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CODEX multiplexed tissue imaging with DNA-conjugated antibodies

Abstract

Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity.

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Fig. 1: Key components of the CODEX technology.
Fig. 2: CODEX pipeline.
Fig. 3: Human FFPE tissues imaged with CODEX.
Fig. 4: Evolution of the CODEX technology.
Fig. 5: Timing for the key elements of a CODEX experiment.
Fig. 6: Components required for the CODEX experiment.
Fig. 7: Parameters for processing raw microscope images by using the CODEX Uploader.
Fig. 8: Starting parameters for performing single-cell segmentation on the uploaded data by using the CODEX Segmenter.
Fig. 9: Cleanup gating of segmented data.
Fig. 10: Starting parameters for performing cell-type annotation on cleaned data by using VorteX.
Fig. 11: Validation of CODEX antibody staining.
Fig. 12: Results from a CODEX multicycle experiment of human FFPE tonsil.

Data availability

We have uploaded both the concatenated CODEX imaging montage of the tissue (https://doi.org/10.6084/m9.figshare.12986981) and single-cell segmented data (https://doi.org/10.6084/m9.figshare.12986099) used in this paper to figshare (https://figshare.com/). The size of the raw imaging data is too large to be stored in a public repository and will therefore be stored in a private cloud-based server. Access to these data will be provided by the corresponding authors upon request.

Code availability

The image processing and data analysis tools presented in this article are available at https://github.com/nolanlab. The code used in this protocol has been peer reviewed.

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Acknowledgements

We thank Angelica Trejo, Han Chen, Kenyi Donoso, Nilanjan Mukherjee and Gustavo Vazquez for excellent technical assistance and Dr. Xavier Rovira-Clavé for critical comments on the manuscript. This work was supported by the U.S. National Institutes of Health (2U19AI057229-16, 5P01HL10879707, 5R01GM10983604, 5R33CA18365403, 5U01AI101984-07, 5UH2AR06767604, 5R01CA19665703, 5U54CA20997103, 5F99CA212231-02, 1F32CA233203-01, 5U01AI140498-02, 1U54HG010426-01, 5U19AI100627-07, 1R01HL120724-01A1, R33CA183692, R01HL128173-04, 5P01AI131374-02, 5UG3DK114937-02, 1U19AI135976-01, IDIQ17X149, 1U2CCA233238-01 and 1U2CCA233195-01); the U.S. Department of Defense (W81XWH-14-1-0180 and W81XWH-12-1-0591); the U.S. Food and Drug Administration (HHSF223201610018C and DSTL/AGR/00980/01); Cancer Research UK (C27165/A29073); the Bill and Melinda Gates Foundation (OPP1113682); the Cancer Research Institute; the Parker Institute for Cancer Immunotherapy; the Kenneth Rainin Foundation (2018-575); the Silicon Valley Community Foundation (2017-175329 and 2017-177799-5022); the Beckman Center for Molecular and Genetic Medicine; Juno Therapeutics, Inc. (122401); Pfizer, Inc. (123214); Celgene, Inc. (133826 and 134073); Vaxart, Inc. (137364); and the Rachford & Carlotta A. Harris Endowed Chair (to G.P.N.). C.M.S. was supported by the Swiss National Science Foundation (P300PB_171189 and P400PM_183915) and the Lady Tata Memorial Trust, London, UK. D.P. was supported by an NIH T32 Fellowship (AR007422), an NIH F32 Fellowship (CA233203), a Stanford Dean’s Postdoctoral Fellowship, a Stanford Cancer Institute Fellowship and Stanford’s Dermatology Department. J.W.H. was supported by an NIH T32 Fellowship (T32CA196585) and an American Cancer Society—Roaring Fork Valley Postdoctoral Fellowship (PF-20-032-01-CSM). Parts of Fig. 2 were created with BioRender.com.

Author information

Authors and Affiliations

Authors

Contributions

S.B., D.P., J.K.-D., N.S., Y.G. and C.M.S. developed the original experimental protocol in the G.P.N. laboratory. S.B., D.P., J.W.H. and C.M.S. performed experiments and analyzed data. V.G.V., N.S. and Y.G. developed the software used. S.B., D.P. and J.W.H. wrote the manuscript and created figures, with input from C.M.S. and G.P.N. All authors revised the manuscript and accepted its final version.

Corresponding authors

Correspondence to Christian M. Schürch or Garry P. Nolan.

Ethics declarations

Competing interests

J.K.-D. is an employee of Akoya Biosciences, Inc. N.S. is an employee of Becton Dickinson, Inc. G.P.N. received research grants from Pfizer, Inc.; Vaxart, Inc.; Celgene, Inc.; and Juno Therapeutics, Inc. during the course of this work. N.S., Y.G. and G.P.N. are inventors on US patent 9909167, granted to Stanford University, that covers some aspects of the technology described in this paper. J.K.-D., N.S., Y.G. and G.P.N. have equity in and/or are scientific advisory board members of Akoya Biosciences, Inc. C.M.S. is a scientific advisor to Enable Medicine, Inc. The other authors declare no competing interests.

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Key reference using this protocol

Schürch, C. M. et al. Cell 182, 1341–1359.e19 (2020): https://doi.org/10.1016/j.cell.2020.07.005

Extended data

Extended Data Fig. 1 Tissue morphology conserved over multicycle imaging.

One tile of the CODEX multicycle for the human tonsil was segmented by using the CODEXSegm software and the nuclear stain for cycle 1 or cycle 26 (nuclear images on the top; segmentation masks on the bottom). For both instances, 2,464 cells were identified. Scale bar, 100 µm.

Extended Data Fig. 2 Analysis of healthy and cancerous tissue morphology during multicycle imaging.

a, H&E image of a healthy spleen. Scale bar, 200 µm. b, Corresponding nuclear (Hoechst) stained image and cellular segmentation (Seg) mask. Scale bar, 200 µm. c, Zoomed-in nuclear stained image and cellular segmentation mask from cycle 2. Scale bar, 20 µm (Extended Data Applied Sciences 28 April 2020). d, Zoomed-in nuclear stained image and cellular segmentation mask from cycle 22. Scale bar, 200 µm. e, Line plot of total cell count per tissue microarray core, measured at cycles 1, 2, 8, 14 and 22, for five healthy tissues. Lines are colored according to the corresponding legend. f, H&E image of stomach cancer. Scale bar, 200 µm. g, Corresponding nuclear (Hoechst) stained image and cellular segmentation mask. Scale bar, 200 µm. h, Zoomed-in nuclear stained image and cellular segmentation mask from cycle 2. Scale bar, 20 µm. i, Zoomed-in nuclear stained image and cellular segmentation mask from cycle 22. Scale bar, 200 µm. j, Line plot of total cell count per tissue microarray core, measured at cycles 1, 2, 8, 14 and 22, for five cancer tissues. Lines are colored according to the corresponding legend.

Supplementary information

Supplementary Information

Supplementary Notes 1–4 and Supplementary Figs. 1–3.

Reporting Summary

Supplementary Tables

Supplementary Table 1 (tab 1): oligonucleotide sequences; Supplementary Table 2 (tab 2): antibodies, clones and manufacturers; Supplemental Table 3 (tab 3): CODEX multicycle antibody panel

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Black, S., Phillips, D., Hickey, J.W. et al. CODEX multiplexed tissue imaging with DNA-conjugated antibodies. Nat Protoc 16, 3802–3835 (2021). https://doi.org/10.1038/s41596-021-00556-8

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