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Abstract Background Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. Methods We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. Results The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. Conclusions This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

Despite advances in cancer therapies, nanomedicine approaches including the treatment of glioblastoma (GBM), the most common, aggressive brain tumor, remains inefficient. These failures are likely attributable to the complex and not yet completely known biology of this tumor, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential, and resistance to radiation and chemotherapy. The intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key feature in tumor progression and in particular in the re-growth of GBM after surgery. In this context, we firstly identified structural differences of TnTs formed by U87-MG cells, as model of GBM cells, in comparison with those formed by normal human astrocytes (NHA), used as a model of healthy cells. Successively, we have studied the possibility to exploit U87-MG TnTs as drug-delivery channels in cancer therapy, using liposomes composed of cholesterol/sphingomyelin and surface functionalized with mApoE and chlorotoxin peptides (Mf-LIP) as nanovehicle model. The results showed that U87-MG cells formed almost exclusively thick and long protrusions, whereas NHA formed more thin and short TnTs. Considering that thick TnTs are more efficient in transport of vesicles and organelles, we showed that fluorescent-labeled Mf-LIP can be transported via TnTs between U87-MG cells and with less extent through the protrusions formed by NHA cells. Our results demonstrate that nanotubes are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumor niches that are hardly targeted by simple drug diffusion in the brain parenchyma. Moreover, the differences identified in TnTs formed by GBM and NHA cells can be exploited to increase treatment precision and specificity.

Background: A reduced effective local concentration significantly contributes to the unsatisfactory therapeutic results of epirubicin in gastric cancer. Mesenchymal stem cells exhibit targeted chemotaxis towards solid tumors and form tunneling nanotubes with tumor cells, facilitating the delivery of various substances. This study demonstrates the novelty of mesenchymal stem cells in releasing epirubicin into gastric cancer cells through tunneling nanotubes., Objective: Epirubicin delivery to gastric cancer cells using mesenchymal stem cells., Methods: In vitro transwell migration assays, live cell tracking, and in vivo targeting assays were used to demonstrate the chemotaxis of mesenchymal stem cells towards gastric cancer. We verified the targeted chemotaxis of mesenchymal stem cells towards gastric cancer cells and the epirubicin loading ability using a high-content imaging system (Equipment type:Operetta CLS). Additionally, tunneling nanotube formation and the targeted release of epirubicin-loaded mesenchymal stem cells co-cultured with gastric cancer cells through mesenchymal stem cell-tunneling nanotubes into gastric cancer cells was observed using Operetta CLS., Results: Mesenchymal stem cells demonstrated targeted chemotaxis towards gastric cancer, with effective epirubicin loading and tolerance. Co-culturing induced tunneling nanotube formation between these cells. Epirubicin-loaded mesenchymal stem cells were released into gastric cancer cells through tunneling nanotubes, significantly increasing their non-viability compared to the negative control group (p < 0.05)., Conclusions: We identified a novel approach for precisely targeting epirubicin release in gastric cancer cells. Therefore, mesenchymal stem cell-tunneling nanotubes could serve as a potential tool for targeted delivery of drugs, enhancing their chemotherapeutic effects in cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ischemic stroke is the leading cause of death and disability worldwide, with increasing incidence and mortality, imposing a significant social and economic burden on patients and their families. However, cerebral vascular occlusion leads to acute loss of neurons and destruction of synaptic structures. The limited treatment options cannot adequately address intra-neuronal mitochondrial dysfunction due to stroke. Therefore, stem cell-derived mitochondria transplantation plays an important role in neuronal protection and recovery after stroke, when combined with the intracranial and extracranial immunoregulatory effects of stem cell therapy, revealing the mechanism of transferred mitochondria in stem cells in protecting neurological function among chronic-phase ischemic stroke by affecting the endogenous apoptotic pathway of neuronal cells. This research elaborated on the mitochondrial dysfunction in neurons after ischemic stroke, followed by human bone marrow mesenchymal stem cells (hBMSC) rescued damaged neurons by mitochondrial transfer through tunneling nanotubes (TNTs), and the immunomodulatory effect of the preferential transfer of stem cells to the spleen when transplanted into the body.which created an immune environment for nerve repair, as well as improved neurological recovery after the chronic phase of stroke. This review is expected to provide a novel idea for applying intracranial stem cell transplantation in chronic-phase ischemic stroke treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

A highly interconnected network of diverse brain regions is necessary for the precise execution of human behaviors, including cognitive, psychiatric, and motor functions. Unfortunately, degeneration of specific brain regions causes several neurodegenerative disorders, but the mechanisms that elicit selective neuronal vulnerability remain unclear. This knowledge gap greatly hinders the development of effective mechanism-based therapies, despite the desperate need for new treatments. Here, we emphasize the importance of the Rhes (Ras homolog-enriched in the striatum) protein as an emerging therapeutic target. Rhes, an atypical small GTPase with a SUMO (small ubiquitin-like modifier) E3-ligase activity, modulates biological processes such as dopaminergic transmission, alters gene expression, and acts as an inhibitor of motor stimuli in the brain striatum. Mutations in the Rhes gene have also been identified in selected patients with autism and schizophrenia. Moreover, Rhes SUMOylates pathogenic form of mutant huntingtin (mHTT) and tau, enhancing their solubility and cell toxicity in Huntington's disease and tauopathy models. Notably, Rhes uses membrane projections resembling tunneling nanotubes to transport mHTT between cells and Rhes deletion diminishes mHTT spread in the brain. Thus, we predict that effective strategies aimed at diminishing brain Rhes levels will prevent or minimize the abnormalities that occur in HD and tauopathies and potentially in other brain disorders. We review the emerging technologies that enable specific targeting of Rhes in the brain to develop effective disease-modifying therapeutics., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Many kinds of nanoparticles and organic dyes as fluorescent probes have been used in the stimulated emission depletion (STED) nanoscopy. Due to high toxicity, photobleaching and non-water solubility, these fluorescent probes are hard to apply in living cell imaging. Here, we report a new fluorescence carbon dots (FNCDs) with high photoluminescence quantum yield (56%), low toxicity, anti-photobleaching and good water-solubility that suitable for live-cell imaging can be obtained by doping fluorine element. Moreover, the FNCDs can stain the nucleolus and tunneling nanotubes (TNTs) in the living cell. More importantly, for STED nanoscopy imaging, the FNCDs effectively depleted background signals and improved imaging resolution. Furthermore, the lateral resolution of single FNCDs size under the STED nanoscopy is up to 22.1 nm for FNCDs deposited on a glass slide was obtained. And because of their good water dispersibility, the higher resolution of single FNCDs size in the nucleolus of a living cell can be up to 19.7 nm. After the image optimization steps, the fine fluorescence images of TNTs diameter with ca. 75 nm resolution is obtained living cell, yielding a threefold enhancement compared with that in confocal imaging. Additionally, the FNCDs show excellent photobleaching resistance after 1,000 scan cycles in the STED model. All results show that FNCDs have significant potential for application in STED nanoscopy. [ABSTRACT FROM AUTHOR]

Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here, we find that striatal-enriched protein, Rhes, is a critical regulator of mitophagy and striatal vulnerability in brain. In vivo interactome and density fractionation reveal that Rhes coimmunoprecipitates and cosediments with mitochondrial and lysosomal proteins. Live-cell imaging of cultured striatal neuronal cell line shows Rhes surrounds globular mitochondria, recruits lysosomes, and ultimately degrades mitochondria. In the presence of 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, Rhes disrupts mitochondrial membrane potential (ΔΨm) and promotes excessive mitophagy and cell death. Ultrastructural analysis reveals that systemic injection of 3-NP in mice promotes globular mitochondria, accumulation of mitophagosomes, and striatal lesion only in the wild-type (WT), but not in the Rhes knockout (KO), striatum, suggesting that Rhes is critical for mitophagy and neuronal death in vivo. Mechanistically, Rhes requires Nix (BNIP3L), a known receptor of mitophagy, to disrupt ΔΨm and promote mitophagy and cell death. Rhes interacts with Nix via SUMO E3-ligase domain, and Nix depletion totally abrogates Rhes-mediated mitophagy and cell death in the cultured striatal neuronal cell line. Finally, we find that Rhes, which travels from cell to cell via tunneling nanotube (TNT)-like cellular protrusions, interacts with dysfunctional mitochondria in the neighboring cell in a Nix-dependent manner. Collectively, Rhes is a major regulator of mitophagy via Nix, which may determine striatal vulnerability in the brain. [ABSTRACT FROM AUTHOR]

Cell membrane nanotubes, variously referred to as tunneling nanotubes and cytonemes, are currently the focus of much interest. They are of ancient origin, as indicated by their opportunistic use for cell invasion by pathogens, including bacteria and virus, and by their employment in bacterial networking. They play a significant role in cancer invasion and in the explanation of glioblastoma resistance to treatment. Their structure and properties have been investigated with optical tweezers. They have been detected in vivo. Their role in the immune system was early verified. Very recently, it was shown that they share many properties with nerve synapses, including the roles of glutamate and Ca ions. Similar features have also been observed in primitive plants. These results support the conjecture that, besides their roles in immunology, developmental biology and cancer, cell membrane nanotubes are the ancestors of the nervous system. [ABSTRACT FROM AUTHOR]

We report a protocol for simultaneous triple labelling of intermediate filaments, microtubules and actin filaments. The described procedure offers an optimal preservation of the structure and antigenicity of individual representatives of cytoskeletal elements and is applicable for labelling of tissue samples and cultured cells. Namely, we demonstrate that using this protocol the cytoskeletal elements are well-preserved and detectable in the whole mount urinary bladder tissue pieces, cryosections of the urinary bladder, and in cultured normal and cancer urothelial cells including their delicate intercellular connections such as tunneling nanotubes (TnTs). The protocol uncovers for the first time the co-distribution of actin filaments, intermediate filaments and microtubules in TnTs, which were up to now known as mono- or bi-cytoskeletal structures. Presented triple labelling protocol provides an efficient tool for studying co-distribution of actin filaments, intermediate filaments, and microtubules and therefore offers new insights into their cellular and tissue distribution. [ABSTRACT FROM AUTHOR]

Tunneling nanotube (TNT)-mediated cell communication play pivotal roles in a series of physiological and pathological processes in multicellular organism. This study was designed to investigate the existence of TNTs between EPCs and osteoclast precursors and evaluate their effects on the differentiation of osteoclast precursors. For these purposes, EPCs and osteoclast precursors (RAW264.7 cells) were stained with different fluorescent dyes before direct co-culture; then, the co-cultured cells were sorted by fluorescence activated cell sorter (FACS), and the differentiation of co-cultured RAW264.7 cells was evaluated. The results showed that the differentiation potential of RAW264.7 cells was significantly inhibited after their co-culture with EPCs. Additionally, the expression of macrophage migration inhibitory factor (MIF) was up-regulated in RAW264.7 cells after co-culture. Moreover, the MIF inhibitor ISO-1 could rescue the formation of TRAP-positive multinuclear osteoclasts and the expression of osteoclastogenesis-associated genes in the co-cultured RAW264.7 cells. The present study demonstrates that EPCs can affect the differentiation of osteoclast precursors through the TNT-like structures formed across these two types of cells and might inform new therapeutic strategies for osteolytic diseases. [ABSTRACT FROM AUTHOR]

Highlights from the article: Compared to differentiated cells, many tumor cells have an altered energy metabolism. Most studies on the metabolism of cancer cells have focused on the investigation of a single type of intra-tumoral cell, although recent studies have described a more complex scene, where the tumor ecosystem via metabolic symbiosis plays a critical role in cancer progression ([9], [10], [29]). This transport through TNTs between malignant and stromal cells can modify the gene regulation and metabolism of TME cells, playing a critical role in tumor growth and metastasis, as well as in resistance to different treatments. Metabolic synergy between cancer and stroma cells is a driver of cancer aggressiveness and it has been shown that lactate is an essential metabolic intermediate between TME cells, fueling the oxidative metabolism of oxygenated tumor cells.

