Complementary Scanning Transmission Ion Microscopy and Live-Cell Confocal Microscopy Reveal Tunneling Nanotube Structure in U87 MG and LN229 Glioblastoma Cells.

Tunneling nanotubes (TNTs) are tiny membrane tunnels that allow cells to rapidly exchange signals, organelles, and biological substances. This efficient form of cellular communication is associated with the aggressive nature of cancers, such as glioblastoma. The structure of TNTs, particularly their cytoskeletal composition and connection to the cell body, is of significant interest because it can reveal information about TNT stability, active transport mechanisms, and functionality. In this study, we investigated the TNT structure of two glioblastoma cell lines, U87 MG and LN229, using two distinct imaging techniques: live-cell confocal microscopy and scanning transmission ion microscopy (STIM). In particular, we demonstrate for the first time that it is feasible to obtain areal density information on label-free biological samples at high resolution from the ion energy loss using STIM. This technique, therefore, enables thickness mapping in the z-direction and the study of detailed ultrastructural features of TNTs, including cytoskeletal structures and cargo transport. Our live-cell confocal microscopy study revealed varying levels of detectable actin and tubulin in the TNTs of both cell lines. Using both imaging modalities, we identified various morphologies of TNT terminals, including anchor-, funnel-, and node-like endings. These morphologies could indicate functional heterogeneity of the TNTs.