Cytonemes in Tumourigenesis
Increasing evidence during the past two decades shows that cells interconnect and communicate through cytonemes. These cytoskeleton-driven extensions of specialized membrane territories have emerged as a novel alternative for cell to cell communication that are involved in development, physiology, and disease. Several recent studies have shown that signalling pathways mediated by cytonemes during development, are essential for certain tumoral cell types progression.
In Drosophila wing disc EGFR and RET tumour models, cytoneme formation is required to receive signals from the neighbouring cells. Genetic ablation of cytonemes prevents tumour progression, restores apico-basal polarity, and improves survival.
Furthermore, cytonemes in the Drosophila glial cells are essential for glioblastoma progression as they alter Wg/Fz1 signalling between glia and neurons. Research on cytoneme formation, maintenance, and cell signalling mechanisms will help to better understand not only physiological developmental processes and tissue homeostasis but also cancer progression.
The nature of these interconnecting structures and their similarities with epithelial cytonemes are currently under debate. Cytonemes have been proposed to mediate communication between neoplastic cells and cells in their microenvironment . In a Drosophila wing disc tumor model utilizing ectopic expression of the epidermal growth factor receptor (EGFR) and receptor protein-tyrosine kinase (Ret) oncogenes, cytoneme formation is required to receive signals from the neighboring cells. Genetic ablation of cytonemes prevents tumor progression, restores apico-basal polarity, and improves survival . This recently established system serves as an optimal platform for novel pharmaceutical approaches against cancer progression in vivo. The authors identified pharmacological combinations against cytoneme-mediated oncogenic signals that prevent tumor progression and improve life span. The value of flies as a valid platform for human disease has accumulated evidences that favor this model for future preclinical studies. In particular, the high cost of testing single or combined pharmacological treatments in mice is several orders of magnitude higher than Drosophila based platforms, which has made preclinical trials risky and challenging. The molecular basis underlying cytonemes, the signals transduced by cytonemes, and the implications in tumorigenesis are hot topics for human disease that open novel avenues for potential future treatments.
In addition, the discovery of tunneling nanotubes (TNTs) brings a novel class of thin and long membrane protrusions that connect benign tumor cells . These protrusions form complex networks that mediate the selective transfer of vesicles, organelles, and small molecules . TNTs are a common phenomenon in different cell types and tissues that increase under pathological conditions, such as infections, cancer, or neurodegenerative diseases . One considerable limitation to the study of TNTs is the fragility of these structures that makes TNTs difficult to preserve after fixation of tissues. This brought a controversy about their existence in vivo. However, intravital techniques enabled the study of TNTs in live animals , which revealed that TNTs are indeed relevant cellular structures in vivo.
In vivo microscopy methods have been used in recent years to study in detail cellular features of cancer cells. A recent study showed that TNTs are induced by stress in prostate cancer and they had a role in mediating intercellular communication that confer stress adaptive cell survival and treatment resistance on the tumoral cells . Additionally, pancreatic cancer cells show TNTs and their formation is stimulated after chemotherapy exposure . Furthermore, TNTs are involved in the communication between tumor cells and macrophages to promote macrophage-dependent tumor cell invasion both in vitro and in an in vivo zebrafish model . Interestingly, colorectal cancer cells have the ability to form locomotory and invasive filopodia that promote invasion and metastasis, and this is suppressed by the phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP) . Related to colorectal cancer, leucine-rich-repeat containing G-protein-coupled receptor 5 (Lgr5), which labels crypt stem cells, represents the cell of origin in gastrointestinal cancers , and Lgr5 promotes the formation of cytonemes in mammalian cells suggesting a possible role for cytonemes in gastrointestinal cancer cell survival, invasion, and metastasis . Exo70, a key component of the Exocyst complex, induces extensive actin membrane protrusions resembling filopodia and blocking Exo70 function inhibits invadopodia formation . Exo70 expression is upregulated in colon cancer samples and its expression is positively correlated with tumor size, invasion depth, and distant metastasis. Colon cancer patients with higher Exo70 expression have a poorer clinical outcome than those with lower Exo70 expression .
