Visualizing the ultrastructures in tunneling nanotubes between cancer cells using focus ion beam/scanning electron microscopy
Chia-Wei Lee1*, Chia-Chen Kuo1, Huei-Jyuan Pan1, Chia-Ning Shen2, Chau-Hwang Lee1,3
1Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan
2Genomics Research Center, Academia Sinica, Taipei, Taiwan
3Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan
* Presenter:Chia-Wei Lee,
Membrane tunneling nanotubes (TNTs) have been noticed as a special way of long-distance cell–cell communications [1, 2]. Various intracellular substances can be transported through TNTs, including proteins, microRNAs, vesicles, and mitochondria [2]. However, owing to the nanometer dimensions of TNTs, the inner structures have rarely been understood. Recently, with the progressions of super-resolution optical microscopy, scientists have discovered many cellular structures much smaller than the diffraction limit [3]. However, the efficiency of fluorescence labeling largely determines the imaging contents of current super-resolution techniques. Therefore, a more comprehensive view revealing all the components in a field of view which may provide various clues to understand more biological details is still very desirable.
In this study, we treated the pancreatic cancer cells PANC-1 with macrophage conditioned medium (MaCM) to induce TNTs formations. By sequentially slicing a single TNT with focus ion beam/scanning electronic microscope (FIB/SEM), we found that the TNT is not just a tunnel but with heavily packed tiny structures. Parts of them were microtubule and actin filaments which were confirmed with confocal fluorescence microscopy. We also found several voids across a single tube, which might be the locations of microvesicles. Recently researchers reported that mitochondria–lysosome contacts regulate mitochondrial and lysosomal dynamics bi-directionally [4]. We also confirmed the existence of lysosomes and mitochondria in the TNTs. These organelle transportations could be a stress response of the cancer cells to the MaCM.

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2. Marzo, L.; Gousset, K.; Zurzolo, C., Front. Physiol. 2012, 3, 72.
3. Sigal, Y. M.; Zhou, R.; Zhuang, X., Science 2018, 361 (6405), 880-887.
4. Wong, Y. C.; Ysselstein, D.; Krainc, D., Nature 2018, 554, 382-386.

Keywords: focus ion beam, tunneling nanotubes, macrophage conditioned medium, mitochondria