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The role of intercellular communication is increasingly recognized as being critical to tumoral invasion, metastasis, and development of resistance to therapy. The recent discovery of cellular protrusions - tumour microtubes - connecting cancer cells in gliomas, and tunneling nanotubes in several other forms of cancer, sheds light on a novel mechanism for molecular networking. Interrupting and disrupting vital lines of intercellular cross-talk via these membranous cellular tubes has strong potential as a novel form of cancer-directed therapy.

Formation of a nephron depends on reciprocal signaling of different morphogens between epithelial and mesenchymal cells within the renal stem/progenitor cell niche. Previously, it has been surmised that a close proximity exists between both involved cell types and that morphogens are transported between them by diffusion. However, actual morphological data illustrate that mesenchymal and epithelial stem/progenitor cell bodies are separated by a striking interface. Special fixation of specimens by glutaraldehyde (GA) solution including cupromeronic blue, ruthenium red, or tannic acid for electron microscopy depicts that the interface is not void but filled in extended areas by textured extracellular matrix. Surprisingly, projections of mesenchymal cells cross the interface to contact epithelial cells. At those sites the plasma membranes of a mesenchymal and an epithelial cell are connected via tunneling nanotubes. Regarding detected morphological features in combination with involved morphogens, their transport cannot longer be explained solely by diffusion. Instead, it has to be sorted according to biophysical properties of morphogens and to detected environment. Thus, the new working hypothesis is that morphogens with good solubility such as glial cell line-derived neurotrophic factor (GDNF) or fibroblast growth factors (FGFs) are transported by diffusion. Morphogens with minor solubility such as bone morphogenetic proteins (BMPs) are secreted and stored for delivery on demand in illustrated extracellular matrix. In contrast, morphogens with poor solubility such as Wnts are transported in mesenchymal cell projections along the plasma membrane or via illustrated tunneling nanotubes. However, the presence of an intercellular route between mesenchymal and epithelial stem/progenitor cells by tunneling nanotubes also makes it possible that all morphogens are transported this way.

Cross-dressing involves the transfer of preformed functional peptide-MHC complexes from the surface of donor cells to recipient cells, such as dendritic cells (DCs). These cross-dressed cells might eventually present the intact, unprocessed peptide-MHC complexes to T lymphocytes. In this review we will discuss some recent findings concerning the intercellular transfer of preformed MHC complexes and the possible mechanisms by which the transfer may occur. We will report evidences showing that both MHC class I and MHC class II functional complexes might be transferred, highlighting the physiological relevance of these cross-dressed cells for the presentation of exogenous antigens to both cytotoxic and helper T lymphocytes., (Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background Information: Tunneling nanotubes (TnTs) are thin plasma membrane bridges mediating transfers of materials and signals between cells. Heterogeneity of heterocellular and homocellular TnTs is largely described but ultrafine imaging of these light-sensitive floating nanometric structures represents a real challenge in microscopy. We propose here imaging strategies designed to dissect structural and dynamic aspects of TnT formation and function in fixed or living PC12 cells., Results: Through time-gated Continuous Wave STimulated Emission Depletion (gCW STED) nanoscopy associated with deconvolution, we provided nanoscale details of membrane and cytoskeleton organisations in two subtypes of TnTs, namely type 1 TnT (TnT1) and type 2 TnT (TnT2). In fixed PC12 cells, TnT1 (length, several tens of micrometres; diameter, 100-650 nm) exhibited a large trumpet-shaped origin, a clear cytosolic tunnel and different bud-shaped connections from closed-ended to open-ended tips. TnT1 contained both actin and tubulin. TnT2 (length, max 20 μm, diameter, 70-200 nm) only contained actin without clear cytosolic tunnel. In living PC12 cells, we observed through gCW STED additional details, unrevealed so far, including a filament spindle emerging from an organising centre at the origin of TnT1 and branched or bulbous attachments of TnT2. However, the power of depletion laser in STED nanoscopy was deleterious for TnTs and prolonged time-lapse experiments were almost prohibited. By circumventing the hazard of photoxicity, we were able to monitor dynamics of bud-shaped tips and intercellular transfer of wheat germ agglutinin labelled cellular elements through time-gated confocal microscopy., Conclusions: Our work identified new structural characteristics of two subtypes of TnTs in PC12 cells as well as dynamics of formation and transfer through complementary imaging methods combined with image processing. Therefore, we could achieve maximum lateral resolution and sample preservation during acquisitions to reveal new insights into TnT studies., Significance: Due to large disparity of TnT-like structures in neuronal, immune, cancer or epithelial cells, high- and superresolution approaches can be utilised for full characterisation of these yet poorly understood routes of cell-to-cell communication., (© 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrP(Sc). The intercellular transfer of PrP(Sc) is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrP(Sc) can move between cells. Here we investigate further the details of this mechanism and show that PrP(Sc) travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs.

Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multisystemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS gene). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon coculture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins., (© 2014 AlphaMed Press.)

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.

