Abstract
Understanding cell–cell interactions is critical in most, if not all, research fields in biology. Nevertheless, studying intercellular crosstalk in vivo remains a relevant challenge, due mainly to the difficulty in spatially locating the surroundings of particular cells in the tissue. Cherry-niche is a powerful new method that enables cells expressing a fluorescent protein to label their surrounding cells, facilitating their specific isolation from the whole tissue as live cells. We previously applied Cherry-niche in cancer research to study the tumor microenvironment (TME) in metastasis. Here we describe how to generate cancer cells with the ability to label their neighboring cells (within the tumor niche) by transferring a liposoluble fluorescent protein. Live niche cells can be isolated and compared with cells distant from the tumor bulk, using a variety of ex vivo approaches. As previously shown, this system has the potential to identify novel components in the TME and improve our understanding of their local interactions. Importantly, Cherry-niche can also be applied to study potential cell–cell interactions due to in vivo proximity in research fields beyond cancer. This protocol takes 2–3 weeks to generate the labeling cells and 1–2 weeks to test their labeling ability.
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Acknowledgements
We thank the Biological Resources Unit, the Flow Cytometry Unit, the Experimental Histopathology Unit and the Cell Services Unit at the Francis Crick Institute for technical support. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001112), the UK Medical Research Council (FC001112), and the Wellcome Trust (FC001112), and European Research Council grant (ERC CoG-H2020-725492); L.O. was also funded by a Barts Charity Lectureship (grant no. MGU045). D.B. was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001045), the UK Medical Research Council (FC001045) and the Wellcome Trust (FC001045); D.P. was the recipient of a Junior EHA Fellowship. C.L.C. was supported by Cancer Research UK (CRUK PFA C36195/A26770) and the European Research Council (ERC STG 337066); D.D. was the recipient of an FCT fellowship (SFRH/BD/52195/2013). NESTIN-GFP mice were a kind gift from G. Enikolopov (Stony Brook University).
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L.O. designed the protocol, performed most of the experiments, analyzed the data and wrote the manuscript. E.N. and V.L.B. performed the experiments on liver metastasis and analyzed the data. D.P. and A.W. generated the leukemic labeling ML-1 cells and performed the experiments reported with those cells. I.K. performed the proliferation and the gel contraction experiments and analyzed the data. D.D. and C.L.C. ran pilot experiments to validate the labeling system, which helped with the troubleshooting, and critically read the manuscript. D.B. supervised the experiments with the leukemic cells. I.M. supervised the study and critically revised the manuscript.
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Peer review information Nature Protocols thanks Lance Liotta and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Ombrato, L. et al. Nature 572, 603–608 (2019): https://doi.org/10.1038/s41586-019-1487-6
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Supplementary Figs. 1–4.
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Ombrato, L., Nolan, E., Passaro, D. et al. Generation of neighbor-labeling cells to study intercellular interactions in vivo. Nat Protoc 16, 872–892 (2021). https://doi.org/10.1038/s41596-020-00438-5
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DOI: https://doi.org/10.1038/s41596-020-00438-5
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