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.
Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions.