Cellular and Molecular Networking Within the Ecosystem of Cancer Cell Communication via Tunneling Nanotubes

Abstract

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.

Keywords: angiogenesis; cancer ecosystems; cancer pathophysiology; intercellular communication; intercellular transfer; tumor microenvironment; tumor microtubes; tunneling nanotubes.

Figure 1

Hypoxia induces TNT formation in colon cancer cells, independent of alterations in cell proliferation. Graphs demonstrate changes in average number of TNTs at 24 h, 48 h, and 72 h (top row) under normoxic vs. hypoxic conditions and average optical density as a surrogate for cell proliferation at the same time points (bottom row) for the colon cancer cell lines DLD-1, HCT-116, and SW-480 and for the comparison of the fibroblast cell line NIH 3T3. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 2

TNTs induced by hypoxia stimulate a potential positive feedback loop by mediating intercellular transfer of HIF-1α and VEGF. Heterotypic forms of TNTs include malignant cell-endothelial cell TNT formation, as well as potential TNT formation among clusters of platelets. (A) Composite of images from a time-lapse series demonstrating intercellular transfer of GFP-tagged HIF-1α via a TNT that connects SKOV3 ovarian cancer cells. (B) SKOV3 cells expressing GFP-VEGF form TNTs that transfer VEGF. (C) HUVECs stained with DiI connected via a long TNT. (D) Heterotypic TNT formation between a HUVEC (left) and a breast cancer cell (MCF-7, on the right). (E) A cluster of platelets cultured in vitro forming many fine pseudopodia-like protrusions representing potential TNTs. (F) Schematic demonstrating potential interplay among microthrombi formed by platelets and/or RBCs communicating via TNTs, in the same ecosystem as malignant cells communicating with TNTs. Scale bars = 100 μm. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 3

Quantification of the intercellular interactions that occur via TNTs: in search of a Dunbar's number for TNTs. We analyzed a 40-h time-lapse set of images of a coculture of Mg63.2 osteosarcoma cells with hFOB osteoblast cells. (A) The duration of TNTs is relatively short, as nearly all of the TNTs that were formed lasted for 1 hour or less. (B) The absolute number of TNTs increases after 10 h in culture, as does the ratio of number of cells with TNTs (C). All of these images are presented in video form in Supplementary Video 1. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 4

Scanning electron microscopy imaging of TNTs in MSTO-211H malignant mesothelioma cells. (A) Electron micrograph revealing multiple insertion points vs. points of extrusion of TNTs in the membrane of a MSTO cell (Scale bar = 2 μm; Magnification 14.62 k X; width = 20.52 μm). (B) A TNT is seen on the left, and on the right is a TNT-like protrusion vs. filopodia/invadopodial extension. An extracellular vesicle is overlying the extension on the right (Scale bar = 1 μm; Magnification 24.69 k X; width = 12.15 μm). Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 5

Melanoma cells that overexpress CSPG4 form TNTs at a higher rate over time. We quantified TNTs between WM1552C-CSPG4 and WM1552C-mock cells at 24 h, 48 h, and 72 h. The average number of TNTs per time point is shown (Top) as well as cell proliferation measured as average optical density (Bottom). Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 6

Knockdown of the UNC-45A myosin motor protein chaperone diminishes TNT formation, but this finding is limited in duration. Upper left: western blot demonstrating effective knockdown of UNC-45A using shRNA. Based on these results, shRNA #2 was used in our study. Upper right: quantitation of effective UNC45-A knockdown by shRNA #1 and #2. Lower left: mean number of TNTs at 24 h, 48 h, and 72 h in wild type SKOV3 ovarian cancer cells compared with cells transfected with the scramble and UNC-45A shRNA #2. Lower right: mean cell count for the same conditions and time points. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 7

Cancer cells forming more TNTs demonstrate lower expression of hENT1. (A) hENT1 expression in S2013 pancreatic cancer cells, A270 ovarian cancer cells, and LOVO colon cancer cells with and without TNTs. (B) Corresponding images for each cell line are located beneath each graph. Cells were stained with immunofluorescent antibody that marks the expression of hENT1, and the expression was quantified as described. Scale bars = 50 μm for each of these panels. (C) The larger image on the bottom demonstrates a particularly long and curved TNT (indicated by white arrows) connecting hENT1-expressing S2013 cells. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 8

Connexin expression may be inversely correlated with TNT formation, and its expression co-localizes most prominently at the base and/or tips of TNTs. This experiment was performed using malignant mesothelioma cell lines MSTO-211H and VAMT. The upper panel shows representative images following fluorophore-tagged connexin staining of both cells lines. The lower panels show graphs of connexin expression in cells forming no TNTs (blue column) compared with those forming TNTs (red column). MSTO cell data are in the lower left graph, and VAMT data are in the lower right. Scale bars = 10 μm. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Figure 9

Schematic of cross talk via hypoxia-induced TNTs that facilitate the intercellular transfer of cargo. TNTs facilitate dynamic interplay that has implications in angiogenesis and other processes that are critical to the ecosystem.