Treatment with tumor-treating fields (TTFields) suppresses intercellular tunneling nanotube formation in vitro and upregulates immuno-oncologic biomarkers in vivo in malignant mesothelioma

Our plain-English summary of Treatment with tumor-treating fields (TTFields) suppresses intercellular tunneling nanotube formation in vitro and upregulates immuno-oncologic biomarkers in vivo in malignant mesothelioma, by A. Sarkari, S. Korenfeld, K. Deniz, et al (eLife, 2023).

The study

• Authors: A. Sarkari, S. Korenfeld, K. Deniz, K. Ladner, P. Wong, S. Padmanabhan, R.I. Vogel, L.A. Sherer, N. Courtemanche, C. Steer, K. Wainer-Katsir, E. Lou
• Journal: eLife
• Published: 2023;12
• Cited by: 14 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Disruption of intercellular communication within tumors is emerging as a novel potential strategy for cancer-directed therapy. Tumor-Treating Fields (TTFields) therapy is a treatment modality that has itself emerged over the past decade in active clinical use for patients with glioblastoma and malignant mesothelioma, based on the principle of using low-intensity alternating electric fields to disrupt microtubules in cancer cells undergoing mitosis. There is a need to identify other cellular and molecular effects of this treatment approach that could explain reported increased overall survival when TTFields are added to standard systemic agents. Tunneling nanotube (TNTs) are cell-contact-dependent filamentous-actin-based cellular protrusions that can connect two or more cells at long-range. They are upregulated in cancer, facilitating cell growth, differentiation, and in the case of invasive cancer phenotypes, a more chemoresistant phenotype. To determine whether TNTs present a potential therapeutic target for TTFields, we applied TTFields to malignant pleural mesothelioma (MPM) cells forming TNTs in vitro. TTFields at 1.0 V/cm significantly suppressed TNT formation in biphasic subtype MPM, but not sarcomatoid MPM, independent of effects on cell number. TTFields did not significantly affect function of TNTs assessed by measuring intercellular transport of mitochondrial cargo via intact TNTs. We further leveraged a spatial transcriptomic approach to characterize TTFields-induced changes to molecular profiles in vivo using an animal model of MPM. We discovered TTFields induced upregulation of immuno-oncologic biomarkers with simultaneous downregulation of pathways associated with cell hyperproliferation, invasion, and other critical regulators of oncogenic growth. Several molecular classes and pathways coincide with markers that we and others have found to be differentially expressed in cancer cell TNTs, including MPM specifically. We visualized short TNTs in the dense stromatous tumor material selected as regions of interest for spatial genomic assessment. Superimposing these regions of interest from spatial genomics over the plane of TNT clusters imaged in intact tissue is a new method that we designate Spatial Profiling of Tunneling nanoTubes (SPOTT). In sum, these results position TNTs as potential therapeutic targets for TTFields-directed cancer treatment strategies. We also identified the ability of TTFields to remodel the tumor microenvironment landscape at the molecular level, thereby presenting a potential novel strategy for converting tumors at the cellular level from 'cold' to 'hot' for potential response to immunotherapeutic drugs.

© 2023, Sarkari et al.

In plain English

This laboratory study tested tumor-treating fields (TTFields), low-intensity alternating electric fields, on malignant pleural mesothelioma. It did not study asbestos exposure. The authors applied TTFields to two human mesothelioma cell lines, MSTO-211H (biphasic) and VAMT (sarcomatoid), and counted tunneling nanotubes, which are thin tube-like links between cells. At 1.0 V/cm and 200 kHz, TTFields significantly reduced nanotube formation in MSTO-211H cells at 48 hours (p=0.018), but the difference was not present at 72 hours. VAMT cells showed no significant differences. In cultures seeded with 40,000 cells, viability stayed above 95% in both treated and control groups. There was no observable change in how fast mitochondria moved through nanotubes: the average was 3.32 µm/min in controls and 3.43 µm/min with TTFields. In eight mice with mesothelioma tumors treated with TTFields or a heat sham, 22 of 1812 genes analyzed were differentially expressed. The authors reported that immune-related genes such as HLA-DRB rose, while TNC, FST and HGF fell. The authors concluded that nanotubes are a potential therapeutic target for TTFields. They noted it is uncertain why only one cell line responded.