In particular, glioblastoma (GB) cells produce long cellular protrusions at the invasive edge of the tumor that scan the surrounding area and interconnect tumor cells. These protrusions are F-actin based and form a complex network that interconnects GB cells; therefore, they are named tumor microtubes (TMs) . TMs contribute to invasion and proliferation, resulting in effective brain colonization by GB cells. Moreover, TMs constitute a multicellular network that connects GB cells over long distances (up to 500 µm length) . This study found that Growth Associated Protein-43 (GAP43) is essential for the development of TMs and the tumor cell network associated with GB progression, and it drives TM-dependent tumor cell invasion, proliferation, interconnection, and radioresistance. TMs share many characteristics with cytonemes, they are actin-based projections and they are marked by several cytoneme markers, including Ihog, LifeActin, Moesin (GMA), glycosylphosphatidyl-inositol (GPI), myosin light chain (MLC), and the nonmuscle type 2 myosin, spaghetti squash (sqh). Moreover, this study  showed in a Drosophila glioma model that the glioma network is dependent on the fly GAP43-like gene (igloo, igl), as has been described in human tumor cells. The glioma network does not develop upon igl silencing. TMs stability in GB is sensitive to GAP43 expression in tumoral cells. Moreover, downregulating Neuroglian (Nrg), which is known to prevent epithelial cytoneme formation, resulted in a reduction of the TM-like processes in GB . Moreover, TMs accumulate Frizzled1 receptor (Fz1) that mediates Wingless (Wg) signaling (Figure 1) . Thus, there are molecular and functional similarities between cytonemes and TMs; however, the term cytoneme is used for physiological situations, and TMs is restricted to the tumoral condition.
Figure 1. Cytonemes in tumourigenesis. Glioma cells produce a network of tumor microtubes that grow to reach and enwrap neighboring neurons. Increased glia–neuron membrane contacts facilitate neuronal Wg sequestering mediated by glioma Frizzled1 receptor accumulated in the tumor microtubes .
TMs and TNTs share some structural features, but TMs are more stable, longer, and thicker (2 µm). In addition, TMs in human cells provide functional coordination to GB cells and facilitate cell repair, brain infiltration, and offer resistance to radiotherapy through dilution of Ca+2 intracellular peaks , which thereby increases the aggressiveness of GB.
- Sol Fereres; Ryo Hatori; Makiko Hatori; Thomas B. Kornberg; Cytoneme-mediated signaling essential for tumorigenesis.. PLOS Genetics 2019, 15, e1008415, 10.1371/journal.pgen.1008415.
- Amin Rustom; Rainer Saffrich; Paul Walther; Ivanka Markovic; Hans-Hermann Gerdes; Nanotubular Highways for Intercellular Organelle Transport. Science 2004, 303, 1007-1010, 10.1126/science.1093133.
- Jennifer Ariazi; Andrew Benowitz; Vern De Biasi; Monique L. Den Boer; Stéphanie Cherqui; Haifeng Cui; Nathalie Douillet; Eliseo A. Eugenin; David Favre; Spencer Goodman; et al. Tunneling Nanotubes and Gap Junctions–Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions. Frontiers in Neuroscience 2017, 10, 333, 10.3389/fnmol.2017.00333.
- George Okafo; Lisa Prevedel; Eliseo Eugenin; Tunneling nanotubes (TNT) mediate long-range gap junctional communication: Implications for HIV cell to cell spread.. Scientific Reports 2017, 7, 16660, 10.1038/s41598-017-16600-1.
- Markus Rehberg; Katharina Nekolla; Sabine Sellner; Marc Praetner; Karina Mildner; Dagmar Zeuschner; Fritz Krombach; Intercellular Transport of Nanomaterials is Mediated by Membrane Nanotubes In Vivo. Small 2016, 12, 1882-1890, 10.1002/smll.201503606.
