Molecular, morphological and functional properties of tunnelling nanotubes between normal and cancer urothelial cells: New insights from the in vitro model mimicking the situation after surgical removal of the urothelial tumor

Abstract

Tunnelling nanotubes (TNTs) are membranous connections that represent a unique type of intercellular communication in different cell types. They are associated with cell physiology and cancer pathology. The possible existence of tunnelling nanotubes communication between urothelial cancer and normal cells has not yet been elucidated. Therefore, we analyzed TNTs formed by T24 cells (human invasive cancer urothelial cells) and normal porcine urothelial (NPU) cells, which serve as surrogate models for healthy human urothelial cells. Monocultures and cocultures of NPU and T24 cells were established and analyzed using live-cell imaging, optical tweezers, fluorescence microscopy, and scanning electron microscopy. TNTs of NPU cells differed significantly from tunnelling nanotubes of T24 cells in number, length, diameter, lipid composition, and elastic properties. Membrane domains enriched in cholesterol/sphingomyelin were present in tunnelling nanotubes of T24 cells but not in NPU cells. The tunnelling nanotubes in T24 cells were also easier to bend than the tunnelling nanotubes in NPU cells. The tunnelling nanotubes of both cell types were predominantly tricytoskeletal, and contained actin filaments, intermediate filaments, and microtubules, as well as the motor proteins myosin Va, dynein, and kinesin 5B. Mitochondria were transported within tunnelling nanotubes in living cells, and were colocalized with microtubules and the microtubule-associated protein dynamin 2. In cocultures, heterocellular tunnelling nanotubes were formed between NPU cells and T24 cells and vice versa. The presence of connexin 43 at the end of urothelial tunnelling nanotubes suggests a junctional connection and the involvement of tunnelling nanotube in signal transduction. In this study, we established a novel urothelial cancer-normal coculture model and showed cells in the minority tend to form tunnelling nanotubes with cells in the majority. The condition with cancer cells in the minority is an attractive model to mimic the situation after surgical resection with remaining cancer cells and may help to understand cancer progression and recurrence. Our results shed light on the biological activity of tunnelling nanotubes and have the potential to advance the search for anticancer drugs that target tunnelling nanotubes.

Cooperation of Various Cytoskeletal Components Orchestrates Intercellular Spread of Mitochondria between B-Lymphoma Cells through Tunnelling Nanotubes

“…Due to the application of a lower concentration (10 µM) of nocodazole, only 13% of the TNTs tore (Figure 6A), and their diameter was significantly reduced after only 5 min of treatment, which became more pronounced with an increasing duration of incubation in the presence of nocodazole (Figure 6B,C and Table 1). On the other hand, nocodazole treatment with different time periods did not affect the length of TNTs (Figure 6D and Table 1) or the shape of B-lymphoma cells (Figure 6E and Table 1), suggesting that the microtubular network plays a crucial role not only in some transport processes but also in the mechanical stability and lifetime of TNTs, consistent with previous publications [6,45,51,60]. The arrangement of the main cytoskeletal elements of B-lymphoma-cell TNTs further supports that the presence of microtubules may contribute to the stability of TNTs.…”

“…Due to its involvement in neurological diseases, mitochondrial transport is one of the most widely studied processes in NT research. Based on these investigations, the direction of mitochondria transport may vary, and depending on the cell type, it can be either unidirectional, where mitochondria are transported from healthy cells to damaged cells via NTs supporting cell survival (e.g., from pericytes to astrocytes, in PC12 and normal porcine urothelial /NPU/ cells, or from bone marrow stromal cells to acute myeloid leukaemic cells to improve resistance against chemotherapy) [24,45,70,71] or bidirectional (e.g., between human umbilical vein endothelial cells /HUVECs/ and mesenchymal stem cells /MSCs/, or between Jurkat cells and MSCs [72][73][74][75][76]. The main molecular components of mitochondrial delivery have already been identified; however, since these results are often contradictory, the exact mechanism of their transfer is still unclear.…”

“…The lack of complete inhibition suggested the potential involvement of other motor proteins that facilitate the bidirectional transport, enabling mitochondria to be transported in the opposite direction through TNTs. To explore this possibility, the activity of myosin II, V, and VI, motor proteins reported to be typically involved in actin-based transport processes, were also blocked [45,58,59]. The inhibition of myosin VI significantly reduced the transport velocity (23.19 ± 14.17 nm/s).…”

“…The dysfunction of mitochondria leads to the accumulation of various reactive oxygen species (ROS), including NADPH oxidase and hydrogen peroxide (H 2 O 2 ), which are widely recognised triggers for NT formation [43]. Some studies suggest that mitochondria are transported through NTs that contain microtubules [24,44,45], but mitochondrial delivery has also been observed via NTs that contain only actin [46], indicating that this question has remained unclear.…”

CD13 Activation Assembles Signaling Complexes that Promote the Formation of Tunneling Nanotubes in Endothelial Cells

“…These were also appreciably longer than in CD13 KO cells (15 μm vs. 10 μm) (Figure 1D, consistent with TNT-like protrusions (Chakraborty et al ., 2023). Since TNTs can be either actin or microtubule-based (Resnik et al ., 2022), we treated WT and CD13 KO KSECs with microtubule (nocodazole) or actin (Cytochalasin B, D) polymerization inhibitors or the Cdc42 GTPase inhibitor (ML141) and measured the TNT index. We found that treatment with actin and Cdc42, but not microtubule inhibitors significantly decreased TNT formation in WT KSECs (Figure 1E, F), indicating that the KSEC protrusions are largely actin-driven, consistent with actin-based endothelial TNTs (Hashimoto et al ., 2016; Dupont et al ., 2018; Belian et al ., 2023).…”