Real-Time Observation of Tunneling Nanotube Dynamics and Its Mediated Intracellular ROS Regulation Using SICM and SECM

TNT model of PDAC cells through doxorubicin (DOX) treatment. We employed scanning ion conductance microscopy (SICM) to in situ visualize and track the formation and cleavage dynamics of TNTs between live PDAC cells as well as the mitochondria transfer via TNTs. We further used scanning electrochemical microscopy (SECM) to monitor the intracellular reactive oxygen species (ROS) levels of the PDAC cells after TNT formation and mitochondria transfer. We observed that the TNTs formed during cell dislodgement, with topographic diversity of diameters primarily ranging from 300 to 800 nm and heights between 1 and 5 μm and a four-step procedure for TNT formation and cleavage (including initial cell-cell contact, TNT formation, elongation, and cleavage with a Y-shaped structure), which has not been reported before. In addition, we found a significant decrease in the intracellular ROS levels of the TNT-connected PDAC cells. Our work demonstrates that TNT-mediated mitochondria transfer alleviates intracellular oxidative stress in PDAC cells, offering valuable insights into the role of TNTs in maintaining cellular redox homeostasis and potential implications for cancer progression and treatment resistance.