Tunneling nanotubes (TNTs) are intercellular structures that allow for the passage of vesicles, organelles, genomic material, pathogenic proteins and pathogens. The unconventional actin molecular motor protein Myosin-X (Myo10) is a known inducer of TNTs in neuronal cells, yet its role in other cell types has not been examined. The Nef HIV-1 accessory protein is critical for HIV-1 pathogenesis and can self-disseminate in culture via TNTs. Understanding its intercellular spreading mechanism could reveal ways to control its damaging effects during HIV-1 infection. Our goal in this study was to characterize the intercellular transport mechanism of Nef from macrophages to T cells. We demonstrate that Nef increases TNTs in a Myo10-dependent manner in macrophages and observed the transfer of Nef via TNTs from macrophages to T cells. To quantify this transfer mechanism, we established an indirect flow cytometry assay. Since Nef expression in T cells down-regulates the surface receptor CD4, we correlated the decrease in CD4 to the transfer of Nef between these cells. Thus, we co-cultured macrophages expressing varying levels of Nef with a T cell line expressing high levels of CD4 and quantified the changes in CD4 surface expression resulting from Nef transfer. We demonstrate that Nef transfer occurs via a cell-to-cell dependent mechanism that directly correlates with the presence of Myo10-dependent TNTs. Thus, we show that Nef can regulate Myo10 expression, thereby inducing TNT formation, resulting in its own transfer from macrophages to T cells. In addition, we demonstrate that up-regulation of Myo10 induced by Nef also occurs in human monocyte derived macrophages during HIV-1 infection. [ABSTRACT FROM AUTHOR]

Tunneling nanotubes (TNTs) are newly discovered tubular structures between two distant cells that facilitate the intercellular exchange of signals and components. Recent reports show that mesenchymal stem cells (MSCs) can rescue injured target cells and promote recovery from a variety of stresses via TNT-mediated mitochondrial transfer. In this study, we explored how TNTs form between bone marrow MSCs and endothelial cells (ECs) by using a human umbilical cord vein endothelial cell (HUVEC) model. TNT formation between MSCs and HUVECs could be induced by treating HUVECs with cytarabine (Ara-C), and human bone marrow mesenchymal stem cells (hBMMSCs) could transfer mitochondria to injured HUVECs through TNTs. Mitochondrial transfer from hBMMSCs to HUVECs via TNTs rescued the injured HUVECs by reducing apoptosis, promoting proliferation and restoring the transmembrane migration ability as well as the capillary angiogenic capacity of HUVECs. This study provides novel insights into the cell–cell communication between MSCs and ECs and supports the results of prior studies indicating that ECs promote hematopoietic regeneration. An improved understanding of MSC-EC cross-talk will promote the development of MSC-directed strategies for improving EC function and hematopoietic system regeneration following myelosuppressive and myeloablative injuries. [ABSTRACT FROM AUTHOR]

Abstract Mesenchymal stem cells can be used as a novel treatment of ischemic stroke, but their therapeutic effect and mechanism of action require further evaluation. Mitochondrial dysfunction has core functions in ischemia–reperfusion stroke injury. Our recent research has demonstrated that mesenchymal stem cells can transfer their functional mitochondria to injured endothelial cells via tunneling nanotubes in vitro, resulting in the rescue of aerobic respiration and protection of endothelial cells from apoptosis. Therefore, we presume that the mechanisms of mitochondrial protection may be involved in stem cell-mediated rescue of injured cerebral microvasculature and recovery from ischemic stroke. In this study, the middle cerebral artery occlusion and reperfusion surgery were conducted on rats, and mesenchymal stem cells were then engrafted into the injured cerebrovascular system. Our results showed that the host cells of injured cerebral microvasculature accepted the mitochondria transferred from the transplanted stem cells, thereby resulting in significantly improving in mitochondrial activity of injured microvasculature, enhancing angiogenesis, reducing infarct volume, and improving functional recovery. Our data provided the evidence that stem cells can rescue damaged cerebrovascular system in stroke through a mechanism not yet identified. Highlights • Stem cell grafting may rescue damaged cerebrovascular system and promote recovery from ischemic stroke. • Injured cerebral microvasculatures accept the mitochondria transferred from the transplanted stem cells. • Mitochondrial protection mechanism may be involved in stem cell-based therapies for ischemic stroke. [ABSTRACT FROM AUTHOR]

Intercellular communication is essential for the development and maintenance of multicellular organisms. Tunneling nanotubes (TNTs) are a recently recognized means of long and short distance communication between a wide variety of cell types. TNTs are transient filamentous membrane protrusions that connect cytoplasm of neighboring or distant cells. Cytoskeleton fiber‐mediated transport of various cargoes occurs through these tubules. These cargoes range from small ions to whole organelles. TNTs have been shown to contribute not only to embryonic development and maintenance of homeostasis, but also to the spread of infectious particles and resistance to therapies. These functions in the development and progression of cancer and infectious disease have sparked increasing scrutiny of TNTs, as their contribution to disease progression lends them a promising therapeutic target. Herein, we summarize the current knowledge of TNT structure and formation as well as the role of TNTs in pathology, focusing on viral, prion, and malignant disease. We then discuss the therapeutic possibilities of TNTs in light of their varied functions. Despite recent progress in the growing field of TNT research, more studies are needed to precisely understand the role of TNTs in pathological conditions and to develop novel therapeutic strategies. A schematic representation of various cellular cargoes transported by tunneling nanotubes (TNTs). nanotubes [ABSTRACT FROM AUTHOR]

