CODEX multiplexed tissue imaging with DNA-conjugated antibodies

Christian M. Schürch, Sarah Black, Darci Phillips, John W. Hickey, Julia Kennedy-Darling, Vishal G. Venkataraaman, Nikolay Samusik, Yury Goltsev, Garry P. Nolan

Published: 2021-07-01 DOI: 10.1038/s41596-021-00556-8

Extended

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.

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References

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Supplementary information

Goltsev, Y. et al. Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell 174, 968–981.e15 (2018).
Schürch, C. M. et al. Coordinated cellular neighborhoods orchestrate antitumoral immunity at the colorectal cancer invasive front. Cell 182, 1341–1359.e19 (2020).
Kennedy-Darling, J. et al. Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction. Eur. J. Immunol. 51, 1262–1277 (2021).
Huang, W., Hennrick, K. & Drew, S. A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays. Hum. Pathol. 44, 29–38 (2013).
Lin, J. R. et al. Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes. eLife 7, 31657 (2018).
Gerdes, M. J. et al. Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. Proc. Natl Acad. Sci. USA 110, 11982–11987 (2013).
Schubert, W. et al. Analyzing proteome topology and function by automated multidimensional fluorescence microscopy. Nat. Biotechnol. 24, 1270–1278 (2006).
Gut, G., Herrmann, M. D. & Pelkmans, L. Multiplexed protein maps link subcellular organization to cellular states. Science 361, eaar7042 (2018).
Agasti, S. S. et al. DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging. Chem. Sci. 8, 3080–3091 (2017).
Wang, Y. et al. Rapid sequential in situ multiplexing with DNA exchange imaging in neuronal cells and tissues. Nano Lett. 17, 6131–6139 (2017).
Saka, S. K. et al. Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues. Nat. Biotechnol. 37, 1080–1090 (2019).
Giesen, C. et al. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. Methods 11, 417–422 (2014).
Angelo, M. et al. Multiplexed ion beam imaging of human breast tumors. Nat. Med. 20, 436–442 (2014).
Keren, L. et al. A structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging. Cell 174, 1373–1387.e19 (2018).
Wang, X. et al. Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 361, eaat5691 (2018).
Moffitt, J. R. et al. Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region. Science 362, eaau5324 (2018).
Moffitt, J. R. et al. High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proc. Natl Acad. Sci. USA 113, 14456–14461 (2016).
Wei, L. et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017).
Zhou, W., Han, Y., Beliveau, B. J. & Gao, X. Combining Qdot nanotechnology and DNA nanotechnology for sensitive single-cell imaging. Adv. Mater. 32, e1908410 (2020).
Bendall, S. C., Nolan, G. P., Roederer, M. & Chattopadhyay, P. K. A deep profiler’s guide to cytometry. Trends Immunol. 33, 323–332 (2012).
Hartmann, F. J. et al. Comprehensive immune monitoring of clinical trials to advance human immunotherapy. Cell Rep. 28, 819–831.e4 (2019).
Gomariz, A. et al. Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy. Nat. Commun. 9, 2532 (2018).
Phillips, D. et al. Highly multiplexed phenotyping of immunoregulatory proteins in the tumor microenvironment by CODEX tissue imaging. Front. Immunol. 12, 687673 (2021).
Newell, E. W. et al. Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization. Nat. Biotechnol. 31, 623–629 (2013).
Samusik, N., Good, Z., Spitzer, M. H., Davis, K. L. & Nolan, G. P. Automated mapping of phenotype space with single-cell data. Nat. Methods 13, 493–496 (2016).
Moen, E. et al. Deep learning for cellular image analysis. Nat. Methods 16, 1233–1246 (2019).
Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods 18, 100–106 (2021).
Blondel, V. D., Guillaume, J.-L., Lambiotte, R. & Lefebvre, E. Fast unfolding of communities in large networks. J. Stat. Mech. 2008, P10008 (2008).
Wu, D. et al. Comparison between UMAP and t-SNE for multiplex-immunofluorescence derived single-cell data from tissue sections. Preprint at bioRxiv https://doi.org/10.1101/549659 (2019).
Kishi, J. Y. et al. SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues. Nat. Methods 16, 533–544 (2019).
Frei, A. P. et al. Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nat. Methods 13, 269–275 (2016).
Schulz, D. et al. Simultaneous multiplexed imaging of mRNA and proteins with subcellular resolution in breast cancer tissue samples by mass cytometry. Cell Syst. 6, 25–36.e5 (2018).
Li, W., Germain, R. N. & Gerner, M. Y. Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D). Proc. Natl Acad. Sci. USA 114, E7321–E7330 (2017).
Ueda, H. R. et al. Tissue clearing and its applications in neuroscience. Nat. Rev. Neurosci. 21, 61–79 (2020).
Wassie, A. T., Zhao, Y. & Boyden, E. S. Expansion microscopy: principles and uses in biological research. Nat. Methods 16, 33–41 (2019).
Winter, P. W. & Shroff, H. Faster fluorescence microscopy: advances in high speed biological imaging. Curr. Opin. Chem. Biol. 20, 46–53 (2014).
Leng, E. et al. Signature maps for automatic identification of prostate cancer from colorimetric analysis of H&E- and IHC-stained histopathological specimens. Sci. Rep. 9, 6992 (2019).
Wu, P. H. et al. Single-cell morphology encodes metastatic potential. Sci. Adv. 6, eaaw6938 (2020).
Goldblum, J. R., Lamps, L. W., McKenney, J. K. & Myers, J. L. Rosai and Ackerman’s Surgical Pathology 11th edn, Vol. 1 (Elsevier, 2018).
Uhlen, M. et al. Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol. 28, 1248–1250 (2010).

CODEX multiplexed tissue imaging with DNA-conjugated antibodies

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