- Alexander Kretschmer; Fan Zhang; Syam Prakash Somasekharan; Charan Tse; Lauren Leachman; Anna Gleave; Brian Li; Ivan Asmaro; Teresa Huang; Leszek Kotula; et al. Stress-induced tunneling nanotubes support treatment adaptation in prostate cancer.. Scientific Reports 2019, 9, 7826, 10.1038/s41598-019-44346-5.
- Snider Desir; Patrick O’Hare; Rachel Isaksson Vogel; William Sperduto; Akshat Sarkari; Elizabeth L. Dickson; Phillip Wong; Andrew C. Nelson; Yuman Fong; Clifford J. Steer; et al. Chemotherapy-Induced Tunneling Nanotubes Mediate Intercellular Drug Efflux in Pancreatic Cancer.. Scientific Reports 2018, 8, 9484, 10.1038/s41598-018-27649-x.
- Samer J. Hanna; Kessler McCoy-Simandle; Edison Leung; Alessandro Genna; John Condeelis; Dianne Cox; Tunneling nanotubes, a novel mode of tumor cell–macrophage communication in tumor cell invasion. Journal of Cell Science 2019, 132, jcs223321, 10.1242/jcs.223321.
- David S. Zuzga; Joshua Pelta-Heller; Peng Li; Alessandro Bombonati; Scott A. Waldman; Giovanni M. Pitari; Phosphorylation of vasodilator-stimulated phosphoprotein Ser239 suppresses filopodia and invadopodia in colon cancer.. International Journal of Cancer 2011, 130, 2539-48, 10.1002/ijc.26257.
- Nick Barker; Johan H. Van Es; Jeroen Kuipers; Pekka Kujala; Maaike Van Den Born; Miranda Cozijnsen; Andrea Haegebarth; Jeroen Korving; Harry Begthel; Peter J. Peters; et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007, 449, 1003-1007, 10.1038/nature06196.
- Joshua C. Snyder; Lauren K. Rochelle; Sébastien Marion; H. Kim Lyerly; Larry S. Barak; Marc G. Caron; Lgr4 and Lgr5 drive the formation of long actin-rich cytoneme-like membrane protrusions.. Journal of Cell Science 2015, 128, 1230-40, 10.1242/jcs.166322.
- Jianglan Liu; Yuting Zhao; Yujie Sun; Bing He; Changsong Yang; Tatyana Svitkina; Yale E. Goldman; Wei Guo; Exo70 stimulates the Arp2/3 complex for lamellipodia formation and directional cell migration.. Current Biology 2012, 22, 1510-5, 10.1016/j.cub.2012.05.055.
- Li Xiao; Kaifeng Zheng; Xia Lv; Jihuan Hou; Liang Xu; Yujie Zhao; Fei Song; YaQiong Fan; Hanwei Cao; Wenqing Zhang; et al. Exo70 is an independent prognostic factor in colon cancer.. Scientific Reports 2017, 7, 5039, 10.1038/s41598-017-05308-x.
- Matthias Osswald; Erik Jung; Felix Sahm; Gergely Solecki; Varun Venkataramani; Jonas Blaes; Sophie Weil; Heinz Horstmann; Benedikt Wiestler; Mustafa Syed; et al. Brain tumour cells interconnect to a functional and resistant network. Nature 2015, 528, 93-98, 10.1038/nature16071.
- Marta Portela; Varun Venkataramani; Natasha Fahey-Lozano; Esther Seco; Maria Losada-Perez; Frank Winkler; Sergio Casas-Tinto; Marta Portela Esteban; WNT vampirization by glioblastoma leads to tumor growth and neurodegeneration. bioRxiv 2018, 1, 428953, 10.1101/428953.
- Sergio Casas-Tintó; Marta Portela; Cytonemes, Their Formation, Regulation, and Roles in Signaling and Communication in Tumorigenesis. International Journal of Molecular Sciences 2019, 20, 5641, 10.3390/ijms20225641.