Tunneling nanotubes (TNTs) are intercellular conduits which meet the communication needs of non-adjacent cells situated in the same tissue but at distances up to a few hundred microns. TNTs are unique type of membrane protrusion which contain F-actin and freely hover over substratum in the extracellular space to connect the distant cells. TNTs, known to form through actin remodeling mechanisms, are intercellular bridges that connect cytoplasm of two cells, and facilitate the transfer of organelles, molecules, and pathogens among the cells. In tumor microenvironment, TNTs act as communication channel among cancer, normal, and immune cells to facilitate the transfer of calcium waves, mitochondria, lysosomes, and proteins, which in turn contribute to the survival, metastasis, and chemo-resistance in cancer cells. Recently, TNTs were shown to mediate the transfer of nanoparticles, drugs, and viruses between cells, suggesting that TNTs could be exploited as a potential route for delivery of anti-cancer agents and oncolytic viruses to the target cells. The present review discusses the emerging concepts and role of TNTs in the context of chemo- and radio-resistance with implications in the cancer therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Mitochondria, cytoplasmic organelles originated from endosymbiotic bacteria, can be metaphorically described using “Janus bifrons” image, due to their involvement in life, providing cellular energy and resulting essential even for stem cells, but playing a key role also in cell death. Mitochondria own a maternally inherited genome and are the site of aerobic respiration; they can produce proteins, nucleotides, lipids, steroids and heme and result involved in iron homeostasis. Moreover, mitochondria can generate free radicals, break down waste products and represent the primary source of cellular heat. The size and shape of mitochondria depend on the intracellular metabolic status, from tubular presentation to a blob form in case of irreversible damage. Each mitochondrion carries different sets of DNA; when one set accumulates mutations, it can be replaced by another. It has been widely demonstrated that mitochondrial disorders are involved in many pathologies, including autism, multiple endocrinopathies, diabetes, Alzheimer’s disease, ataxia, Barth’s syndrome, myopathy, and even aging and cancer. Human population is characterized by different mitochondrial DNA haplogroups reflecting the mutations accumulated and useful to characterize genetic diversity. The mitochondrial role also results relevant in pregnancy, providing information about maternal-fetal dyad in physiological and in pathological conditions. Recent evidence suggests that an intriguing bidirectional inter-talk exists between microbiota and mitochondria, influencing cellular homeostasis and metabolism. A recently demonstrated mitochondrial property is the possibility to be transferred from a donor cell to a recipient cell, through a system of tunneling nanotubes. Recently, a promising integrated approach involving omics sophisticate technologies has been applied in mitochondrial pathophysiology. This is still at an early stage, and further studies will clarify such complex genotype-phenotype relationships. In conclusion, mitochondria are not simple energetic organelles but represent dynamic structures communicating with the cell nucleus and even with other cells, influencing metabolism and their targets’ functions. More detailed knowledge of their involvement in disease, even though a combined omics approach, could represent a chance for new therapies.

Tunneling nanotubes (TNT) are dynamic connections between cells, which represent a novel route for cell-to-cell communication. A growing body of evidence points TNT towards a role for intercellular exchanges of signals, molecules, organelles, and pathogens, involving them in a diverse array of functions. TNT form among several cell types, including neuronal cells, epithelial cells, and almost all immune cells. In myeloid cells (e.g., macrophages, dendritic cells, and osteoclasts), intercellular communication via TNT contributes to their differentiation and immune functions. Importantly, TNT enable myeloid cells to communicate with a targeted neighboring or distant cell, as well as with other cell types, therefore creating a complex variety of cellular exchanges. TNT also contribute to pathogen spread as they serve as “corridors” from a cell to another. Herein, we addressed the complexity of the definition and in vitro characterization of TNT in innate immune cells, the different processes involved in their formation, and their relevance in vivo. We also assess our current understanding of how TNT participate in immune surveillance and the spread of pathogens, with a particular interest for HIV-1. Overall, despite recent progress in this growing research field, we highlight that further investigation is needed to better unveil the role of TNT in both physiological and pathological conditions.

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., Competing Interests: AS, SK, KD, KL, PW, SP, RV, LS, NC, CS No competing interests declared, KW Employee of Novocure Ltd, EL has received honoraria and travel expenses for lab-based research talks, and equipment for laboratory-based research, Novocure, Ltd, 2018-2021. Parts of the work presented in this manuscript were performed in collaboration with Novocure, the company that provided the equipment (inovitro and inovitro live) used to apply TTFields, (© 2023, Sarkari et al.)