Cell-to-cell information exchange mediated by membrane protrusions in tunneling nanotubes (TNTs) has been widely described in distinct cell lines. Here, we describe a new form of direct intercellular communication in a murine macrophage-like cell line that is mediated by pseudopodial fusions that form over scraped plastic tissue culture surfaces along scratch lines. These structures are capable of forming intercellular, tunnel-like channels (inter-pseudopodial axis connections) that can be differentiated from TNTs based on length, thickness, tandem arrangement along an axis, pseudopodial origin and permanency. These channels were able to exchange membrane lipids and contain particles 0.5 μm or lesser in diameter between cells and might represent an additional biological function of pseudopodia.

Tunneling nanotubes are important membrane channels for cell-to-cell communication. In this study, we investigated the effect of the microenvironment on nanotubular structures by preparing a three-dimensional silk fibroin micropillar structure. In previous reports, tunneling nanotubes were described as stretched membrane channels between interconnected cells at their nearest distance. They hover freely in the cell culture medium and do not contact with the substratum. Interestingly, the micropillars could provide supporting points for nanotubular connection on silk fibroin films, where nanotubular structure formed a stable anchor at contact points. Consequently, the extension direction of nanotubular structure was affected by the micropillar topography. This result suggests that the hovering tunneling nanotubes in the culture medium will come into contact with the raised roadblock on the substrates during long-distance extension. These findings imply that the surface microtopography of biomaterials have an important influence on cell communication mediated by tunneling nanotubes.

Effective cell-to-cell communication is critical for the survival of both unicellular and multicellular organisms. In multicellular plants, direct cell coupling across the cell wall boundaries is mediated by long membrane-lined cytoplasmic bridges, the plasmodesmata. Exciting recent discoveries suggest that the occurrence of such membrane-lined intercellular channels is not unique to plant lineages but more prevalent across biological kingdoms than previously assumed. Striking functional analogies exist among those channels, in that not only do they all facilitate the exchange of various forms of macromolecules, but also they are exploited by some opportunistic pathogens to spread infection from one host cell to another. However, host cells may have also evolved strategies to offset such exploitation of the critical cellular infrastructure by the pathogen. Indeed, recent studies support an emerging paradigm that cellular connectivity via plasmodesmata plays an important role in innate immune responses. Preliminary hypotheses are proposed as to how various regulatory mechanisms integrating plasmodesmata into immune signaling pathways may have evolved., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The application of polymeric nanoparticles (NPs) has a promising future for targeting and delivering drugs into the central nervous system (CNS). However, the fate of NPs once entered in the brain after crossing the blood-brain barrier (BBB) and taken up into neuronal cells is a neglected area of study. Thus, here, we investigate the possible mechanisms of a cell-to-cell transport of poly-lactide-co-glycolide (PLGA) NPs modified with a glycopeptide (g7-NPs), already demonstrated to be able to cross the BBB after in vivo administration in rodents. We also tested antibody (Ab) -modified g7-NPs both in vitro and in vivo to investigate the possibility of specific targeting. Our results show that g7-NPs can be transported intra- and inter-cellularly within vesicles after vesicular internalization. Moreover, cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. The transport is dependent on F-actin and can be increased by induction of TNT-like structures overexpressing M-Sec, a central factor and inducer of TNT formation. Moreover, cell-to-cell transport occurs independently from NP surface modification with antibodies. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration of NPs in mice., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mesenchymal stem cells (MSC) are known to repair broken heart tissues primarily through a paracrine fashion while emerging evidence indicate that MSC can communicate with cardiomyocytes (CM) through tunneling nanotubes (TNT). Nevertheless, no link has been so far established between these two processes. Here, we addressed whether cell-to-cell communication processes between MSC and suffering cardiomyocytes and more particularly those involving TNT control the MSC paracrine regenerative function. In the attempt to mimic in vitro an injured heart microenvironment, we developed a species mismatch coculture system consisting of terminally differentiated CM from mouse in a distressed state and human multipotent adipose derived stem cells (hMADS). In this setting, we found that crosstalk between hMADS and CM through TNT altered the secretion by hMADS of cardioprotective soluble factors such as VEGF, HGF, SDF-1α, and MCP-3 and thereby maximized the capacity of stem cells to promote angiogenesis and chemotaxis of bone marrow multipotent cells. Additionally, engraftment experiments into mouse infarcted hearts revealed that in vitro preconditioning of hMADS with cardiomyocytes increased the cell therapy efficacy of naïve stem cells. In particular, in comparison with hearts treated with stem cells alone, those treated with cocultured ones exhibited greater cardiac function recovery associated with higher angiogenesis and homing of bone marrow progenitor cells at the infarction site. In conclusion, our findings established the first relationship between the paracrine regenerative action of MSC and the nanotubular crosstalk with CM and emphasize that ex vivo manipulation of these communication processes might be of interest for optimizing current cardiac cell therapies., (© 2013 AlphaMed Press.)

