Intercellular communication plays a critical role in the ever-evolving landscape of invasive cancers. Recent studies have elucidated the potential role of tunneling nanotubes (TNTs) in this function. TNTs are long, filamentous, actin-based cell protrusions that mediate direct cell-to-cell communication between malignant cells. In this study, we investigated the formation of TNTs in response to variable concentrations of the chemotherapeutic drug doxorubicin, which is used extensively in the treatment of cancer patients. Doxorubicin stimulated an increased formation of TNTs in pancreatic cancer cells, and this occurred in a dose-dependent fashion. Furthermore, TNTs facilitated the intercellular redistribution of this drug between connected cells in both pancreatic and ovarian cancer systems in vitro. To provide supportive evidence for the relevance of TNTs in pancreatic cancer in vivo, we performed multiphoton fluorescence microscopy and imaged TNTs in tumor specimens resected from three human patients with pancreatic adenocarcinoma, and one with neuroendocrine carcinoma. In sum, TNT formation was upregulated in aggressive forms of pancreatic carcinoma, was further stimulated after chemotherapy exposure, and acted as a novel method for drug efflux. These findings implicate TNTs as a potential novel mechanism of drug resistance in chemorefractory forms of cancer.

Intercellular communication is vital to the ecosystem of cancer cell organization and invasion. Identification of key cellular cargo and their varied modes of transport are important considerations in understanding the basic mechanisms of cancer cell growth. Gap junctions, exosomes, and apoptotic bodies play key roles as physical modalities in mediating intercellular transport. Tunneling nanotubes (TNTs)—narrow actin-based cytoplasmic extensions—are unique structures that facilitate direct, long distance cell-to-cell transport of cargo, including microRNAs, mitochondria, and a variety of other sub cellular components. The transport of cargo via TNTs occurs between malignant and stromal cells and can lead to changes in gene regulation that propagate the cancer phenotype. More notably, the transfer of these varied molecules almost invariably plays a critical role in the communication between cancer cells themselves in an effort to resist death by chemotherapy and promote the growth and metastases of the primary oncogenic cell. The more traditional definition of “Systems Biology” is the computational and mathematical modeling of complex biological systems. The concept, however, is now used more widely in biology for a variety of contexts, including interdisciplinary fields of study that focus on complex interactions within biological systems and how these interactions give rise to the function and behavior of such systems. In fact, it is imperative to understand and reconstruct components in their native context rather than examining them separately. The long-term objective of evaluating cancer ecosystems in their proper context is to better diagnose, classify, and more accurately predict the outcome of cancer treatment. Communication is essential for the advancement and evolution of the tumor ecosystem. This interplay results in cancer progression. As key mediators of intercellular communication within the tumor ecosystem, TNTs are the central topic of this article.

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Tumor Treating Fields (TTFields) is a novel therapeutic strategy that uses alternating electric fields to disrupt mitosis in actively dividing cells through exertion of dielectrophoretic force and dipole alignment on microtubule subunits. However, the additional effects of TTFields on cellular morphology and communication remain unclear. Tunneling nanotubes (TNTs) are ultrafine F-actin-based protrusions that facilitate intercellular communication through cell-cell contact, including efficient transport of molecular cargo that accelerate invasive potential and chemoresistance. We hypothesized that by creating dielectrophoretic force on polar actin subunits, treatment with TTFields would lead to sustained disruption or prevention of formation of MPM TNTs. TTFields (200 kHz) were applied at 0.5 or 1.0 V/cm to VAMT and MSTO MPM cell lines using the Inovitro system (Novocure). TNT index (average # of TNTs/cell) was determined at 0, 24, 48, and 72 hours of TTFields application. At the 72 hour period, TTFields were discontinued and assessment for recovery of TNT formation was performed after an additional 24 hours. Cell viability was determined by staining with NucGreen 488 dye. We also used time-lapse microscopy with concurrent application of TTFields and the chemotherapeutic agents cisplatin and pemetrexed to analyze effects on TNTs and functional cargo transfer. Application of continuous TTFields at 1.0 V/cm, but not at 0.5 V/cm, suppressed TNT formation by 48.9% in MSTO (p=0.005). This suppression was achieved at the 48-hour time point and was independent of cell proliferation. No significant differences in TNT index were noted for VAMT. Cell viability was consistently above 95% at all time points for both cell lines at the stated frequency and intensities. Cargo transfer rates were lower in experimental groups treated with TTFields and either cisplatin, pemetrexed, or both. Here, we show that treatment with TTFields suppresses formation of TNTs between MSTO cells, but not VAMT, suggesting additional factors that may determine susceptibility to TTFields treatment. Additionally, TTFields treatment of MSTO decreased the function of TNTs in these cells, as demonstrated by lower cargo transfer rates. In sum, these data identify effects of TTFields on TNTs as a novel mechanism for this therapeutic modality. Citation Format: Akshat Sarkari, Sophie Korenfeld, Katherine Ladner, Phillip Wong, Antonia Martinez, Eyal Dor-On, Moshe Giladi, Amrinder Nain, Emil Lou. Tumor treating fields induce cellular and morphologic changes including disruption of intercellular communication networks in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2011.