Tunneling nanotubes (TNTs) are long F-actin-positive plasma membrane bridges connecting distant cells, allowing the intercellular transfer of cellular cargoes, and are found to be involved in glioblastoma (GBM) intercellular crosstalk. Glial fibrillary acid protein (GFAP) is a key intermediate filament protein of glial cells involved in cytoskeleton remodeling and linked to GBM progression. Whether GFAP plays a role in TNT structure and function in GBM is unknown. Here, analyzing F-actin and GFAP localization by laser-scan confocal microscopy followed by 3D reconstruction (3D-LSCM) and mitochondria dynamic by live-cell time-lapse fluorescence microscopy, we show the presence of GFAP in TNTs containing functional mitochondria connecting distant human GBM cells. Taking advantage of super-resolution 3D-LSCM, we show the presence of GFAP-positive TNT-like structures in resected human GBM as well. Using H 2 O 2 or the pro-apoptotic toxin staurosporine (STS), we show that GFAP-positive TNTs strongly increase during oxidative stress and apoptosis in the GBM cell line. Culturing GBM cells with STS-treated GBM cells, we show that STS triggers the formation of GFAP-positive TNTs between them. Finally, we provide evidence that mitochondria co-localize with GFAP at the tip of close-ended GFAP-positive TNTs and inside receiving STS-GBM cells. Summarizing, here we found that GFAP is a structural component of TNTs generated by GBM cells, that GFAP-positive TNTs are upregulated in response to oxidative stress and pro-apoptotic stress, and that GFAP interacts with mitochondria during the intercellular transfer. These findings contribute to elucidate the molecular structure of TNTs generated by GBM cells, highlighting the structural role of GFAP in TNTs and suggesting a functional role of this intermediate filament component in the intercellular mitochondria transfer between GBM cells in response to pro-apoptotic stimuli., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer ME declared a shared affiliation with the authors DC, MS, and FP to the handling editor at the time of review., (Copyright © 2023 Simone, Capobianco, Di Palma, Binda, Legnani, Vescovi, Svelto and Pisani.)

Abstract The LST1 gene is located in the MHC class III cluster between the MHC class I and II regions. While most genes in this cluster have been sufficiently characterised, a definitive function and expression pattern for LST1 still remains elusive. In the present review we describe its promotor, gene organisation, splice variants and expression in human tissues, cell lines and cancer. We focus on LST1 expression in inflammation and discuss known correlations with autoimmune diseases and cancer. Current data on LST1 polymorphisms and their known associations with pathologies are also discussed in detail. We summarize the potential functions that have been described for the full-length LST1 protein including its function as a transmembrane adaptor protein with inhibitory signal transduction and its role as a membrane scaffold facilitating the formation of tunnelling nanotubes. We also discuss further potential functions by compiling all known LST1-interacting proteins. Furthermore, we address knowledge gaps and conflictive issues regarding disease association, non-hematopoietic expression and the discrepancy between RNA and protein expression data. [ABSTRACT FROM AUTHOR]

Various types of intercellular connections that are essential for communication between cells are often utilized by pathogens. Recently, a new type of cellular connection, consisting of long, thin, actin-rich membrane extensions named tunneling nanotubes (TNTs), has been shown to play an important role in cell-to-cell spread of HIV and influenza virus. In the present report, we show that TNTs are frequently formed by cells infected by an alphaherpesvirus, bovine herpesvirus 1 (BoHV-1). Viral proteins, such as envelope glycoprotein E (gE), capsid protein VP26, and tegument protein Us3, as well as cellular organelles (mitochondria) were detected by immunofluorescence and live-cell imaging of nanotubes formed by bovine primary fibroblasts and oropharynx cells (KOP cells). Time-lapse confocal studies of live cells infected with fluorescently labeled viruses showed that viral particles were transmitted via TNTs. This transfer also occurred in the presence of neutralizing antibodies, which prevented free entry of BoHV-1. We conclude that TNT formation contributes to successful cell-to-cell spread of BoHV-1 and demonstrate for the first time the participation of membrane nanotubes in intercellular transfer of a herpesvirus in live cells. [ABSTRACT FROM AUTHOR]

A recently discovered key role of reactive oxygen species (ROS) in mitochondrial traffic has opened a wide alley for studying the interactions between cells, including stem cells. Since its discovery in 2006, intercellular mitochondria transport has been intensively studied in different cellular models as a basis for cell therapy, since the potential of replacing malfunctioning organelles appears to be very promising. In this study, we explored the transfer of mitochondria from multipotent mesenchymal stem cells (MMSC) to neural cells and analyzed its efficacy under normal conditions and upon induction of mitochondrial damage. We found that mitochondria were transferred from the MMSC to astrocytes in a more efficient manner when the astrocytes were exposed to ischemic damage associated with elevated ROS levels. Such transport of mitochondria restored the bioenergetics of the recipient cells and stimulated their proliferation. The introduction of MMSC with overexpressed Miro1 in animals that had undergone an experimental stroke led to significantly improved recovery of neurological functions. Our data suggest that mitochondrial impairment in differentiated cells can be compensated by receiving healthy mitochondria from MMSC.We demonstrate a key role of Miro1, which promotes the mitochondrial transfer from MMSC and suggest that the genetic modification of stem cells can improve the therapies for the injured brain. [ABSTRACT FROM AUTHOR]

Tunneling nanotubes (TNT) are dynamic connections between cells, which represent a novel route for cell-to-cell communication. A growing body of evidence points TNT towards a role for intercellular exchanges of signals, molecules, organelles, and pathogens, involving them in a diverse array of functions. TNT form among several cell types, including neuronal cells, epithelial cells, and almost all immune cells. In myeloid cells (e.g., macrophages, dendritic cells, and osteoclasts), intercellular communication via TNT contributes to their differentiation and immune functions. Importantly, TNT enable myeloid cells to communicate with a targeted neighboring or distant cell, as well as with other cell types, therefore creating a complex variety of cellular exchanges. TNT also contribute to pathogen spread as they serve as "corridors" from a cell to another. Herein, we addressed the complexity of the definition and in vitro characterization of TNT in innate immune cells, the different processes involved in their formation, and their relevance in vivo. We also assess our current understanding of how TNT participate in immune surveillance and the spread of pathogens, with a particular interest for HIV-1. Overall, despite recent progress in this growing research field, we highlight that further investigation is needed to better unveil the role of TNT in both physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Intercellular communication is pivotal in various stages of cancer progression. For smart and effective communication, cancer cells employ diverse modes of messaging that may be further fine-tuned by the microenvironmental changes. Extracellular matrix (ECM) stiffening due to excess deposition and crosslinking of collagen is one of the crucial tumor-microenvironmental changes that influence a plethora of cellular processes, including cell-cell communication. We herein studied the crosstalk between exosomes and tunneling nanotubes (TNT), the two distinct means of cell-cell communication under varying ECM-stiffness conditions. We show that exosomes promote the formation of tunneling nanotubes in breast cancer cells, which results in cellular internet. Interestingly, exosomes drastically increased the fraction of cells connected by TNT; however, they elicited no effect on the number of TNTs per pair of connected cells or the length of TNT. The observed pro-TNT effects of exosomes were found to be ECM-stiffness dependent. ECM-stiffness tuned exosomes were found to promote TNT formation predominantly via the 'cell dislodgment model'. At the molecular level, exosomal thrombospondin-1 was identified as a critical pro-TNT factor. These findings underline the influence of ECM stiffening on two diverse modes of cell communication and their interdependence, which may have significant implications in cancer biomedical research. [Display omitted] • Exosomes promote the formation of cellular internet by promoting TNT formation. • Pro-TNT effects of exosomes are dependent on extracellular matrix stiffness. • ECM Stiffness-tuned exosomes promote TNT formation majorly by cell dislodgement model. • The cargo transport along TNTs is faster in exosomes-induced TNTs. • Exosomal thrombospondin-1 plays a vital role in stimulating TNT formation in breast cancer cells. [ABSTRACT FROM AUTHOR]

