Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies


Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

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Fig. 1: Development of the DIIFCO imaging method.
Fig. 2: RNA detection in intact fresh-frozen human tumours with DIIFCO.
Fig. 3: RNA detection in intact human FFPE tumours with DIIFCO.
Fig. 4: RNA and protein detection in intact human tumours with DIIFCO.
Fig. 5: RNA and protein detection in human-derived organoids.
Fig. 6: Singe-cell analysis of RNAs and proteins in intact human tumours.
Fig. 7: Quantitative cell-by-cell analysis in intact human breast tumours.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding authors on reasonable request.

Code availability

The custom code used in this study is available at GitHub (


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We thank A. Östman for discussions. This study was supported by the Swedish Research Council (grant nos. 2009-3364, 2013-3189 and 2017-00815, to P.U.), the Swedish Cancer Society (grant nos. CAN 2016-801, 19 0544 Pj and 19 0545 Us, to P.U.), the Swedish Childhood Cancer Foundation (grant no. PR2018-0123, to P.U.), the Swedish Brain Foundation (grant nos. FO2018-0209, to P.U., and FO2018-0281, to A.F.), the Olle Engkvist foundation (to P.U.), the David and Astrid Hagelén Foundation (to N.T.), the Karolinska Institutet Research Foundation (to N.T., S.K. and P.U.), the Takeda Science Foundation (to N.T.), Grant-in-Aid for Scientific Research (KAKENHI 18H04906, 18K19482 and 19H03792, to N.T.), the Kobayashi Foundation for Cancer Research (to N.T.), the Keio Gijuku Academic Development Funds (to N.T.), the Scandinavia-Japan Sasakawa Foundation (to N.T., K.F. and S.K.), and the Wenner-Gren Foundation (to S.K.). The light-sheet microscopy infrastructure used in this research received grants from the Strategic Research Area in Neuroscience (StratNeuro) and the Strategic Research Area in Stem Cells and Regenerative Medicine (StratRegen), supported by the Swedish government.

Author information




N.T., S.K. and P.U. conceived the study and designed experiments. N.T., S.K., D.K., K.F., L.L. and K.M. performed the experiments and data analyses. N.T., S.K. and D.K. performed 3D image processing and 3D data analyses. A.M., P.K., S.R., C.L., L.K., N.N., K.M., J.H. and C.S. provided mouse and human tumour samples. T.M., O.K., R.P., A.F. and H.C. provided human organoids. M.O. and A.M. provided conceptual advice on clinical aspects. S.K., C.S., L.L. and H.C. reviewed and commented on the manuscript. N.T. and P.U. performed data interpretation and wrote the manuscript. P.U. supervised the research.

Corresponding authors

Correspondence to Nobuyuki Tanaka or Per Uhlén.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and captions for Supplementary Videos 1–6.

Reporting Summary

Supplementary Video 1

Volume rendering of a mouse embryo.

Supplementary Video 2

Cell-by-cell analysis of human breast cancer tissue.

Supplementary Video 3

Parvalbumin RNA and protein staining of a mouse cortical section.

Supplementary Video 4

Volume rendering of a human organoid.

Supplementary Video 5

Cell-by-cell analysis of human colon cancer tissue.

Supplementary Video 6

Spatial niche analysis of human breast cancer tissue.

Supplementary Dataset 1

Key resources.

Supplementary Dataset 2

Probe sequences and accession numbers of the targeted RNAs.

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Tanaka, N., Kanatani, S., Kaczynska, D. et al. Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies. Nat Biomed Eng 4, 875–888 (2020).

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