Investigating Tunneling Nanotubes in Cancer Cells: Guidelines for Structural and Functional Studies through Cell Imaging

Abstract

By allowing insured communication between cancer cells themselves and with the neighboring stromal cells, tunneling nanotubes (TNTs) are involved in the multistep process of cancer development from tumorigenesis to the treatment resistance. However, despite their critical role in the biology of cancer, the study of the TNTs has been announced challenging due to not only the absence of a specific biomarker but also the fragile and transitory nature of their structure and the fact that they are hovering freely above the substratum. Here, we proposed to review guidelines to follow for studying the structure and functionality of TNTs in tumoral neuroendocrine cells (PC12) and nontumorigenic human bronchial epithelial cells (HBEC-3, H28). In particular, we reported how crucial is it (i) to consider the culture conditions (culture surface, cell density), (ii) to visualize the formation of TNTs in living cells (mechanisms of formation, 3D representation), and (iii) to identify the cytoskeleton components and the associated elements (categories, origin, tip, and formation/transport) in the TNTs. We also focused on the input of high-resolution cell imaging approaches including Stimulated Emission Depletion (STED) nanoscopy, Transmitted and Scanning Electron Microscopies (TEM and SEM). In addition, we underlined the important role of the organelles in the mechanisms of TNT formation and transfer between the cancer cells. Finally, new biological models for the identification of the TNTs between cancer cells and stromal cells (liquid air interface, ex vivo, in vivo) and the clinical considerations will also be discussed.

Nucleolin regulates 14‐3‐3ζ mRNA and promotes cofilin phosphorylation to induce tunneling nanotube formation

“…We used three approaches to distinguish TNTs from filopodia as per accepted criteria in the field: 9,31 (a) TNTs “hang” between two cells, unlike filopodia which grow along the substratum. We performed “depth coding” from confocal z ‐stack reconstructions and considered only those structures as TNTs that appear approximately 3‐4 µm above the substratum, as shown in the example for U2OS cells in Figure S3.…”

“…9 A peculiar and unique feature of TNTs in cell culture is that they "hang" between the connected cells without contacting the substratum. [9][10][11] F-actin fibers are essential cytoskeletal constituents of TNTs, and are sometimes also supported by microtubules and/or intermediate filaments. 12 Microtubule-based TNTs are thicker in diameter, providing more stability and longevity to the nanotube.…”

“…The second type (“TNT2”) consists of shorter and thicker TNTs, which usually form through cell‐cell dislodgement and mediate close range intercellular communication 9 . A peculiar and unique feature of TNTs in cell culture is that they “hang” between the connected cells without contacting the substratum 9‐11 . F‐actin fibers are essential cytoskeletal constituents of TNTs, and are sometimes also supported by microtubules and/or intermediate filaments 12 .…”

“…One type (“TNT1”) constitutes longer TNTs that connect distant cells, with the nanotubes being narrower and usually formed by the initial protrusion of filopodia‐like projections from cell surfaces. The second type (“TNT2”) consists of shorter and thicker TNTs, which usually form through cell‐cell dislodgement and mediate close range intercellular communication 9 . A peculiar and unique feature of TNTs in cell culture is that they “hang” between the connected cells without contacting the substratum 9‐11 .…”

Direct Transfer of Mesoporous Silica Nanoparticles between Macrophages and Cancer Cells

“…For example, at immunological synapses, immune molecules such as MHC class I and class II proteins and costimulatory molecules undergo intercellular membrane transfer from antigen presenting cells (APC) to T cells [ 12 , 13 ]. Cells can communicate remotely via biovesicle transfer [ 14 , 15 , 16 ] but also by direct physical connections through open cytoplasmic channels that include tunneling nanotubes (TNTs) [ 17 , 18 , 19 , 20 , 21 ]. TNT formation can occur either de novo from filopodia-like protrusions, or during detachment of adjacent cells, with both processes being F-actin-dependent [ 22 ].…”