Direct cell-to-cell communication is crucial for the survival of cells in stressful situations such as during or after radiation exposure. This communication can lead to non-targeted effects, where non-treated or non-infected cells show effects induced by signal transduction from non-healthy cells or vice versa. In the last 15 years, tunneling nanotubes (TNTs) were identified as membrane connections between cells which facilitate the transfer of several cargoes and signals. TNTs were identified in various cell types and serve as promoter of treatment resistance e.g. in chemotherapy treatment of cancer. Here, we discuss our current understanding of how to differentiate tunneling nanotubes from other direct cellular connections and their role in the stress reaction of cellular networks. We also provide a perspective on how the capability of cells to form such networks is related to the ability to surpass stress and how this can be used to study radioresistance of cancer cells.

Cellular communication plays a crucial role in the coordination and organization of cancer cells. Especially processes such as uncontrolled cell growth, invasion, and therapy resistance (development), which are features of very malignant tumors like glioblastomas, are supported by an efficient cell-to-cell communication in the tumor environment. One powerful way for cells to communicate are tunneling nanotubes (TNTs). These tiny membrane tunnels interconnect cells over long distances and serve as highways for information exchange between distant cells. Here, we study the response of cellular communication via TNTs in U87 glioblastoma cells to homogeneous irradiation with α-particles as a stress factor. We describe the development of TNT networks in certain time steps after irradiation using confocal live-cell imaging and suggest an evaluation method to characterize these communication networks. Our results show that irradiated cells establish their network faster and have more cell-to-cell connections with high TNT content than sham-irradiated controls within the first 24 h. These findings suggest that there is an additional trigger upon radiation damage which results in fast and intensive network formation by TNTs as a radiation damage response mechanism.

Purpose: High doses of ionizing radiation in radiotherapy can elicit undesirable side effects to the skin. Proton minibeam radiotherapy (pMBRT) may circumvent such limitations due to tissue-sparing effects observed at the macro scale. Here, we mapped DNA damage dynamics in a 3D tissue context at the sub-cellular level. Methods: Epidermis models were irradiated with planar proton minibeams of 66 µm, 408 µm and 920 µm widths and inter-beam-distances of 2.5 mm at an average dose of 2 Gy using the scanning-ion-microscope SNAKE in Garching, GER. γ-H2AX + 53BP1 and cleaved-caspase-3 immunostaining revealed dsDNA damage and cell death, respectively, in time courses from 0.5 to 72 h after irradiation. Results: Focused 66 µm pMBRT induced sharply localized severe DNA damage (pan-γ-H2AX) in cells at the dose peaks, while damage in the dose valleys was similar to sham control. pMBRT with 408 µm and 920 µm minibeams induced DSB foci in all cells. At 72 h after irradiation, DNA damage had reached sham levels, indicating successful DNA repair. Increased frequencies of active-caspase-3 and pan-γ-H2AX-positive cells revealed incipient cell death at late time points. Conclusions: The spatially confined distribution of DNA damage appears to underlie the tissue-sparing effect after focused pMBRT. Thus, pMBRT may be the method of choice in radiotherapy to reduce side effects to the skin.

Tunneling nanotubes (TNTs) are fine, nanometer-sized membrane connections between distant cells that provide an efficient communication tool for cellular organization. TNTs are thought to play a critical role in cellular behavior, particularly in cancer cells. The treatment of aggressive cancers such as glioblastoma remains challenging due to their high potential for developing therapy resistance, high infiltration rates, uncontrolled cell growth, and other aggressive features. A better understanding of the cellular organization via cellular communication through TNTs could help to find new therapeutic approaches. In this study, we investigate the properties of TNTs in two glioblastoma cell lines, U87 MG and LN229, including measurements of their diameter by high-resolution live-cell stimulated emission depletion (STED) microscopy and an analysis of their length, morphology, lifetime, and formation by live-cell confocal microscopy. In addition, we discuss how these fine compounds can ideally be studied microscopically. In particular, we show which membrane-labeling method is suitable for studying TNTs in glioblastoma cells and demonstrate that live-cell studies should be preferred to explore the role of TNTs in cellular behavior. Our observations on TNT formation in glioblastoma cells suggest that TNTs could be involved in cell migration and serve as guidance.

Intercellular communication in the tumor microenvironment has a major impact on therapeutic outcomes. In particular, highly aggressive cancers such as glioblastoma benefit from efficient cellular communication by developing resistance to therapy or by infiltrating surrounding tissue to escape radiotherapy. Tiny membrane tunnels, such as tunneling nanotubes (TNTs), can serve as an effective communication tool for tumor cells. With these structures, the cells can build a large network that allows them to react and adapt to their environment as quickly as possible, thereby combating the therapy. In this study, we investigated the effects of chemotherapeutic agents on TNT networks in U87 MG cells to determine if they affect the number of TNTs and, consequently, possible therapeutic outcomes. The effect of drugs on TNT stability may be related to their effects on actin and tubulin, which are known stabilizers of membrane structures. Therefore, we also analyzed the cytoskeletal content of TNTs in U87 MG cells and examined the effect of the actin polymerization inhibitor cytochalasin B (CytoB) on TNT formation and networks. CytoB reduced the number of TNTs in this study, suggesting that actin, as a cytoskeletal component, is required for developing TNT networks. In contrast, the chemotherapeutic agents temozolomide (TMZ) and cytarabine (AraC) do not affect TNTs and actin content. As TNT networks are closely linked to increased therapy resistance of certain tumors, we believe that the lack of suppression of TNTs is a major problem in current glioblastoma therapy and should be a target for further research in the future.