In this study, we demonstrated that hypoxic conditions stimulated an increase in tunneling nanotube (TNT) formation in chemoresistant ovarian cancer cells (SKOV3, C200). We found that suppressing the mTOR pathway using either everolimus or metformin led to suppression of TNT formation in vitro, verifying TNTs as a potential target for cancer-directed therapy. Additionally, TNT formation was detected in co-cultures including between platinum-resistant SKOV3 cells, between SKOV3 cells and platinum-chemosensitive A2780 cells, and between SKOV3 cells cultured with benign ovarian epithelial (IOSE) cells; these findings indicate that TNTs are novel conduits for malignant cell interactions and tumor cell interactions with other cells in the microenvironment. When chemoresistant C200 and parent chemosensitive A2780 cells were co-cultured, chemoresistant cells displayed a higher likelihood of TNT formation to each other than to chemosensitive malignant or benign epithelial cells. Hypoxia-induced TNT formation represents a potential mechanism for intercellular communication in ovarian cancer and other forms of invasive refractory cancers.

Malignant pleural mesothelioma is a particularly aggressive and locally invasive malignancy with a poor prognosis despite advances in understanding of cancer cell biology and development of new therapies. At the cellular level, cultured mesothelioma cells present a mesenchymal appearance and a strong capacity for local cellular invasion. One important but underexplored area of mesothelioma cell biology is intercellular communication. Our group has previously characterized in multiple histological subtypes of mesothelioma a unique cellular protrusion known as tunneling nanotubes (TnTs). TnTs are long, actin filament-based, narrow cytoplasmic extensions that are non-adherent when cultured in vitro and are capable of shuttling cellular cargo between connected cells. Our prior work confirmed the presence of nanotube structures in tumors resected from patients with human mesothelioma. In our current study, we quantified the number of TnTs/cell among various mesothelioma subtypes and normal mesothelial cells using confocal microscopic techniques. We also examined changes in TnT length over time in comparison to cell proliferation. We further examined potential approaches to the in vivo study of TnTs in animal models of cancer. We have developed novel approaches to study TnTs in aggressive solid tumor malignancies and define fundamental characteristics of TnTs in malignant mesothelioma. There is mounting evidence that TnTs play an important role in intercellular communication in mesothelioma and thus merit further investigation of their role in vivo.

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.

Mutated forms of the RAS oncogene drive 30% of all cancers, but they cannot be targeted therapeutically using currently available drugs. The molecular and cellular mechanisms that create a heterogenous tumor environment harboring both mutant and wild-type RAS have not been elucidated. In this study, we examined horizontal transfer of mutant KRAS (Kirsten Rat Sarcoma Virus) between colorectal cancer (CRC) cells via a direct form of cell-to-cell communication called tunneling nanotubes (TNTs). TNT formation was significantly higher in CRC cell lines expressing mutant KRAS than CRC cell lines expressing wild-type RAS; this effect was most pronounced in metastatic CRC cell lines with both mutant KRAS and deficiency in mismatch repair proteins. Using inverted and confocal fluorescence time-lapse and fluorescence recovery after photobleaching (FRAP)-based microscopy, we observed GFP-tagged mutant KRASG12D protein trafficking between CRC cells through TNTs within a span of seconds to several minutes. Notably, acquisition of mutant KRAS increased Extracellular Signal-regulated Kinase (ERK) phosphorylation and upregulated tunneling nanotube formation in recipient wildtype CRC cells. In conclusion, these findings suggest that intercellular horizontal transfer of RAS can occur by TNTs. We propose that intercellular transfer of mutant RAS can potentially induce intratumoral heterogeneity and result in a more invasive phenotype in recipient cells.