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Multiplexed single-cell analysis of organoid signaling networks

Abstract

Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type–specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type–specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for >40 antibodies from millions of cells across 126 organoid cultures in a single MC run.

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Fig. 1: Organoids are high-dimensional systems.
Fig. 2: TOBis MC protocol overview.
Fig. 3: TOBis multiplexing overview.
Fig. 4: TOBis MC barcoding fidelity.
Fig. 5: TOBis MC for organoid co-cultures.
Fig. 6: TOBis MC data analysis using CyGNAL.

Data availability

All raw data, processed data and working illustrations are available as a Community Cytobank project (https://community.cytobank.org/cytobank/experiments#project-id=1334).

Code availability

The latest CyGNAL pipeline is available at https://github.com/TAPE-Lab/CyGNAL. CyGNAL version 0.2.1 as described in this publication can be found at https://github.com/TAPE-Lab/CyGNAL/releases/tag/v0.2.1. The OT-2 barcode preparation code is available at https://github.com/TAPE-Lab/OT-2-Automated-Barcode-Pipetting. The code in this paper has been peer reviewed.

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Acknowledgements

We are extremely grateful to L. Dow for sharing murine colonic organoids, M. Garnett and H. Francies for sharing PDOs, O. Ornatsky for providing 196cisplatin, S. Acton for providing murine tissue for fibroblast and macrophage isolation and A. Taylor and S. Guldin for OT-2 access and advice. We thank the UCL CI Flow-Core for CyTOF support. This work was supported by Cancer Research UK (C60693/A23783), the Cancer Research UK UCL Centre (C416/A25145), the Cancer Research UK City of London Centre (C7893/A26233), the UCLH Biomedical Research Centre (BRC422), The Royal Society (RSG\R1\180234) and The Rosetrees Trust (A1989).

Author information

Authors and Affiliations

Authors

Contributions

J.S. developed TOBis, designed rare earth metal-conjugated antibody panels and performed MC analysis. X.Q. designed and performed organoid and MC experiments, analyzed the data and wrote the manuscript. F.C.R. developed CyGNAL and wrote the manuscript. P.V. and M.R.Z. performed organoid and MC experiments. Y.J.B. and M.N. developed TeMal reagents. C.J.T. designed the study, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Christopher J. Tape.

Ethics declarations

Competing interests

M.N. has pending intellectual property on the use of TeMal reagents for mass cytometry applications, which has been licensed to Fluidigm Corporation.

Additional information

Peer review information Nature Protocols thanks Kara L. Davis, Ozgun Gokce and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key reference using this protocol

Qin, X. et al. Nat. Methods 17, 335–342 (2020): https://doi.org/10.1038/s41592-020-0737-8

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Supplementary Tables 1 and 2 and Supplementary Method.

Reporting Summary

Supplementary Table 3

Debarcoding key for 35-plex TOBis

Supplementary Table 4

Debarcoding key for 126-plex TOBis

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Sufi, J., Qin, X., Rodriguez, F.C. et al. Multiplexed single-cell analysis of organoid signaling networks. Nat Protoc 16, 4897–4918 (2021). https://doi.org/10.1038/s41596-021-00603-4

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