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: Laboratory study: experiments on mesothelioma cell lines in culture, cell-free actin experiments, and an animal experiment in mice with gene-expression (spatial transcriptomic) analysis of tumors
• Who was studied: Two human malignant pleural mesothelioma cell lines (MSTO-211H, biphasic; VAMT, sarcomatoid), and eight mice injected with AB1 mouse mesothelioma cells (four treated with TTFields, four with heat sham)
• Exposure: Tumor-treating fields (alternating electric fields), mainly at 1.0 V/cm and 200 kHz, with 150 kHz, 400 kHz and 0.5 V/cm also tested; some cells also received cisplatin (160 nM) and pemetrexed (24 nM). No asbestos exposure was studied.
• Main results:
  • In frequency testing on MSTO-211H cells, TTFields at 200 kHz applied in one direction gave fewer nanotubes than control by 24 hours (p=0.004), and fewer than two-direction application at 150 kHz (p=0.005).
  • TTFields at 1.0 V/cm significantly suppressed nanotube formation in the biphasic MSTO-211H cell line 48 hours after treatment began.
  • In MSTO-211H cells treated at 1.0 V/cm and 200 kHz in two directions, the difference in nanotube formation was statistically significant at 48 hours (p=0.018), but was not present at 72 hours.
  • In VAMT (sarcomatoid) cells at 1.0 V/cm, there were no significant differences between treated and control cells in nanotube formation or cell growth.
  • When 40,000 MSTO-211H cells were seeded one day before treatment, viability was above 95% in both control and treated groups, at both intensities and at all time points.
  • When cells were plated at lower density, the TTFields-treated group had an 80% reduction in cell count compared with control by 72 to 96 hours (p=0.003).
  • In cell-free experiments, TTFields made no difference to actin polymerization.
  • In MSTO-211H cells, mitochondria moved through nanotubes at an average of 3.32 µm/min in controls and 3.43 µm/min with TTFields. The authors saw no observable effect at the intensity and frequency tested.
  • Combining TTFields with cisplatin gave more pronounced nanotube suppression in MSTO-211H cells over 72 hours. This result came from a single experiment (n=1).
  • In mouse tumors, 22 of 1812 genes analyzed were differentially expressed between TTFields-treated and heat-sham tumors.
  • In TTFields-treated mouse tumors compared with heat controls, the most prominently downregulated genes were TNC, FST and HGF.
  • In TTFields-treated mouse tumors compared with heat controls, the most prominently upregulated genes were HLA-DRB, LYZ, COL1A1 and COL1A2.
  • Nanotube-like protrusions were seen in both treated and control mouse tumors. They were shorter than those in human tumor specimens and much shorter than those seen in cell culture.
• The authors' conclusion: The authors concluded that tunneling nanotubes are a potential therapeutic target for TTFields in malignant pleural mesothelioma, and possibly in other cancers.
• Limitations the authors state:
  • It is uncertain which factors are necessary for nanotube formation, and why the biphasic cell line responded to TTFields while the sarcomatoid line did not.
  • The 22 mm coverslip size forced a trade-off between plating cells too densely and plating too few cells to grow well.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Biomarker, Mesothelioma, Overall Survival, Pleural Mesothelioma, Biphasic Mesothelioma, Sarcomatoid, Tumor Microenvironment.

Related studies

• Expert guidance on prophylaxis and treatment of dermatologic adverse events with Tumor Treating Fields (TTFields) therapy in the thoracic region — M.J. Anadkat, M. Lacouture, et al. (2022)
• Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma — E. Lou, S. Fujisawa, et al. (2012)
• The transcriptomic fingerprint of cancer response to Tumor Treating Fields (TTFields) — K. Wainer-Katsir, A. Haber, et al. (2025)
• Efficacy and immunogenic effects of Tumor Treating Fields (TTFields) in preclinical models of pancreatic ductal adenocarcinoma, with and without gemcitabine/nab-paclitaxel — T. Kan, T. Haj Khalil, et al. (2026)
• Intercellular communication in malignant pleural mesothelioma: properties of tunneling nanotubes — J.W. Ady, S. Desir, et al. (2014)
• Tumor Treating Fields (TTFields) Hinder Cancer Cell Motility through Regulation of Microtubule and Acting Dynamics — T. Voloshin, R.S. Schneiderman, et al. (2020)

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