Abstract
Membrane nanotubes are cytosolic protrusions with diameters <1 µm that extend between cells separated by tens of µm. They mediate several forms of intercellular communication and are upregulated in diverse diseases. Difficulties in visualizing and studying nanotubes within intact tissues have, however, prompted skepticism regarding their in vivo relevance, and most studies have been confined to cell culture systems. Here, we introduce lattice-light sheet imaging of MDA-MB-231 human breast cancer cells genetically engineered to brightly express membrane–targeted GFP as a promising approach to visualize membrane nanotubes in vitro and in situ. We demonstrate that cultured cells form multiple nanotubes that mediate intercellular communication of Ca2+ signals and actively traffic GFP-tagged membrane vesicles along their length. Furthermore, we directly visualize nanotubes in situ, interconnecting breast cancer cells in live acute brain slices from an experimental mouse model of breast cancer brain metastasis. This amenable experimental system should facilitate the transition of the study of intercellular communication by membrane nanotubes from cell culture to the whole animal.
Highlights
Cell-to-cell communication is vital for coordinating the development and proper functioning of tissues and organs
We introduce the use of lattice-light sheet microscopy (LLSM) to image membrane nanotubes in vitro and in situ
To visualize membrane nanotubes we employed a human breast cancer cell line[15] expressing a bright fluorescent genetically-encoded GFP membrane marker. We show these cells form multiple nanotubes in culture, which functionally mediate the propagation of Ca2+ signals between cells, and actively traffic GFP-tagged membrane aggregates along their length. Using this cell line in a well-established model of brain cancer metastasis in the mouse[15,16,17] we show the membrane marker is retained after several weeks in vivo and, utilizing high-resolution LLSM of acute live brain slices, we visualize membrane nanotubes interconnecting cells within dense brain metastatic lesions
Summary
MDA-231 cells form multiple nanotubes in culture. MDA-MB-231Br2 GFP cells (MDA-231GFP). We further observed similar membrane protrusions extending from the upper parts of MDA-231GFP cells and forming footlike contacts to the cover glass (length 12.5 ± 6 μm, mean ± s.d, n = 113; width 423 ± 35 nm FWHM, mean ± s.d, n = 6, Fig. 1b,d). We utilized the stable integration of plasma membrane targeted GFP into the genome of MDA-231 cells as a permanent membrane marker to enable high-resolution LLSM imaging within brain metastases. We observed long (13.85 ± 6 μm, mean ± s.d, n = 26), thin (457 ± 30 nm, mean ± s.d, n = 10) nanotube-like structures interconnecting MDA-231GFP cells within brain slices (Fig. 3a–c and Movie 3); characteristics closely similar to those of nanotubes in culture (cf Fig. 1). A deeper understanding of potential interactions between cancer cells mediated by membrane nanotubes may illuminate new avenues of research and potential therapies
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