The intracellular fate of nanosized drug delivery systems is still not well understood. Various internalization pathways have been discovered, but knowledge of their intracellular trafficking is still incomplete. The aim of this study was to examine the internalization, pathways, and positioning taken by solid lipid nanoparticles (SLNs) in cells. SLNs were fluorescence labeled with a newly synthesized fluorescent probe, 14-DACA. The probe was strongly incorporated into the nanoparticle core under the influence of its long lipophilic chain, enabling superior visualization of SLNs under complex and dynamic intracellular conditions. The intracellular distribution of SLNs was studied qualitatively using a co-localization technique and quantitatively using fluorescence intensity profiles. SLNs were seen inside the cells as distinct bright blue dots that underwent dynamic movement and were finally positioned in the proximity of the nucleus. A few SLNs were shown to be present in mitochondria and between actin filaments, but none in the cell nucleus or lysosomes. SLNs are here reported to be present in tunneling nanotubes (TNTs), which could be a new route of SLN transfer between cells. More TNTs were observed in cells treated with SLNs. The presence of TNTs was additionally confirmed by atomic force microscopy analysis, which indicated that treated cells were more rough than control cells. Detailed investigation of the subcellular localization of SLNs and the evidence for their transfer and distribution via TNTs to the cells, which are not in direct contact with the source of SLNs, are important for understanding the mechanism of targeted drug delivery. Understanding the possible intercellular distribution of SLNs via TNTs can significantly influence approaches to treating organelle-specific diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Tunneling nanotubes are actin-based cytoplasmic extensions that function as intercellular channels in a wide variety of cell types.There is a renewed and keen interest in the examination of modes of intercellular communication in cells of all types, especially in the field of cancer biology. Tunneling nanotubes -which in the literature have also been referred to as "membrane nanotubes," "'intercellular' or 'epithelial' bridges," or "cytoplasmic extensions" - are under active investigation for their role in facilitating direct intercellular communication. These structures have not, until recently, been scrutinized as a unique and previously unrecognized form of direct cell-to-cell transmission of cellular cargo in the context of human cancer. Our recent study of tunneling nanotubes in human malignant pleural mesothelioma and lung adenocarcinomas demonstrated efficient transfer of cellular contents, including proteins, Golgi vesicles, and mitochondria, between cells derived from several well-established cancer cell lines. Further, we provided effective demonstration that such nanotubes can form between primary malignant cells from human patients. For the first time, we also demonstrated the in vivo relevance of these structures in humans, having effectively imaged nanotubes in intact solid tumors from patients. Here we provide further analysis and discussion on our findings, and offer a prospective 'road map' for studying tunneling nanotubes in the context of human cancer. We hope that further understanding of the mechanisms, methods of transfer, and particularly the role of nanotubes in tumor-stromal cross-talk will lead to identification of new selective targets for cancer therapeutics.

Cell-to-cell communication and exchange of materials are vital processes in multicellular organisms during cell development, cell repair, and cell survival. In neuronal and immunological cells, intercellular transmission between neighboring cells occurs via different complex junctions or synapses. Recently, long distance intercellular connections in mammalian cells called tunneling nanotubes (TNTs) have been described. These structures have been found in numerous cell types and shown to transfer signals and cytosolic materials between distant cells, suggesting that they might play a prominent role in intercellular trafficking. However, these cellular connections are very heterogeneous in both structure and function, giving rise to more questions than answers as to their nature and role as intercellular conduits. To better understand and characterize the functions of TNTs, we have highlighted here the latest discoveries regarding the formation, structure, and role of TNTs in cell-to-cell spreading of various signals and materials. We first gathered information regarding their formation with an emphasis on the triggering mechanisms observed, such as stress and potentially important proteins and/or signaling pathways. We then describe the various types of transfer mechanisms, in relation to signals and cargoes that have been shown recently to take advantage of these structures for intercellular transfer. Because a number of pathogens were shown to use these membrane bridges to spread between cells we also draw attention to specific studies that point toward a role for TNTs in pathogen spreading. In particular we discuss the possible role that TNTs might play in prion spreading, and speculate on their role in neurological diseases in general.

Cell-to-cell communication is the basis of all biology in multicellular organisms, allowing evolution of complex forms and viability in dynamic environments. Though biochemical interactions occur over distances, physical continuity remains the most direct means of cellular interactions. Cellular bridging through thin cytoplasmic channels-plasmodesmata in plants and tunneling nanotubes in animals-creates direct routes for transfer of signals and components, even pathogens, between cells. Recently, two new cellular connections, designated epithelial (EP) bridges, were discovered and found to be structurally distinct from other cellular channels. The first EP bridge type facilitates material transport between cells similar to plasmodesmata and tunneling nanotubes, the second EP bridge type mediates migration of cells between EP cell masses representing a novel form of cell migration. Here, we compare the structures and functions of EP bridges with other cellular channels and discuss biochemical and cellular interactions involved in EP bridge formation. Potential roles for EP bridges in health and disease are also presented.