The process of intercellular communication seems to have been a highly conserved evolutionary process. Higher eukaryotes use several means of intercellular communication to address both the changing physiological demands of the body and to fight against diseases. In recent years, there has been an increasing interest in understanding how cell-derived nanovesicles, known as extracellular vesicles (EVs), can function as normal paracrine mediators of intercellular communication, but can also elicit disease progression and may be used for innovative therapies. Over the last decade, a large body of evidence has accumulated to show that cells use cytoplasmic extensions comprising open-ended channels called tunneling nanotubes (TNTs) to connect cells at a long distance and facilitate the exchange of cytoplasmic material. TNTs are a different means of communication to classical gap junctions or cell fusions; since they are characterized by long distance bridging that transfers cytoplasmic organelles and intracellular vesicles between cells and represent the process of heteroplasmy. The role of EVs in cell communication is relatively well-understood, but how TNTs fit into this process is just emerging. The aim of this review is to describe the relationship between TNTs and EVs, and to discuss the synergies between these two crucial processes in the context of normal cellular cross-talk, physiological roles, modulation of immune responses, development of diseases, and their combinatory effects in tissue repair. At the present time this review appears to be the first summary of the implications of the overlapping roles of TNTs and EVs. We believe that a better appreciation of these parallel processes will improve our understanding on how these nanoscale conduits can be utilized as novel tools for targeted therapies.

The cellular response to the recombinant NS1 protein of West Nile virus (NS1WNV) was studied using three different cell types: Vero E6 simian epithelial cells, SH-SY5Y human neuroblastoma cells, and U-87MG human astrocytoma cells. Cells were exposed to two different forms of NS1WNV: (i) the exogenous secreted form, sNS1WNV, added to the extracellular milieu; and (ii) the endogenous NS1WNV, the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1WNV varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells. Addition of sNS1WNV to the cell culture medium resulted in significant remodeling of the actin filament network in Vero E6 cells. This effect was not observed in SH-SY5Y and U-87MG cells, implying that the cellular uptake of sNS1WNV and actin network remodeling were dependent on cell type. In the three cell types, NS1WNV-expressing cells formed filamentous projections reminiscent of tunneling nanotubes (TNTs). These TNT-like projections were found to contain actin and NS1WNV proteins. Interestingly, similar actin-rich, TNT-like filaments containing NS1WNV and the viral envelope glycoprotein EWNV were also observed in WNV-infected Vero E6 cells.

Tunneling nanotubes (TNTs) are thin membranous tubes that interconnect cells, representing a novel route of cell-to-cell communication and spreading of pathogens. TNTs form between many cell types, yet their inception mechanisms remain elusive. We review in this study general concepts related to the formation and stability of membranous tubular structures with a focus on a deviatoric elasticity model of membrane nanodomains. We review experimental evidence that tubular structures initiate from local membrane bending facilitated by laterally distributed proteins or anisotropic membrane nanodomains. We further discuss the numerical results of several theoretical and simulation models of nanodomain segregation suggesting the mechanisms of TNT inception and stability. We discuss the coupling of nanodomain segregation with the action of protruding cytoskeletal forces, which are mostly provided in eukaryotic cells by the polymerization of f-actin, and review recent inception mechanisms of TNTs in relation to motor proteins.

Many lines of evidence suggest that the Parkinson's disease (PD)-related protein a-synudein (a-SYN) can propagate from cell to cell in a prion-like manner. However, the cellular mechanisms behind the spreading remain elusive. Here, we show that human astrocytes derived from embryonic stem cells actively transfer aggregated a-SYN to nearby astrocytes via direct contact and tunneling nanotubes (TNTs). Failure in the astrocytes' lysosomal digestion of excess a-SYN oligomers results in a-SYN deposits in the trans-Golgi network followed by endoplasmic reticulum swelling and mitochondrial disturbances. The stressed astrocytes respond by conspicuously sending out TNTs, enabling intercellular transfer of a-SYN to healthy astrocytes, which in return deliver mitochondria, indicating a TNTmediated rescue mechanism. Using a pharmacological approach to inhibit TNT formation, we abolished the transfer of both a-SYN and mitochondria. T ogether, our results highlight the role of astrocytes in a-SYN cell-to-cell transfer, identifying possible pathophysiological events in the PD brain that could be of therapeutic relevance. [ABSTRACT FROM AUTHOR]

Background: Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. Methods: We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. Results: The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. Conclusions: This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication. [ABSTRACT FROM AUTHOR]

Tunneling nanotubes (TNTs) are long bridge-like structures that connect eukaryotic cells and mediate intercellular communication. We found earlier that the conserved alphaherpesvirus US3 protein kinase induces long cell projections that contact distant cells and promote intercellular virus spread. In this report, we show that the US3-induced cell projections constitute TNTs. In addition, we report that US3-induced TNTs mediate intercellular transport of information (e.g., green fluorescent protein [GFP]) in the absence of other viral proteins. US3-induced TNTs are remarkably stable compared to most TNTs described in the literature. In line with this, US3-induced TNTs were found to contain stabilized (acetylated and detyrosinated) microtubules. Transmission electron microscopy showed that virus particles are individually transported in membrane-bound vesicles in US3-induced TNTs and are released along the TNT and at the contact area between a TNT and the adjacent cell. Contact between US3-induced TNTs and acceptor cells is very stable, which correlated with a marked enrichment in adherens junction components beta-catenin and E-cadherin at the contact area. These data provide new structural insights into US3-induced TNTs and how they may contribute to intercellular communication and alphaherpesvirus spread. IMPORTANCE Tunneling nanotubes (TNT) represent an important and yet still poorly understood mode of long-distance intercellular communication. We and others reported earlier that the conserved alphaherpesvirus US3 protein kinase induces long cellular protrusions in infected and transfected cells. Here, we show that US3- induced cell projections constitute TNTs, based on structural properties and transport of biomolecules. In addition, we report on different particular characteristics of US3-induced TNTs that help to explain their remarkable stability compared to physiological TNTs. In addition, transmission electron microscopy assays indicate that, in infected cells, virions travel in the US3-induced TNTs in membranous transport vesicles and leave the TNT via exocytosis. These data generate new fundamental insights into the biology of (US3-induced) TNTs and into how they may contribute to intercellular virus spread and communication. [ABSTRACT FROM AUTHOR]

Macrophages are important cells of the innate immune system. They exhibit a high plasticity in phenotypes and play a major role in healing by initiating the early inflammatory reactions via the pro-inflammatory M1 phenotype. The anti-inflammatory M2 phenotype is assumed to induce regenerative processes and vascularization in subsequent tissue repair. Especially for regenerative processes, their interplay with multipotent human mesenchymal stromal cells (hMSCs) is decisive. Accordingly, in vitro co-culture models of these cell types are an important starting point for unraveling regenerative mechanisms. In our study, we compared direct co-culture, transwell-systems, and the use of conditioned medium to investigate the mitochondria transfer between the two cell types and the influence of hMSCs' presence on the phagocytic activity of macrophages. Using flow cytometry and fluorescence microscopy, we visualized the transfer of mitochondria in both directions: from hMSCs to macrophages and most notably also vice versa. Both cell types release mitochondria and internalize them in direct contact via tunneling nanotubes, as well as in indirect contact due to extracellular vesicles (EVs). Mitochondria were non-directionally released into the medium and could be transferred via conditioned medium. After three hours of direct and indirect co-culture, the majority of the cells showed a mitochondrial uptake. Co-cultivation also led to an increase of phagocytic activity of macrophages, with the highest phagocytic rate after 48 h and most pronounced in direct co-cultivation. [ABSTRACT FROM AUTHOR]

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton's jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine. [ABSTRACT FROM AUTHOR]

Osteoclasts are multinucleated bone-resorbing cells with a dynamic actin cytoskeleton. Osteoclasts are derived from circulating mononuclear precursors. Confocal and stimulated emission depletion (STED) super-resolution microscopy was used to investigate peripheral blood-derived human osteoclasts cultured on glass surfaces. STED and confocal microscopy demonstrated that the actin was curved and branched, for instance, in the vicinity of membrane ruffles. The overall architecture of the curved actin network extended from the podosomes to the top of the cell. The other novel finding was that a micrometer-level tube containing actin bridged the osteoclasts well above the level of the culture glass. The actin filaments of the tubes originated from the network of curved actin often surrounding a group of nuclei. Furthermore, nuclei were occasionally located inside the tubes. Our findings demonstrated the accumulation of c-Src, cortactin, cofilin, and actin around nuclei suggesting their role in nuclear processes such as the locomotion of nuclei. ARP2/3 labeling was abundant at the substratum level of osteoclasts and in the branched actin network, where it localized to the branching points. We speculate that the actin-containing tubes of osteoclasts may provide a means of transportation of nuclei, e.g., during the fusion of osteoclasts. These novel findings can pave the way for future studies aiming at the elucidation of the differentiation of multinucleated osteoclasts. [ABSTRACT FROM AUTHOR]

Infiltrating macrophages in tumor microenvironment, through their secreted cytokines and growth factors, regulate several processes of cancer progression such as cancer cell survival, proliferation, invasion, metastasis and angiogenesis. Recently, intercellular cytoplasmic bridges between cancer cells referred as tunneling nanotubes (TNTs) have been recognized as novel mode of intercellular communication between cancer cells. In this study, we investigated the effect of inflammatory mediators present in conditioned medium derived from macrophages on the formation of TNTs in breast adenocarcinoma cells MCF-7. Results show that treatment with macrophage conditioned medium (MɸCM) not only enhanced TNT formation between cells but also stimulated the release of independently migrating viable cytoplasmic fragments, referred to as microplasts, from MCF-7 cells. Time lapse microscopy revealed that microplasts were released from parent cancer cells in extracellular space through formation of TNT-like structures. Mitochondria, vesicles and cytoplasm could be transferred from parent cell body to microplasts through connecting TNTs. The microplasts could also be resorbed into the parent cell body by retraction of the connecting TNTs. Microplast formation inhibited in presence cell migration inhibitor, cytochalasin-B. Notably by utilizing migratory machinery within microplasts, distantly located MCF-7 cells formed several TNT based intercellular connections, leading to formation of physically connected network of cells. Together, these results demonstrate novel role of TNTs in microplast formation, novel modes of TNT formation mediated by microplasts and stimulatory effect of MɸCM on cellular network formation in MCF-7 cells mediated through enhanced TNT and microplast formation. [ABSTRACT FROM AUTHOR]

