Treatment with tumor-treating fields (TTFields) suppresses intercellular tunneling nanotube formation in vitro and upregulates immuno-oncologic biomarkers in vivo in malignant mesothelioma
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Curated by eLife
eLife assessment
Overall, this is an interesting topic of study, and the conclusions could be of relevance more broadly. However, mechanistic support, limited TTF frequencies/timing, and visual support of the quantitative data would be critical in order to provide convincing and rigorous support for this interesting concept.
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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.
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eLife assessment
Overall, this is an interesting topic of study, and the conclusions could be of relevance more broadly. However, mechanistic support, limited TTF frequencies/timing, and visual support of the quantitative data would be critical in order to provide convincing and rigorous support for this interesting concept.
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Reviewer #1 (Public Review):
This study is based on the hypothesis that tumor treating fields, a form of cancer therapy that exposes tumors to alternating electrical fields, has an effect on tunneling microtubes, fine actin-rich protrusions that connect cancer cells and allow intercellular communication, contributing to the tumor microenvironment and therapeutic resistance. This is an interesting hypothesis and may be of importance. To prove their hypothesis better data presentation and mechanistic studies are needed, as it is not clear based on this study how the proposed effect is working.
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Reviewer #2 (Public Review):
The authors tested TTFields' effect on TNT formation in two mesothelioma cell lines, MSTO-211H and VMAT. The MSTO-211H is a biphasic cell line with epithelioid and sarcomatoid features while VMAT only has sarcomatoid morphology. They treated their cell lines at 150 or 200 kHz either unidirectionally or bidirectionally. The experiments took place within 72 hours of plating, after which the cells will become confluent on coverslips and their TNT formation drops.
Under these experimental conditions, they found: (i) Unidirectional is more effective than bidirectional TTFields in reducing TNT formation, (ii) TNT formation was markedly reduced after 48 hours of treatment in MSTO-211H but not VMAT cells, (iii) no difference in actin polymerization or actin filament bundling after one hour of TTFields treatment, …
Reviewer #2 (Public Review):
The authors tested TTFields' effect on TNT formation in two mesothelioma cell lines, MSTO-211H and VMAT. The MSTO-211H is a biphasic cell line with epithelioid and sarcomatoid features while VMAT only has sarcomatoid morphology. They treated their cell lines at 150 or 200 kHz either unidirectionally or bidirectionally. The experiments took place within 72 hours of plating, after which the cells will become confluent on coverslips and their TNT formation drops.
Under these experimental conditions, they found: (i) Unidirectional is more effective than bidirectional TTFields in reducing TNT formation, (ii) TNT formation was markedly reduced after 48 hours of treatment in MSTO-211H but not VMAT cells, (iii) no difference in actin polymerization or actin filament bundling after one hour of TTFields treatment, (iv) reduced TNT formation when TTFields were combined with cisplatin but not with both cisplatin and pemetrexed, (v) analysis TNT cargo transport using markers of gondolas and mitochondria did not show changes in transport velocity, and (vi) in vivo spatial transcriptomic analysis revealed EMT markers and immunogenic markers.
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Reviewer #3 (Public Review):
Sarkari et al. describe the effects of TTFields on inter-cellular communication structures called tunneling nanotubes in malignant pleural mesothelioma cells. Recent studies have implicated these F-actin-based nanotubes in promoting malignant transformation and biology by allowing long-range communications between malignant cells. The authors suggest that TTFields disrupt these structures by impacting the expression of genes involved in nanotube formation and cell proliferation. Although TTFields are thought to affect tubulin-based structures, recent studies suggest that TTFields also impact actin-based structures. Therefore, the authors' findings are in keeping with this new understanding. They also found that TTFields upregulated marker genes in immunity. This is one of the first studies that implicate …
Reviewer #3 (Public Review):
Sarkari et al. describe the effects of TTFields on inter-cellular communication structures called tunneling nanotubes in malignant pleural mesothelioma cells. Recent studies have implicated these F-actin-based nanotubes in promoting malignant transformation and biology by allowing long-range communications between malignant cells. The authors suggest that TTFields disrupt these structures by impacting the expression of genes involved in nanotube formation and cell proliferation. Although TTFields are thought to affect tubulin-based structures, recent studies suggest that TTFields also impact actin-based structures. Therefore, the authors' findings are in keeping with this new understanding. They also found that TTFields upregulated marker genes in immunity. This is one of the first studies that implicate TTFields in these tunneling nanotube structures. Overall, the study adds to our understanding of TTFields on various cellular structures. However, conclusions are only partially supported by the data presented. The study is largely descriptive and there are many areas that need to be addressed to substantively improve the premise and rigors and strengthen the conclusions.
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