Invisible Bridges: Unveiling the Role and Prospects of Tunneling Nanotubes in Cancer Therapy

Abstract

Tunneling nanotubes (TNTs) are essential intercellular communication channels that significantly impact cancer pathophysiology, affecting tumor progression and resistance. This review methodically examines the mechanisms of TNTs formation, their structural characteristics, and their functional roles in material and signal transmission between cells. Highlighting their regulatory functions within the tumor microenvironment, TNTs are crucial for modulating cell survival, proliferation, drug resistance, and immune evasion. The review critically evaluates the therapeutic potential of TNTs, focusing on their applications in targeted drug delivery and gene therapy. It also proposes future research directions to thoroughly understand TNTs biogenesis, identify cell-specific molecular targets, and develop advanced technologies for the real-time monitoring of TNTs. By integrating insights from molecular biology, nanotechnology, and immunology, this review highlights the transformative potential of TNTs in advancing cancer treatment strategies.

Introduction

Tunneling nanotubes (TNTs) are unique intercellular conduits characterized by slender, nanoscale structures formed by cellular membranes, connecting distant cells.¹ Composed primarily of F-actin and suspended within the extracellular matrix, TNTs facilitate the transfer of various substances, including organelles like mitochondria,² proteins,³ and RNA.^4,5 This capability allows cells to share resources, enhancing survival and functional efficiency.⁶ Moreover, TNTs transmit cellular signals such as calcium ion waves and electrical impulses, enhancing communication and coordination between cells.⁷ Playing a crucial role in the pathogenesis of diseases like cancer, neurodegenerative disorders, and viral infections, TNTs offer a novel perspective on the intricate mechanisms of cellular interaction and hold potential as targets for therapeutic strategies.⁸

TNTs are emerging as pivotal players in tumor progression and therapy due to their role in facilitating direct cellular communication and enhancing metabolic functions and antiapoptotic capabilities in tumor cells.⁹ By transferring critical organelles and molecules such as mitochondria and proteins, TNTs not only bolster the invasive and metastatic potential of tumor cells but also assist in the intercellular coordination and cooperation necessary for tumor aggressiveness.¹⁰ Additionally, TNTs contribute to the tumor microenvironment (TME) by transporting factors responsible for chemotherapeutic drug resistance.¹¹⁻¹³ Given their significant involvement in tumor development, targeting TNTs to inhibit their formation or function may represent an effective anticancer strategy.¹⁴ Furthermore, TNTs offer a novel method for delivering therapeutic drugs and genetic material directly to tumor cells, enhancing the specificity and efficacy of treatments while potentially reducing damage to surrounding healthy tissues.¹⁵

TNTs represent a burgeoning field in cancer research, unveiling novel mechanisms of intercellular interaction among tumor cells. Future studies are poised to further delineate the roles of TNTs across various cancer types and to develop TNT-based diagnostic and therapeutic approaches. This review comprehensively examines the molecular composition, structural characteristics, and functional roles of TNTs in material and signal transmission, highlighting their key role in enhancing tumor cell metabolism, drug resistance, and immune evasion. Additionally, it emphasizes the potential of TNTs as drug delivery systems and their promising applications in targeted therapies. Key future research directions include detailed studies of TNTs biogenesis, heterogeneity analysis, interactions with the TME, and assessments of clinical translatability to offer more precise and effective solutions for cancer therapy.

TNTs: Intercellular Biological Bridges—Structural Complexity and Diversity in Material Transfer

TNTs have distinct features that set them apart from other cell protrusions like filopodia, cilia, and cytonemes in both structure and function. TNTs are typically thinner and longer compared to filopodia and invadopodia and are internally supported by an F-actin cytoskeleton.¹ Unlike these other protrusions, TNTs do not adhere to the substratum.¹⁶ More importantly, they have open endings in the plasma membranes of the two connected cells, enabling the direct exchange of a wide variety of cargo between their cytoplasms.³ Additionally, electrical signals transmitted via TNTs are more selective compared to those propagated through gap junctions.¹⁷

TNTs are dynamic yet fragile structures predominantly composed of F-actin. Some TNTs, however, also incorporate microtubules or intermediate filaments, which not only increase their diameter but also enhance their capacity to transport organelles.^18,19 Research on B lymphoma cells has revealed that TNTs facilitate the bidirectional transport of mitochondria, a process orchestrated by the collaborative efforts of various cytoskeletal motor proteins.²⁰ These proteins, which include kinesin and myosin VI, operate along both microtubules and actin filaments, benefiting from the diverse arrangement of the primary actin cytoskeleton within TNTs. Microtubules lend added stability and prolong the lifespan of TNTs, while F-actin provides essential structural support. Ongoing studies have further identified additional structural elements within TNTs, such as glial fibrillary acidic protein (GFAP), found in TNTs of glioblastoma (GBM) cells.²¹ Moreover, research highlights the critical role of paxillin in TNTs formation, with other proteins like EGFR, c-Met, and β1 integrin also localized within these structures.²² Collectively, these findings underscore the structural and compositional diversity of TNTs and illuminate their formation as a complex network mechanism.

TNTs serve as a versatile intercellular communication tool capable of transporting a wide range of substances, including organelles such as mitochondria,^23,24 lysosomes,²⁵ and lipid droplets²⁶ along with vesicles,²⁷ membrane surface receptors,²⁸ proteins,²⁹ viruses,³⁰ RNA,^4,5,31,32 and intercellular signals.^33,34 This diversity of transportable substances has been documented extensively in the literature.^3,6 Among these, the transport of mitochondria has been particularly well-studied. Tumor cells obtain mitochondria from nontumor cells via TNTs, enhancing their metabolic capacity and promoting malignant behaviors.^23,24 Moreover, various types of nucleic acids, including mRNA,⁵ noncoding RNA,⁴ viral RNA,³² and mitochondrial RNA,³¹ can also be transferred via TNTs. This transfer, either unidirectional or bidirectional, involves cytoplasmic continuity, setting it apart from other intercellular transfer mechanisms such as apoptotic bodies and extracellular vesicles.^35,36 Studies using a human–mouse coculture model have demonstrated that full-length mRNA can be transferred between cells through TNTs.³⁷ Transcriptomic analysis shows that the amount of mRNA transferred is closely correlated to the endogenous expression levels of the donor genes. This contact-dependent RNA transfer mechanism can substantially modify the natural transcriptome of the recipient cells, providing new avenues for the in vivo delivery of mRNA-based therapeutics. Additionally, oncogenic KRAS can be transferred between colon cancer cells via TNTs, increasing cellular and tumor heterogeneity.³⁸ Brain-tumor-initiating cells can transfer mitochondria to astrocytes via TNTs, significantly altering the transcriptome of the recipient cells and greatly enhancing their proliferation.⁹

The TME encompasses a diverse array of cells, with TNTs serving as vital communication channels between cancer cells, normal cells, and immune cells. These structures significantly influence cancer cell survival, metastasis, and chemoresistance. Therefore, a deeper exploration of the mechanisms underlying TNTs formation and the factors influencing cargo selection and transfer is essential for enhancing our knowledge of tumor progression and for refining therapeutic approaches.

The Mysteries of TNTs: A Symphony of Environmental, Physical, and Molecular Mechanisms

Environmental Influences: Shaping the Formation of TNTs by External Factors

TNTs have been observed to form in the TME in response to various stressors or stimuli, facilitating cellular adaptation to adverse conditions. Studies have demonstrated that different cancer cell models can spontaneously generate TNTs when cultured in vitro, in contrast to nontumorigenic epithelial cells like HBEC3, which seldom do.³⁹ A range of stress conditions, including oxidative stress,^21,40 pharmacological treatments,⁴¹ nutritional deficiencies,⁴² infections,⁴³ hypoxia,⁴⁴ and radiation exposure,⁴⁵ are known to stimulate TNTs formation. Additionally, specific environmental conditions such as low serum levels, high glucose concentrations, and acidic culture environments also promote the formation of TNTs.⁴⁶ For instance, treatment with pyrrolidone not only results in extensive TNTs networks but also exhibits antiproliferative effects at submicromolar concentrations.⁴¹ Similarly, treatments with 5-fluorouracil, which induces thymidine deficiency, trigger TNTs formation in MCF-7 breast cancer cells due to nutritional deprivation.⁴² In prostate cancer, the blockade of androgen receptors and metabolic stress are shown to induce TNTs formation, with stress-induced proteins such as clusterin and YB-1 localizing within TNTs to facilitate bidirectional transport.⁴⁷ Moreover, conditioned media from macrophages have been shown to induce TNTs formation between tumor cells, suggesting that the formation of TNTs may be a response to immune stress in cancer cells.^10,48,49 These findings underscore the intricate relationship between cellular stress responses and TNTs formation, highlighting the potential of targeting TNTs in developing cancer treatment strategies.

Physics Perspective: Exploring the Theoretical Foundations of TNTs Formation

The formation of TNTs is governed by two primary mechanisms: cellular displacement and protrusion of actin structures. Inhibition of actin polymerization significantly impedes the formation of TNTs, underscoring the vital role of actin in both processes. Additionally, the presence of microtubules is not essential for the formation of TNTs in all cell types, as many TNTs do not incorporate microtubules.^50,51 However, research indicates that TNTs containing both microtubules and F-actin are more robust and stable than those containing only F-actin.⁵²

Theoretical physics has greatly advanced our understanding of TNTs, particularly by predicting their stability and potential formation mechanisms prior to the availability of direct empirical evidence. Theoretical approaches have elucidated how the local curvature of the cell membrane and the distribution of proteins or heterogeneous nanodomains on the membrane initiate TNTs formation.⁵² Furthermore, the polymerization of F-actin and the dynamics of the cytoskeleton are crucial factors. The stability of TNTs is reinforced by membrane continuity, which is closely related to the type of formation—through either cellular displacement or actin protrusion—and is influenced by the mechanical properties of the cell membrane, such as the balance of forces described in elasticity models of the membrane. Specific proteins and lipids play key roles in stabilizing TNTs by regulating the membrane curvature. Recent experimental studies have provided empirical validation of these theoretical models. For instance, Tsai et al. demonstrated that IRSp53 clustering, driven by membrane curvature, recruits the actin polymerase VASP, leading to localized actin filament assembly and membrane protrusion formation.⁵³ This study highlights the crucial role of curvature-sensing proteins like IRSp53 in the initiation of actin-based structures, supporting the theoretical predictions regarding TNTs formation. Similarly, Madarász et al. provided experimental evidence showing that IRSp53 contributes to actin-based force generation within membrane nanotubes.⁵⁴ This supports the theoretical notion that local curvature and protein distribution on the cell membrane are critical factors in TNTs formation. Their work emphasizes the importance of actin dynamics and membrane curvature sensing in maintaining the structural integrity and functionality of TNTs. Future research will benefit from ongoing advancements in the theoretical models of biomembranes and their interactions with biomolecules, further enhancing our understanding of the physical and biological properties of TNTs.

Molecular Mechanisms Underlying TNTs Formation

The formation of TNTs is governed by a complex network of intracellular signals and protein interactions that orchestrate the dynamic reorganization of the cytoskeleton and coordinate activities across various signaling pathways (Table 1 and Figure 1). Additionally, TNTs exhibit significant heterogeneity.^55,56 This intricate orchestration underpins the pivotal roles of TNTs under both physiological functions and pathological conditions.

Table 1. Key Molecular Mechanisms and Signaling Pathways Influencing TNTs Formation.

Molecular Mechanism	Role in TNTs Formation	Reference(s)
Rho family of GTPases (Rac1, Cdc42, RhoA)	Initiate actin reorganization crucial for TNTs biogenesis	(57)
WASP, WAVE2	Effectors of Cdc42 and Rac1, regulate actin polymerization through the Arp2/3 complex	(58)
CDC42/IRSp53/VASP network	Promotes filopodial protrusions but negatively regulates TNTs formation	(58)
Rhes	Promotes TNTs formation	(59−61)
Eps8	Enhances TNTs formation through actin bundling activity	(62)
Ca2+-calmodulin-dependent protein kinase II	Stabilizes F-actin, binds G-actin to extend TNTs’ half-life	(63)
Myosin X	Linked to TNTs formation, but inhibition does not completely prevent TNTs	(56)
Rab family proteins (Rab11a, Rab8a)	Involved in cascade reactions facilitating TNTs formation; Rab8 also transfers transferrin via TNTs	(64), 65
Rab35 and associated proteins (ACAP2, MICAL-L1, ARF6, EHD1)	Regulates TNTs formation through cascading mechanism	(66)
RalGPS2 and interacting proteins (Akt, PDK1, LST1, RalA)	Facilitates nanotube formation, especially in specific cancer cells	(67), 68
M-sec and related pathways (filamin, Cdc42, exocyst complex)	Initiates TNTs formation by promoting membrane protrusions	(55)
Cell adhesion molecules (ICAM1)	Plays a critical role in the stability and functionality of TNTs	(71)
Transcription factor p53	Regulates TNTs formation through pathways involving EGFR, Akt1, PI3K, and mTOR	(72), 73
MARCKS phosphorylation	Regulates TNTs formation through the phosphorylation of its effector domain	(74)
MAPK pathway activated by EGFR	Critical for the formation of TNTs between ovarian cancer cells	(10)
OXER1 receptor activation	Activates TNTs formation through the Gβγ/PKCα/FARP1/Cdc42 axis in the adrenocortical carcinoma cell line	(75)
PI3K/AKT, EPS8 signaling pathways	Critical for promoting TNTs formation	(22), 47
HGF/c-Met/β1-integrin signaling pathway	Regulates TNTs formation in nonsmall cell lung cancer	(76)
Cx43-associated cancer pathways	Plays a significant role in TNTs formation in various cancers	(78)
Ras/MAPK/MEK and Arp2/3 complex pathways	Crucial in regulating TNTs formation	(22)
HGF and EGF coactivation	Both HGF and EGF induce TNTs, with higher rates when combined	(22)
Intracellular free calcium ions	Regulate the balance between the growth and contraction of TNTs, which is essential for effective cargo transport	(28)

Figure 1.

Molecular mechanisms of TNTs formation. (Drawn with Figdraw.)

TNTs formation is prompted by external and internal stimuli, leading to cellular protrusions governed by key cytoskeletal regulators, particularly the Rho family of GTPases such as Rac1, Cdc42, and RhoA.⁵⁷ These proteins are pivotal in initiating actin reorganization critical for TNTs biogenesis. Specifically, Cdc42 and Rac1, through their effector molecules WASP and WAVE2, promote actin polymerization via the Arp2/3 complex, essential for TNTs formation.⁵⁸ Rac1’s presence within TNTs structures and Cdc42’s role in the initiation phase highlight their significant roles. Inhibition of these GTPases can reduce both the quantity and lifespan of TNTs.⁵⁸ Additionally, the CDC42/IRSp53/VASP network, typically promoting filopodial protrusions, paradoxically impairs TNTs formation and vesicular transfer between cells.⁵⁸

Apart from that, Rhes, a homologue of Ras abundantly found in certain neurons, significantly promotes TNTs production. Research indicates that neurons transfected with GFP-Rhes show increased cellular extensions in the striatum compared to those with GFP alone.⁵⁹ Moreover, Rhes can transfer to nearby cells through TNTs, facilitating the transport of proteins like the Huntington disease protein via “TNT-like Rhes tunnels”.⁵⁹ In addition, the SUMO E3-like domain at the C-terminal ubiquitin-like modification and serine 33 in the N-terminal domain of Rhes synergistically induce TNTs production, which can be completely inhibited by cell relaxin D.^60,61

Another actin-regulatory protein, Eps8, enhances TNTs formation through its bundling activity, distinct from its typical capping function.⁶² Furthermore, the β isoform of Ca²⁺-calmodulin-dependent protein kinase II stabilizes F-actin and binds G-actin to extend TNTs’ half-life.⁶³

The formation of TNTs is also influenced by proteins associated with the cytoskeleton. Myosin X is linked to prominent TNTs formation, although inhibiting myosin X does not entirely prevent TNTs formation,⁵⁶ indicating multiple biogenesis mechanisms. Small GTPases of the Rab family, particularly Rab11a and Rab8a, facilitate TNTs formation through cascade reactions, with VAMP3 regulating the process downstream of Rab8a.⁶⁴ Rab8’s role extends to the transfer of transferrin from cancer cells to fibroblasts via TNTs.⁶⁵ Additionally, Rab35 collaborates with ACAP2, MICAL-L1, ARF6, and EHD1 in regulating TNTs formation through a cascading mechanism.⁶⁶ RalGPS2, a RalA GTPase guanine nucleotide exchange factor, interacts with Akt, PDK1, LST1, and RalA to facilitate nanotube formation, notably within specific cancer cell lines such as the urinary epithelial cancer-derived 5637 cell line.^67,68

Membrane-associated proteins and transcription factors also play pivotal roles. M-sec and LST1 are significantly involved in the initiation of TNTs,^67,69 with M-sec activating the small GTPase Ral-A and promoting membrane protrusions through downstream effectors like filamin, Cdc42, and the exocyst complex.⁷⁰ Cell adhesion molecules like ICAM1⁷¹ and transcription factor p53 are critical in stabilizing and regulating TNTs formation, where p53 activation across various cell lines regulates TNTs formation through pathways involving EGFR, Akt1, PI3K, and mTOR.^72,73 Recent research has revealed that the myristoylated alanine-rich C-kinase substrate (MARCKS) becomes a novel regulatory factor for TNTs through the phosphorylation of its effector domain. U87-NP cells exhibit aberrant TNTs, characterized by rodlike structures at their termini. Further investigation into the functionality of TNTs in U87 mutants is necessary to determine whether the lack of phosphorylation in MARCKS results in loss of TNTs functionality.⁷⁴

Current research has also identified novel signaling pathways critical for TNTs formation. The MAPK pathway, activated by EGFR, is essential in ovarian cancer cells,¹⁰ and the activation of the OXER1 receptor in a human adrenocortical carcinoma cell line drives TNTs formation through the Gβγ/PKCα/FARP1/Cdc42 axis.⁷⁵ Additionally, the PI3K/AKT and EPS8 signaling pathways are crucial in promoting TNTs formation,^22,47 along with the HGF/c-Met/β1-integrin pathway in nonsmall cell lung cancer.⁷⁶

For TNTs to successfully transport cargo, not only initiation mechanisms but also mechanisms to maintain stability and to facilitate contraction after transport completion are required. Additionally, the level of intracellular free calcium ions plays a critical role in regulating the balance between the growth and contraction of TNTs.²⁸

In conclusion, the formation of TNTs is governed not only by actin-associated molecules but also by the mechanical forces exerted by cells. These forces emerge from the dynamic interplay between actin cytoskeleton rearrangement, adhesion protein interactions, and cellular migration.⁷⁷ Crucially, the effective coordination of membrane dynamics between donor and recipient cells is vital. These findings highlight the complexity of TNTs formation, which is regulated by a myriad of signaling molecules and pathways contributing to their biogenesis and functionality. Such intricate knowledge is essential for the development of innovative therapeutic strategies, especially in scenarios in which TNTs play a vital role in facilitating direct intercellular communication and material transport.

Exploring the Science of TNTs: Advanced Research Methods and Technological Insights

Table 2 presents a summary of the in vivo and in vitro experimental techniques and methods used for studying TNTs. TNTs, as a unique form of intercellular communication, continue to be revealed through various experimental techniques. One method for observing TNTs involves the use of a modified Transwell assay system. This system effectively excludes the transfer of cytoplasmic diffusible substances between cells, such as exosomes, microvesicles, and gap junctions, reducing exosome transport by over 95%. The use of heparin blockade can further hinder the presumed receptor cells’ uptake of exosomes, thus effectively inhibiting the transfer of extracellular vesicles. This method is particularly suited for studying the selective cellular communication mediated by TNTs.⁷⁹

Table 2. Summary of In Vivo and In Vitro Experimental Techniques and Methods for Studying TNTs.

Method/Technique	Key Features	Primary Use	Reference(s)
Modified transwell assay	Excludes diffusible substances, focuses on TNTs	Studying selective TNT-mediated communication	(79)
gCW STED nanoscopy and time-gated confocal microscopy	Minimizes phototoxicity, monitors dynamic transfers	Observing TNTs structure and cellular interactions	(80)
TP-FLIM-FRET	Combines two-photon excitation with FRET for high precision imaging	Detecting material transfer and protein interactions within TNTs	(81)
High-resolution live-cell STED microscopy	Measures TNTs characteristics like diameter, lifecycle, and morphology	Real-time analysis of TNTs dynamics in tumor models	(82)
Super-resolution live-cell STED nanoscopy	Used under 2D culture conditions to study mitochondrial interactions	Studying mitochondrial heat shock protein functions via TNTs	(84)
Optical tomographic microscopy	Detects transparent objects, no need for staining	Revealing mitochondrial transfer and apoptosis induction via TNTs	(85)
FIB-SEM and CLEM	Ultrastructural analysis of TNTs	Detailed morphological and structural analysis	(86)
3D nanofiber scaffolding	Cultures cells on nanofibers arranged in parallel and cross-linked architectures	Simulating tumor extracellular matrix for studying mesothelioma	(87)
3D bioprinting technology	Constructs complex renal cancer models	Studying TNT-like structures in tumor formation and drug sensitivity	(88)
Microfluidic system for TNTs simulation	Simulates TNTs to study mitochondrial transfers and interactions	Investigating mitochondrial behavior and apoptosis in cancer cells	(85)
SPOTT	Overlays spatial genomics with TNTs imaging	Analyzing TNTs in intact tissues	(14)
Spatiotemporal cell and genome analysis (SaGA)	Utilizes high-resolution imaging and stable fluorescent labeling to observe metabolic heterogeneity	Studying metabolic exchanges and TNTs formation in lung cancer	(89)
Deep learning for TNTs detection	Employs advanced AI to automate detection and quantification of TNTs and connected cells	Enhancing the precision and efficiency of TNTs research	(90)
DNA origami nanostructures (DONs)	Organizes cells in 3D spaces to create biomimetic membrane channels	Organizes cells in 3D spaces to create biomimetic membrane channels	(91)
Novel biomaterials and staining methods	Smart biomaterials for specific TNTs imaging, triple labeling for cytoskeletal studies	Enhancing specificity and visualization of TNTs	(92), 93
In vivo confocal microscopy	High-resolution imaging of living tissues, allows 3D reconstruction	Confirming the presence of TNTs in surgical and biopsy samples	(46)
Immunohistochemical and laser confocal microscopy with 3D reconstruction	Uses markers like 14–3–3γ, GAP43, PCNA, and GFAP; includes 3D imaging of tissue samples	Identifying TNTs and visualizing their interfaces in GBM samples	(44)
Mitochondrial transit observation	Observes mitochondria in transit via staining and imaging	Identifying TNTs in vivo	(23), 96
Costaining of mitochondria with cytoskeletal elements	Uses mitochondrial and cytoskeletal markers to highlight interactions	Identifying TNTs in vivo	(97)
FEI Magellan 400 field-emission scanning electron microscope	Direct visualization of TNTs in fixed samples	Observing TNTs in mouse peritoneal xenograft tumors	(99)
Live fluorescence imaging	Uses cytoplasmic GFP and other fluorescent markers	Visualizing TNTs dynamically within the cellular environment	(98)

Microscopic techniques for observing TNTs include a variety of advanced methods. Gateable continuous-wave stimulated-emission depletion (gCW STED) nanoscopy, related to deconvolution techniques, enables detailed observation of TNTs structures while minimizing phototoxicity. Time-gated confocal microscopy allows monitoring of the dynamic transfer of budding tips and wheat germ agglutinin-labeled cellular components between cells.⁸⁰ Furthermore, combining two-photon excitation fluorescence lifetime imaging (FLIM) and fluorescence resonance energy transfer (FRET) technologies, known as TP-FLIM-FRET, facilitates the detection of material transfer within TNTs and allows high-precision imaging of protein interactions without disrupting the TNTs structure.⁸¹ High-resolution live-cell STED microscopy is used to measure the diameter, length, morphology, lifecycle, and formation process of TNTs in glioblastoma cell lines U87 MG and LN229. These TNTs typically appear linear and form complex communication networks between cells. In U87 cells, the average lifecycle of a TNT is 88 min, while in LN229 cells it is 41 min. The formation of TNTs often coincides with cell movement, such as during the separation of daughter cells postmitosis.⁸² However, it is important to distinguish TNTs from intercellular bridges formed during incomplete cytokinesis, as these structures are morphologically and functionally distinct. While intercellular bridges contain both F-actin and dense microtubule bundles of the midbody, TNTs, despite sometimes containing microtubules, do not exhibit the midbody structure.⁸³ Additionally, super-resolution live-cell STED nanoscopy under 2D culture conditions has been used to study the function of the intercellular connections mediated by TNTs involving mitochondrial heat shock protein 70.⁸⁴ Optical tomographic microscopy, which can detect transparent objects without the need for staining, has revealed 3D-rendered images of breast cancer cells under different treatment conditions. This technique has shown that mitochondria in tumor cells can be transferred via TNTs to adjacent tumor cells, thereby inducing apoptosis.⁸⁵ More complex microscopic techniques, such as FIB-SEM microscopy and correlative light and electron microscopy (CLEM), allow for ultrastructural analysis of TNTs.⁸⁶

In exploring the structure and function of TNTs using in vitro models, Jana et al. cultured and studied malignant mesothelioma cells on three-dimensional scaffolds composed of nanofibers arranged in parallel and cross-linked architectures.⁸⁷ This effectively simulates the natural fibrous structure of the tumor extracellular matrix, overcoming the limitations of traditional two-dimensional cell culture in depicting three-dimensional tumor cell behavior. In 2021, researchers constructed a complex renal cancer model using 3D bioprinting technology, demonstrating the function of TNT-like structures in intercellular communication and providing a highly controllable and reproducible new tool for studying the role of TNTs in tumor formation and cancer drug sensitivity.⁸⁸ However, limitations of this model include the lack of long-term observation of cellular behavior and adaptability studies across various tumor cell types, restricting its broader application. Additionally, researchers have developed a novel method named spatial profiling of tunneling nanotubes (SPOTT), which overlays regions of interest from spatial genomics with imaged clusters of TNTs in intact tissues to analyze TNTs.¹⁴ To further investigate the relationship between TNTs and mitochondria, researchers used a microfluidic system to simulate TNTs and discovered that mitochondria transferred via TNTs release endonuclease G (EndoG), forming so-called “unsealed mitochondria”.⁸⁵ These unsealed mitochondria do not induce cell death by themselves but can activate caspase-3 to induce apoptosis in tumor cells. When used in conjunction with doxorubicin, unsealed mitochondria have a synergistic apoptotic effect, enhancing tumor cell death.

The applications of bioinformatics and AI in cancer research are becoming increasingly important. A recent conference abstract highlighted the use of spatiotemporal cell and genome analysis (SaGA) technology to study metabolic heterogeneity and cooperative behaviors during the collective invasion process of different subgroups in nonsmall cell lung cancer. Researchers employed high-resolution imaging and stable fluorescent labeling to observe metabolic exchanges between subgroups, revealing multiple mechanisms that promote cell invasion. They discovered that certain stressors in the TME can induce the formation of TNTs, serving as a widespread exchange mechanism.⁸⁹ Moreover, the introduction of deep learning approaches facilitates the use of advancements in artificial intelligence to automatically detect and quantify TNTs and connected cells.⁹⁰ The research also utilized DNA origami nanostructures (DONs) to organize homotypic and heterotypic cells in 3D spaces, establishing biomimetic membrane channels. Artificial cell origami clusters (COCs) were used to study different types of cell–cell communication, including TNTs.⁹¹ These technologies not only offer high controllability and stability but also provide a universal tool for synthetic biology and in vitro cellular engineering.

In research focused on labeling TNTs, a novel smart biomaterial has demonstrated its unique value. This material utilizes intermolecular excited-state proton transfer (inter-ESPT) fluorescent probes that can trigger the inter-ESPT process through hydrogen-bonding interactions with proteins, thus specifically observing protein-based TNTs in living cells.⁹² Additionally, CellMask plasma membrane dye has been identified as an optimal choice for studying TNTs, as it uniformly labels cell membranes and provides a stable signal with minimal impact on cells.⁸² Furthermore, a new triple-labeling method—simultaneously marking intermediate filaments, microtubules, and actin filaments—has been reported. This method is suitable not only for cultured cells but also for frozen sections and tissue blocks, providing an effective tool for studying the cytoskeletal structure of TNTs.⁹³ These advancements play a crucial role in enhancing our understanding of the structure and function of TNTs.

On one hand, it is crucial to delve into the molecular composition, structural characteristics, and biophysical properties of TNTs to fully understand their role in intercellular communication. On the other hand, further verification of the existence and function of TNTs in vivo is necessary. Since TNTs were first described by Rustom et al. in 2004⁹⁴ and subsequently discovered in vivo in the cornea of fruit flies in 2008,⁹⁵ numerous studies have confirmed their presence in various tissues. For instance, in 2012, Lou and colleagues used confocal microscopy to perform three-dimensional reconstructions on surgical specimens, confirming the presence of TNTs in human mesothelioma and lung adenocarcinoma tumor samples.⁴⁶ Researchers have employed various staining and imaging techniques, including hematoxylin and eosin staining and quadruple immunohistochemical staining for 14–3–3γ or GAP43 (TNTs markers), PCNA (a proliferation marker), and GFAP (an astrocytic marker). Using laser confocal microscopy and 3D reconstruction techniques, TNTs were primarily observed at the interfaces of GBM samples, further validating the in vivo mechanisms of TNTs.⁴⁴ Additionally, the identification of TNTs in vivo has been facilitated by various techniques, such as observing mitochondria in transit^23,96 and using costaining of mitochondria with actin filaments and microtubules.⁹⁷ The expression of cytoplasmic GFP has also proven effective for visualizing TNTs within the cellular environment.⁹⁸ More direct observation methods include using an FEI Magellan 400 field-emission scanning electron microscope to directly visualize TNTs in fixed samples of mouse peritoneal xenograft tumors.⁹⁹ Organoids are multicellular 3D structures designed to replicate the anatomical and functional aspects of tissues in vitro. Tumor organoids aim to mimic the heterogeneity of tumors observed in vivo. In glioblastoma stem cells isolated from patients, TNTs were observed to form within these tumor organoids.¹⁰⁰ Using the transparency of ocular media, optical in vivo imaging has observed the occurrence of TNTs in the mouse retina and cornea.¹⁰¹ When tissue transparency poses a challenge, alternative experimental approaches are employed. These include implanting cranial glass windows in mice following skull opening and transplanting patient-derived glioblastoma cells. Postimplantation of GFP-expressing glioblastoma cells into the mouse brain, longitudinal in vivo two-photon microscopy is utilized to monitor tumor microtubes and their connections.¹⁰² In conclusion, while the initial identification of TNTs often involved resected tissues, subsequent studies using organoids, murine models, and advanced imaging techniques have robustly demonstrated the in vivo presence of TNTs. These findings collectively support the physiological relevance of TNTs in tumors and carcinomas beyond the confines of surgical artifacts. These methods collectively advance our understanding of TNTs behavior in vivo.

TNTs: A Dual Symphony of Intercellular Bridges in Tumor Progression and Immune Regulation

TNTs play a pivotal role in the proliferation, invasion, and metastasis of tumor cells, largely due to their mediation of material transport. TNTs can form between tumor cells as well as between tumor and nontumor cells, notably including interactions between tumor cells and immune cells. These TNTs can mediate immune responses, potentially promoting or inhibiting antitumor immunity, thus influencing the overall dynamics of tumor progression and the host’s immune defense mechanisms. Table 3 and Figure 2 outline the role of TNTs in tumor progression and immune regulation.

Table 3. Role of TNTs in Tumor Progression and Immune Regulation.

Interaction Type	Cell Types Involved	Impact Category	Description and Impact	Reference(s)
Tumor–tumor interactions	Breast, ovarian, pancreatic, bladder, and colon cancers	Tumor progression and invasiveness	TNTs promote tumor progression by enhancing cell migration, invasion, and epithelial–mesenchymal transition, facilitating oncogene and microRNA transfers that increase tumor aggression and invasiveness	(4), (10), (38), (48), (49), (103−105)
Tumor–nontumor cell interactions	Tumor cell–mesenchymal stem cell	Signal transmission	TNTs between these cells facilitate signaling that triggers cytokine production and other survival-stimulating factors, promoting tumor progression	(13)
	Tumor cells and astrocytes	Mitochondrial transfer and transcriptomic changes	TNTs facilitate mitochondrial transfers causing metabolic enhancements and transcriptomic changes in recipient cells, driving tumor progression	(9), (23), (106)
	Tumor cells and cancer-associated fibroblasts	Metabolic enhancement	TNTs transfer mitochondria-containing materials to cancer cells, boosting ATP production and migration capabilities	(24)
	Tumor cells and pericytes/endothelial cells	Angiogenesis	TNTs contribute to angiogenesis and facilitate material transfers, such as lipid droplets, enhancing vascular proliferation and branching	(26), (108)
	Tumor cells and immune cells (various types)	Immune evasion	TNTs enable immune evasion by transferring mitochondria from lymphocytes to cancer cells, providing metabolic benefits and reducing immune responsiveness	(109), (112)
		Metastasis support	TNTs assist metastasis by aiding tumor cells in crossing endothelial barriers and establishing metastatic foci	(111)
		Immune regulation and activation	TNTs enhance immune functions, such as T cell activation and cytokine production, impacting immunotherapeutic strategies and cytotoxic activities	(113)

Figure 2.

Schematic of TNTs between different cell types in the tumor microenvironment and their role in cancer pathogenesis and therapy. The formation of TNTs between different cell types within the TME is shown, illustrating their role in facilitating intercellular communication and substance exchange. The red box indicates how TNTs can be harnessed to deliver therapeutic agents specifically to cancer cells. The blue box demonstrates how targeting TNTs can be a strategy to interfere with tumor progression and enhance therapeutic efficacy. Abbreviations: TME, tumor microenvironment; TTField, tumor-treating fields. (Drawn using Figdraw.)

Tumor Communication Network: Dynamics of TNTs among Cancer Cells

The formation of TNTs in breast cancer cells promotes the release of microvesicles, enhancing both physical and biochemical connections between tumor cells,⁴⁹ and can further facilitate cell migration and invasion.¹⁰³ In ovarian cancer cells, TNTs induced by macrophages mediate the intercellular transfer of substances, thereby enhancing the tumor cells’ invasiveness, angiogenic potential, proliferation rate, and resistance to therapy.¹⁰ In pancreatic cancer, TNTs formation is associated with cancer cell migration, invasion, and epithelial-mesenchymal transition.⁴⁸ TNTs in glioblastoma may play a guiding role in cell migration,^82,104 contributing to tumor progression and therapeutic resistance.¹⁰⁵ Additionally, the transfer of microRNA-155 mediated by TNTs significantly increases proliferation and invasion capabilities in bladder cancer cells.⁴ Moreover, the intercellular transfer of oncogenic KRAS via TNTs in colon cancer cells enhances tumor heterogeneity and aggressiveness.³⁸ However, in some cases, the presence of TNTs can enhance antitumor immunity. For instance, TNTs can mediate the transfer of the phagocytic signal phosphatidylserine from apoptotic to non-apoptotic cells, thereby labeling living cells with an “eat me” signal that promotes macrophage phagocytosis.³³

Interface Dialogue: TNTs Interactions between Tumor and Nontumor Cells

Interactions between tumor cells and nontumor cells within the TME are crucial for cancer progression. The formation of TNTs between acute lymphoblastic leukemia cells and mesenchymal stem cells facilitates signaling that triggers the production of cytokines and other survival-stimulating factors, thereby promoting tumor progression.¹³ Additionally, TNTs facilitate the transfer of mitochondria from bone marrow stromal cells to tumor cells, enhancing the oxidative phosphorylation in the recipient cells.²³ In GBM, astrocytes transfer nontumor mitochondria via TNTs, promoting GBM cell proliferation and migration, and increasing the tumor’s resistance to drugs.¹⁰⁶ Retrograde mitochondrial signaling also plays a role in cancer progression; recent studies indicate that cancer stem cells can transfer mitochondria to astrocytes through TNTs, causing transcriptomic changes in the recipient cells and significantly enhancing overall tumor progression.⁹

Similarly, the formation of TNTs between tumor cells and endothelial cells enhances tumor invasiveness.¹⁰⁷ Inhibiting the formation of TNTs can induce tumor cells to undergo a transition to a less invasive phenotype. Studies have shown that cancer-associated fibroblasts transfer mitochondrion-containing materials to cancer cells via TNTs. This process increases mitochondrial ATP production within the cancer cells while having minimal impact on ATP production via the glycolytic pathway. Consequently, the 3D migration capability of cancer cells is enhanced. This metabolic symbiosis between tumor and stromal cells may become a new target for cancer therapy in the future.²⁴ TNTs are also associated with angiogenesis in tumor cells. Research has shown that pericytes produce TNTs which facilitate vascular proliferation and branching.¹⁰⁸ Additionally, TNTs connecting endothelial cells contain lipid droplets, and the quantity of these droplets increases following treatment with vascular endothelial growth factor.²⁶

The interactions between tumor cells and immune cells significantly contribute to immune evasion, allowing cancer cells to escape immune surveillance.¹⁰⁹ This complex interaction is further facilitated by TNTs, which enhance the invasive capabilities of tumor cells.¹¹⁰ Research highlights that macrophages can use TNTs to establish connections with intravascular tumor cells, aiding the tumor cells in crossing the endothelial layer and forming metastatic foci in the lungs, revealing a novel mechanism for tumor cell metastasis via TNTs.¹¹¹ Furthermore, TNTs play a crucial role in depleting immune cells by transferring mitochondria from lymphocytes to cancer cells. This not only provides cancer cells with a metabolic advantage but also weakens the immune response.¹¹² Additionally, TNTs are instrumental in regulating key immune cell functions such as activating T cells, promoting cytokine and antibody production, enhancing phagocytic actions, and cytotoxicity.¹¹³ These structures also induce cellular differentiation or reprogramming both in vivo and in vitro, showcasing the potential for new immunotherapeutic or immune cell therapy approaches.

This integration of TNTs into the cancer microenvironment and immune interactions underscores the complexity of cancer progression and the sophisticated evasion strategies that tumors use to thrive. This understanding opens up potential therapeutic targets and strategies to counteract tumor immune evasion and promote more effective immune responses against cancer.

TNTs exhibit diverse structural and functional characteristics across different immune cells, suggesting that various cell types may develop unique mechanisms for the formation, stabilization, and functionality of TNTs. This diversity highlights the complexity of TNT-mediated communication and its potential roles in cellular processes, including immune responses. As research into the functions and mechanisms of TNTs formation progresses,^{57,63,68,75,76,114−116} there is potential for developing new therapeutic strategies that exploit these structures. Understanding the communication mechanisms between immune cells via TNTs is crucial to developing novel immunotherapeutic approaches. This understanding could lead to breakthroughs in how we manipulate the immune system to combat diseases such as cancer, making the study of TNTs a promising area in immunology and oncology research.

Radiation and Chemotherapy Resistance Induced by TNTs: A Covert Survival Strategy of Tumor Cells

TNT-mediated resistance to chemotherapy and radiation in tumors is primarily attributed to the transfer of mitochondria, the propagation of multidrug resistance (MDR) proteins such as P-glycoprotein (P-gp), thymidylate synthase (TS), or multidrug resistance protein 1 (MDR1), as well as other small molecules or vesicles, or the direct mediation of the extrusion of chemotherapeutic drugs.

The exchange of small molecules and even mitochondria mediated by TNTs can promote cancer progression by enhancing resistance to hypoxia and chemotherapy.¹¹⁷ Notably, studies involving mesenchymal stem cells (MSCs) have shown that they can transfer mitochondria to glioma stem cells (GSCs) via TNTs, which boosts GSCs’ resistance to common chemotherapy agents like temozolomide (TMZ). This mitochondrial transfer induces metabolic reprogramming in GSCs and creates a metabolic dependence on exogenous mitochondria. Interestingly, inhibiting orotate production with brequinar (BRQ) can restore the sensitivity of GSCs to TMZ, opening potential for new therapeutic strategies based on the synthetic lethality between TMZ and BRQ.¹¹⁸ Further preclinical research is needed to understand the key mechanisms by which TNT-driven MSCs protect against various diseases, which could lead to clinical studies. The ATP produced by these mitochondria is a key energy source for drug efflux in chemotherapy-resistant tumor cells.¹¹⁹ Mitochondria can be transferred from adipose stem cells (ASCs) to breast cancer cells (BCCs) via TNTs. Under hypoxic conditions, BCCs containing mitochondria from ASCs show reduced HIF-1α expression and increased ATP production, which drives ABC transporter-mediated MDR. Thus, blocking the transfer of mitochondria from ASCs to BCCs could be a novel and effective strategy for treating breast cancer.¹²⁰

Overexpression of membrane P-gp can lead to MDR in tumor cells. Research indicates that TNTs can mediate the transfer of P-gp overexpression resistance from drug-resistant breast cancer cell variants to drug-sensitive parental breast cancer cells. This facilitates survival and increases resistance in sensitive cells when faced with chemotherapeutic agents.¹¹⁵ Additionally, TS and MDR1 are known mechanisms for resistance to the chemotherapy drug 5-FU.^121,122 Studies have shown that 5-FU can induce the formation of TNTs in MCF-7 breast cancer cells, and the presence of TNTs helps the cancer cells survive and increases their resistance to the drug. This resistance may be facilitated by the transfer of TS or MDR1 through TNTs, promoting the survival and drug resistance of cancer cells after chemotherapy.⁴²

The transfer of molecules and vesicles and even the direct mediation of chemotherapeutic drugs through TNTs can lead to chemotherapy resistance. For example, studies in ovarian cancer have shown that TNTs facilitate the transfer of molecules between platinum-sensitive and platinum-resistant ovarian cancer cells as well as between cancer cells and normal ovarian epithelial stromal cells. This includes the transfer of anticancer drugs, suggesting that targeting TNTs to disrupt this communication pathway could offer a new strategy to overcome significant clinical challenges related to platinum resistance.¹²³ In the bone marrow microenvironment, vesicle transfer mediated by TNTs from stromal cells to leukemia cells has been shown to increase resistance to the tyrosine kinase inhibitor imatinib, although the specific components of these vesicles were not detailed in the study.¹²⁴ Furthermore, in ovarian and pancreatic cancer cells, the chemotherapy drug doxorubicin has been found to stimulate the formation of TNTs, leading to drug efflux.¹²⁵ Other research indicates that connections via TNTs can enhance therapeutic resistance in tumor cells, as observed in glioblastoma⁹⁸ and prostate cancer,⁴⁷ although these studies did not specify the substances transported via TNTs. This growing body of research highlights the complex role TNTs play in mediating resistance mechanisms and underscores the potential therapeutic benefits of targeting these structures to improve treatment outcomes in cancer.

Emerging Applications of TNTs in Cancer Therapy: Precision Delivery and Therapeutic Innovation

TNTs are involved in multiple stages of tumor progression, from development to drug resistance. There are mainly two potential therapeutic strategies targeting TNTs: promoting drug delivery through TNTs between cells and inhibiting the formation of TNTs. Table 4 and Figure 2 detail the emerging applications of TNTs in cancer therapy.

Table 4. Emerging Applications of TNTs in Cancer Therapy.

Strategy	Method/Technique	Description	Key Findings and Impact	Reference(s)
Drug Highways: Precision Drug Delivery	Engineered nanoparticles	Use of nanoparticles coupled with receptor antibodies or peptides for targeted drug delivery via TNTs	Enhances therapeutic effects, improves precision, and reduces off-target toxicity	(15), (127−129)
	Direct transport of drugs and viruses	TNTs facilitate direct transport of viruses, antitumor agents, and drugs, enhancing the efficacy of treatments like doxorubicin in cancer cells	Accelerates drug delivery and enhances treatment precision in cancers like ovarian, lung, and pancreatic	(125), (132), (133), (135), (136)
Blocking Transmission: Strategic Inhibition	Pharmacological inhibitors	Includes actin polymerization inhibitors, DNA synthesis inhibitors, mTOR inhibitors, and microtubule inhibitors to prevent TNTs formation	Potential to address tumor drug resistance; further clinical application needed	(11), (109)
	Drugs targeting TNT-associated proteins	Focuses on gene knockdown strategies and targeting proteins associated with TNTs, which are sensitive to mechanical forces	Opens new therapeutic avenues but requires more research to verify clinical safety and efficacy	(23), (63), (141)
	Novel nanotherapeutic platforms	Combines two uniquely designed liposomes targeting cytoskeletal disruption and mitochondrial impairment in tumor cells	Reduces metastatic capability by inhibiting TNTs formation and vasculogenic mimicry	(142)
	Tumor-treating fields (TTFields)	Low-intensity alternating electric fields disrupt microtubules and inhibit TNTs formation in cancer cells	Modulates immune oncological markers and cellular proliferation pathways in mesothelioma	(14), (143)

Drug Highways: Utilizing TNTs for Precision Drug Delivery

This strategy involves designing more sophisticated drug delivery systems that use TNTs for intercellular transfer. Such systems aim to reach and impact distant tumor niches, which are crucial for maintaining the plasticity of cancer stem cells, participating in tumor immune evasion, and enhancing the metastatic potential. Achieving selective drug delivery to tumor sites represents one of the biggest challenges in successful chemotherapy. Several strategies are being explored to tackle this issue, including the use of engineered nanoparticles coupled with receptor antibodies or peptides. These engineered nanoparticles have numerous advantages over traditional drugs and are one of the most researched tools in the field of drug delivery.¹¹⁶ Cells share materials through TNTs and can deliver engineered nanoparticles via these structures in a controlled manner. Understanding the mechanisms that trigger TNT-mediated transport dynamics is crucial. Inhibiting TNTs formation can enhance drug accumulation in target cells and reduce off-target toxicity; conversely, enhancing these connections significantly improves the therapeutic effects of drugs across tissues. The transport rates of various types of nanoparticles within TNTs vary, potentially due to the characteristics of the nanoparticles or cell types, which warrants further investigation. Moreover, the properties of nanoparticles are critical for understanding their exchange between cells and the impact of TNTs on therapeutic outcomes.^116,126 Macrophages transfer mesoporous silica nanoparticles (MSNs) to homotypic or heterotypic cells via TNTs, providing visual evidence of nanoparticle delivery to cancer cells and demonstrating the potential of this mechanism as a drug delivery tool. Homotypic TNTs between macrophages may reflect normal immune surveillance functions, whereas heterotypic membrane connections with cancer cells could be triggered by specific immune responses.¹²⁷ Recent studies have employed fluorescent nanodiamonds (FNDs) as stable tracers and protein carriers, successfully tracking and demonstrating the active transport of proteins and other biomolecules between human cells via TNTs. This highlights the potential applications of novel carbon-based nanomaterials in the intercellular delivery of biomolecules. However, further research is required to determine whether the transported proteins can maintain their activity and how they are released.¹²⁸ The structural differences in TNTs formed between various cell types or between tumor and healthy cells can be exploited to enhance the precision of nanoparticle delivery systems. SiO₂ nanoparticles serve as carriers in a drug delivery system, facilitating the transfer between tumor cells with TNTs playing a crucial role.¹²⁹ Additionally, studies have used surface-functionalized nanoparticle models to transport drugs through TNTs in GBM, utilizing structural differences in TNTs between GBM cells and normal human astrocytes to increase the precision and specificity of treatment.¹⁵

Furthermore, TNTs facilitate the direct transport of viruses, antitumor agents, and chemotherapeutic drugs, playing a crucial role in cancer therapy. TNTs mediate the transfer of oncolytic viral gene products between cells, initiating and sustaining bystander effects.¹³⁰ Research exploring the potential role of mast cells in cancer therapy has shown that primary human mast cells activated via FcεRI form TNTs with tumor cells concurrent with tumor cell death, suggesting that these structures could participate in delivering antitumor mediators to the tumor cells.¹³¹ In recent advancements in cancer therapy, drug delivery has been carried out using traditional carriers such as liposomes, and synthetic nanoparticles primarily enter cells via endocytosis. However, intercellular transfer through TNTs is faster; a novel approach described encapsulates doxorubicin within M1-type macrophages, which is then directly transferred to the cytoplasm of target cells via TNTs, bypassing conventional entry pathways. This direct TNT-mediated transfer not only accelerates drug delivery but also enhances precision, significantly improving treatment outcomes in metastatic ovarian cancer.¹³² Doxorubicin has also been observed to be transported via TNTs in lung and pancreatic cancers.^125,133 Engineered microglial cells carrying paclitaxel use TNTs and extracellular vesicles to transfer their cargo to glioma cells.¹³⁴ Mesenchymal stem cells have been used to release epirubicin into gastric cancer cells via TNTs. Specifically, researchers have discovered that mesenchymal stem cells, attracted to gastric cancer cells, can effectively load and release epirubicin, significantly enhancing anticancer effects. This method provides a new pathway for precisely targeting gastric cancer cells with epirubicin, potentially enhancing the efficacy of chemotherapy.¹³⁵ Researchers have described a selective tumor-penetrating drug called the R11–phalloidin conjugate, which transfers directly between cells via TNTs, avoiding off-target effects. Notably, the transfer from normal cells to tumor cells is unidirectional, as the required channels extend more one-directionally from normal cells to tumor cells. Additionally, this bladder intravesical conjugate exhibited strong anticancer activity in an in situ mouse bladder tumor model and showed good tolerability and biosafety. Overall, this study provides a new selective tumor-penetrating strategy for intravesical cancer therapy in bladder cancer.¹³⁶

Blocking Transmission: Strategic Inhibition of TNTs Formation

Inhibiting the formation of TNTs represents a potential strategy to address tumor drug resistance. Actin and M-Sec, known markers of TNTs, are not specific to these structures.⁷⁰ Current research on pharmacological inhibitors of TNTs primarily includes actin polymerization inhibitors, drugs that inhibit DNA synthesis, and mTOR inhibitors.¹⁰⁹ These inhibitors, such as CK-666,⁸⁶ everolimus,⁴⁶ metformin,⁴⁶ cytochalasin B and D,^50,137 latrunculin B,¹³⁸ cytarabine,¹³⁹ and migrastatin core ether,¹⁴⁰ have shown promise in studies, but most have not yet been applied in preclinical models. Additionally, the actin polymerase inhibitors among these drugs may exhibit cytotoxicity to cells in tissues. Vincristine, which inhibits microtubule formation, also affects TNTs formation, offering an alternative intervention approach.¹¹ Further research is focused on drugs targeting proteins, including gene knockdown to regulate TNTs formation, targeting TNT-associated proteins, and exploiting TNTs’ sensitivity to mechanical forces.^23,63,141 These approaches pave new pathways for research and therapy; however, applying TNT-disrupting drugs clinically remains challenging, necessitating further studies to ensure their efficacy and safety.

Recent research has unveiled a revolutionary nanotherapeutic platform integrating two uniquely designed liposomes: KFCsk@LIP and KTMito@LIP. These liposomes employ a dual strategy targeting the aggressive disruption of the cytoskeletal architecture and defensive mechanisms of tumor cells, leading to the collapse of the cytoskeletal structure and severe impairment of mitochondrial functions. This innovative approach effectively cuts off the pathways for vasculogenic mimicry (VM) and the formation of TNTs, significantly reducing the metastatic capability of tumors.¹⁴² Additionally, tumor-treating fields (TTFields) utilize low-intensity alternating electric fields to disrupt microtubules during mitosis in cancer cells. This treatment has been clinically employed to manage glioblastoma and malignant pleural mesothelioma. SPOTT technology has revealed on a molecular level how TTFields alter the TME. Studies indicate that TTFields inhibit the formation of TNTs in malignant pleural mesothelioma (MPM) cells in vitro and modulate immune oncological biomarkers in vivo while also downregulating pathways associated with cellular proliferation and invasion—key regulators of tumor growth.¹⁴ The SPOTT technique helps identify TNTs as potential therapeutic targets for TTFields-directed cancer treatment strategies. TTFields may disrupt or prevent the formation of TNTs in mesotheliomas, thereby sensitizing these cells to the cytotoxic effects of chemotherapy and TTFields.¹⁴³ TNTs not only support intercellular signaling and material exchange among tumor cells but also may play a role in tumor cell adaptation and resistance to treatment. Thus, targeting TNTs presents a promising new cancer treatment strategy. This approach could not only block intratumoral communication but also potentially reduce the aggressiveness and metastatic capabilities of tumors, offering an effective therapeutic option.¹⁴⁴

TNTs as Potential Targets in Cancer Therapy: Exploring New Frontiers and Challenges

TNTs exhibit significant potential as targets in cancer therapy due to their ability to transport substances such as proteins, RNA, and viral particles between cells. This capability positions them as promising candidates for drug delivery systems, especially within the TME, where they can efficiently transport antitumor drugs or gene therapy vectors. TNTs play a crucial role in the interactions between tumor cells and the surrounding stroma, regulating tumor aggressiveness and migration. Targeting TNTs could disrupt the communication networks of tumor cells, thereby inhibiting their invasiveness and metastatic spread. Additionally, TNTs facilitate the exchange of materials between antigen-presenting cells and effector T cells, potentially enhancing tumor immune responses. This enhancement could improve the immune system’s ability to recognize and eliminate tumor cells, offering a strategic approach to bolster immunotherapy outcomes.

To trigger the formation of TNTs via cell dislodgement within specific cell types or tissues for targeted drug delivery, several approaches can be considered. By manipulating cellular interactions and the TME—such as altering adhesion properties and mechanical stress—TNTs formation can be enhanced.^77,145 Additionally, modulation of biophysical properties, like extracellular matrix stiffness, and biochemical signals, particularly exosomal thrombospondin-1, has been discussed as a key factor in promoting TNTs formation.¹⁴⁶ This comprehensive approach allows for the creation of a favorable environment for TNTs development, offering significant implications for targeted therapies. However, the ubiquity and dynamism of TNTs pose significant challenges for specific targeting. Intervening specifically in the TNTs of tumor cells without affecting the function of normal cells remains a major challenge. Additionally, the structural and functional complexity of TNTs, which varies across different environments and types, complicates research and development efforts. A thorough understanding of the biological characteristics of TNTs is essential for the development of targeted therapies. Interfering with the function of TNTs could impact the material exchange and signaling pathways of normal cells, potentially leading to adverse effects. Therefore, more precise strategies are needed to minimize the impact on normal cellular functions. Given the difficulty in observing TNTs and their fragile structures, current research heavily relies on high-resolution microscopy techniques, which have limitations in clinical applications. Consequently, there is a need to develop simpler and more clinically applicable detection and intervention techniques to advance TNT-targeted cancer therapy.

Future research should delve deeper into the mechanisms of TNTs formation, identifying the molecular and environmental factors that influence their development. This necessitates studies of specific roles in signal transduction pathways and cytoskeletal reorganization. By integrating disciplines such as molecular biology, nanotechnology, and immunology, the development of new molecular probes, labeling techniques, and advanced imaging technologies can facilitate the real-time observation of TNTs dynamics. This will enhance our understanding of TNTs’ roles in tumor progression. Given the structural and functional heterogeneity of TNTs across different tumor cells, understanding this heterogeneity and its impact on TNTs’ functions will be a vital research direction. Furthermore, elucidating the interactions between tumor cells and their microenvironment, including immune and stromal cells—particularly in terms of tumor growth and immune evasion—is essential.

Therapeutically, research should focus on how to target TNTs formation or function specifically without affecting normal cell functionality. Evaluating the long-term safety and potential side effects of such targeted strategies is crucial. Leveraging TNTs for drug delivery could lead to novel therapeutic systems for difficult-to-treat cancers, such as pancreatic cancer and glioblastoma, exploring the potential of TNTs to enhance immunotherapeutic efficacy. For example, regulating TNTs to boost the presentation of tumor antigens can enhance the immune system’s recognition and attack on tumor cells. Combining nanotechnology with TNTs could lead to the design of nanoscale intervention tools or sensors for precise regulation of the TME. By addressing these challenges and exploring new research avenues, TNTs hold the promise of playing a more significant role in cancer therapy.

Conclusion

This review synthesizes the pivotal role of TNTs in tumor cell proliferation, invasion, and metastasis, highlighting their significance in facilitating intercellular material and signal transmission within the TME. TNTs not only play a crucial role in enhancing the metabolic functions and drug resistance of tumor cells but also demonstrate potential as carriers for drug delivery, opening new strategies for cancer therapy. Future research should focus on thoroughly deciphering the biogenesis mechanisms of TNTs, developing cell-specific molecular targets, and advancing real-time monitoring technologies. Additional studies should explore drug delivery systems based on TNTs and their integration with immunotherapy and nanotechnology, which could help overcome the current challenges. Through these research advances, the clinical application prospects of TNTs will be further expanded, providing more precise and effective solutions for cancer treatment.

Glossary

Abbreviations

TNTs

tunneling nanotubes

TME

tumor microenvironment

GFAP

glial fibrillary acidic protein

GBM

glioblastomas

MARCKS

myristoylated alanine-rich C-kinase substrate

HGF

hepatocyte growth factor

EGF

epidermal growth factor

gCW STED

gateable continuous wave stimulated emission depletion

FLIM

fluorescence lifetime imaging

FRET

fluorescence resonance energy transfer

CLEM

correlative light and electron microscopy

SPOTT

spatial profiling of tunneling nanotubes

EndoG

endonuclease G

SaGA

spatiotemporal cell and genome analysis

DON

DNA origami nanostructure

COC

cell origami cluster

inter-ESPT

intermolecular excited-state proton transfer

P-gp

P-glycoprotein

TS

thymidylate synthase

MDR1

multidrug resistance protein 1

MSC

mesenchymal stem cell

GSC

glioma stem cell

TMZ

temozolomide

BRQ

brequinar

ASC

adipose stem cell

BCC

breast cancer cell

MDR

multidrug resistance

MSN

mesoporous silica nanoparticle

FND

fluorescent nanodiamond

VM

vasculogenic mimicry

TTField

tumor-treating field

MPM

malignant pleural mesothelioma

Data Availability Statement

Data are available upon reasonable request.

This work was supported by the Natural Science Foundation of Hebei Province (Grant H2022206539) and the Special Project of Central Government for Local Science and Technology Development of Hebei Province (246Z7703G).

The authors declare no competing financial interest.