the bottom chamber from 30% to 11% with NV1066 alone and from 71% to 18% with NV1066 plus ganciclovir
• The authors' conclusion: The authors concluded that their work gave proof-of-principle that TNTs provide an alternate, long-range route for the bystander effect, carrying oncolytic virus and viral thymidine kinase-activated ganciclovir to noninfected cells
• Limitations the authors state:
  • No drug specifically targets TNTs, so the inhibition control used an actin-destabilizing agent (cytochalasin B) rather than a TNT-specific blocker
  • Other routes of transfer such as exosomes or microvesicles were reduced but not eliminated, and may also contribute
  • The work was in vitro; the authors state the effects still need to be studied in vivo

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Apoptosis, Tumor Microenvironment.

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