The purpose of this study was to evaluate the effects of tumor-treating fields (TTFields) on tunneling nanotubes (TNTs) in malignant mesothelioma. TTFields have emerged as a therapeutic modality for the treatment of glioblastoma, through application of alternating electric fields, which exert dipole alignment and dielectrophoretic force. The dielectrophoretic force is intensified in cellular compartments that are non-uniform in size and shape such as the mitotic furrow. TNTs are filamentous actin protrusions that are conduits for intercellular communication and transport of vital cell signals that stimulate cell growth, invasion, and resistance to treatment. TNTs are highly prevalent in malignant pleural mesothelioma. We hypothesized that by creating dielectrophoretic force, TTFields may disrupt or prevent the formation of mesothelioma TNTs and thus sensitize these cells to the cytotoxic effect of chemotherapy and TTFields. TTFields (1.1-1.6 V/cm; 150-200 kHz) were applied using the inovitro system to VAMT and MSTO malignant mesothelioma cell lines. TNT index (average # of TNTs/cell) was determined at 24, 48, and 72 hours of TTFields application. Cell viability assessment using evaluation of cell count and colony formation assays were performed to determine effects of TTFields either alone or in combination with varying concentrations of cisplatin (range 0-10 µM) or pemetrexed (0-512 nM). In separate experiments, we examined the effects of cell cycle inhibition on TNTs using the compound AZD 5438 (Tocris Biosciences). Application of continuous TTFields suppressed TNT formation by 60% over a 72-hour period. This suppression was achieved by the 24-hour timepoint, and maintained over the 72 hours. Assessment of cell viability showed high rate of cell death at the stated frequencies and intensities. The addition of cisplatin or pemetrexed to TTFields application significantly decreased the cell count. The combination of TTFields and low concentration cisplatin (0.1-1 µM) resulted in enhanced treatment efficacy as evaluated using the colony formation assay. Complete inhibition of the cell cycle paradoxically stimulated a significantly higher number of TNTs. Here, we show that TTFields application suppresses TNTs formation in malignant mesothelioma cell lines. Treatment with TTFields leads to enhanced treatment efficacy when combined with standard of care chemotherapeutic drugs cisplatin and pemetrexed. Additional optimization of the applied TTFields intensities and frequencies may further improve treatment efficacy. The sharp rise in TNTs seen following cell cycle inhibition is a potential cellular stress response to cancer-directed treatment. Suppression of TNT formation, and thus TNT-mediated intercellular communication networks in tumors, is a potential mechanism of TTF efficacy in treatment of mesothelioma, glioblastoma, and other invasive cancers. Citation Format: Akshat Sarkari, Michal Munster, Einav Zeevi, Moshe Giladi, Emil Lou. In vitro application of tumor-treating fields to suppress tunneling nanotubes in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5156.