Tunneling nanotubes ( TNTs) are membranous conduits for direct cell-to-cell communication. Until the past decade, little had been known about their composite structure, function, and mechanisms of action in both normal physiologic conditions as well as in disease states. Now TNTs are attracting increasing interest for their key role(s) in the pathogenesis of disease, including neurodegenerative disorders, inflammatory and infectious diseases, and cancer. The field of TNT biology is still in its infancy, but inroads have been made in determining potential mechanisms and function of these remarkable structures. For example, TNTs function as critical conduits for cellular exchange of information; thus, in cancer, they may play an important role in critical pathophysiologic features of the disease, including cellular invasion, metastasis, and emergence of chemotherapy drug resistance. Although the TNT field is still in a nascent stage, we propose that TNTs can be investigated as novel targets for drug-based treatment of cancer and other diseases. [ABSTRACT FROM AUTHOR]

The field of neuroscience is rapidly progressing, continuously uncovering new insights and discoveries. Among the areas that have shown immense potential in research, tunneling nanotubes (TNTs) have emerged as a promising subject of study. These minute structures act as conduits for the transfer of cellular materials between cells, representing a mechanism of communication that holds great significance. In particular, the interplay facilitated by TNTs among various cell types within the brain, including neurons, astrocytes, oligodendrocytes, glial cells, and microglia, can be essential for the normal development and optimal functioning of this complex organ. The involvement of TNTs in neurodegenerative disorders, such as Alzheimer's disease, Huntington's disease, and Parkinson's disease, has attracted significant attention. These disorders are characterized by the progressive degeneration of neurons and the subsequent decline in brain function. Studies have predicted that TNTs likely play critical roles in the propagation and spread of pathological factors, contributing to the advancement of these diseases. Thus, there is a growing interest in understanding the precise functions and mechanisms of TNTs within the nervous system. This review article, based on our recent work on Rhes-mediated TNTs, aims to explore the functions of TNTs within the brain and investigate their implications for neurodegenerative diseases. Using the knowledge gained from studying TNTs could offer novel opportunities for designing targeted treatments that can stop the progression of neurodegenerative disorders.

Long-range intercellular communication between Central Nervous System (CNS) cells is an essential process for preserving CNS homeostasis. Paracrine signaling, extracellular vesicles, neurotransmitters and synapses are well-known mechanisms involved. A new form of intercellular crosstalk mechanism based on Tunneling Nanotubes (TNTs), suggests a new way to understand how neural cells interact with each other in controlling CNS functions. TNTs are long intercellular bridges that allow the intercellular transfer of cargoes and signals from one cell to another contributing to the control of tissue functionality. CNS cells communicate with each other via TNTs, through which ions, organelles and other signals are exchanged. Unfortunately, almost all these results were obtained through 2D in-vitro models, and fundamental mechanisms underlying TNTs-formation still remain elusive. Consequently, many questions remain open, and TNTs role in CNS remains largely unknown. In this review, we briefly discuss the state of the art regarding TNTs identification and function. We highlight the gaps in the knowledge of TNTs and discuss what is needed to accelerate TNTs-research in CNS-physiology. To this end, it is necessary to: 1) Develop an ad-hoc TNTs-imaging and software-assisted processing tool to improve TNTs-identification and quantification, 2) Identify specific molecular pathways involved into TNTs-formation, 3) Use in-vitro 3D-CNS and animal models to investigate TNTs-role in a more physiological context pushing the limit of live-microscopy techniques. Although there are still many steps to be taken, we believe that the study of TNTs is a new and fascinating frontier that could significantly contribute to deciphering CNS physiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Capobianco, Simone, Svelto and Pisani.)

Polyclonal B cell activation and the resulting hypergammaglobulinemia are a detrimental consequence of visceral leishmaniasis (VL); however, the mechanisms underlying this excessive production of nonprotective antibodies are still poorly understood. Here, we show that a causative agent of VL, Leishmania donovani, induces CD21-dependent formation of tunneling nanotubule (TNT)-like protrusions in B cells. These intercellular connections are used by the parasite to disseminate among cells and propagate B cell activation, and close contact both among the cells and between B cells and parasites is required to achieve this activation. Direct contact between cells and parasites is also observed in vivo , as L. donovani can be detected in the splenic B cell area as early as 14 days postinfection. Interestingly, Leishmania parasites can also glide from macrophages to B cells via TNT-like protrusions. Taken together, our results suggest that, during in vivo infection, B cells may acquire L. donovani from macrophages via TNT-like protrusions, and these connections are subsequently exploited by the parasite to disseminate among B cells, thus propagating B cell activation and ultimately leading to polyclonal B cell activation. IMPORTANCE Leishmania donovani is a causative agent of visceral leishmaniasis, a potentially lethal disease characterized by strong B cell activation and the subsequent excessive production of nonprotective antibodies, which are known to worsen the disease. How Leishmania activates B cells is still unknown, particularly because this parasite mostly resides inside macrophages and would not have access to B cells during infection. In this study, we describe for the first time how the protozoan parasite Leishmania donovani induces and exploits the formation of protrusions that connect B lymphocytes with each other or with macrophages and glides on these structures from one cell to another. In this way, B cells can acquire Leishmania from macrophages and become activated upon contact with the parasites. This activation will then lead to antibody production. These findings provide an explanation for how the parasite may propagate B cell activation during infection., Competing Interests: The authors declare no conflict of interest.