Intratumoral heterogeneity is a critical factor when diagnosing and treating patients with cancer. Marked differences in the genetic and epigenetic backgrounds of cancer cells have been revealed by advances in genome sequencing, yet little is known about the phenotypic landscape and the spatial distribution of intratumoral heterogeneity within solid tumours. Here, we show that three-dimensional light-sheet microscopy of cleared solid tumours can identify unique patterns of phenotypic heterogeneity, in the epithelial-to-mesenchymal transition and in angiogenesis, at single-cell resolution in whole formalin-fixed paraffin-embedded (FFPE) biopsy samples. We also show that cleared FFPE samples can be re-embedded in paraffin after examination for future use, and that our tumour-phenotyping pipeline can determine tumour stage and stratify patient prognosis from clinical samples with higher accuracy than current diagnostic methods, thus facilitating the design of more efficient cancer therapies.

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Download references


The authors would like to thank J. Szumiło, Department of Clinical Pathomorphology, Medical University of Lublin, Lublin, Poland for kindly providing human tissue samples. This study was supported by the Swedish Research Council (grants 2009-3364, 2010-4392 and 2013-3189 to P.U.), the Swedish Cancer Society (grant CAN2013/802 and CAN2016/801 to P.U.), the Swedish Brain Foundation (grant FO2017/0107 to P.U.), the Linnaeus Center in Developmental Biology for Regenerative Medicine (DBRM) (P.U.), a Knut and Alice Wallenberg Foundation Grant to the Center for Live Imaging of Cells at the Karolinska (CLICK) Institutet (P.U.), the Royal Swedish Academy of Sciences (P.U.), the David and Astrid Hagelén Foundation (N.T.), the Takeda Science Foundation (N.T.), the Scandinavia-Japan Sasakawa Foundation (N.T. and S.K.), and the Wenner-Gren Foundation (S.K.). The light-sheet microscopy infrastructure used in this work 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

Author notes

  1. Nobuyuki Tanaka and Shigeaki Kanatani contributed equally to this work.


  1. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden

    • Nobuyuki Tanaka
    • , Shigeaki Kanatani
    • , Dagmara Kaczynska
    • , Lauri Louhivuori
    • , Ayako Miyakawa
    •  & Per Uhlén
  2. Department of Urology, Keio University School of Medicine, Tokyo, 160-8582, Japan

    • Nobuyuki Tanaka
    • , Kazuhiro Matsumoto
    •  & Mototsugu Oya
  3. Department of Biological Sciences, Columbia University, New York, NY, 10027, USA

    • Raju Tomer
  4. Turku Centre for Biotechnology, University of Turku and Faculty of Science and Engineering, Åbo Akademi University, FI-20521, Turku, Finland

    • Cecilia Sahlgren
  5. Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, MB, 5600, The Netherlands

    • Pauliina Kronqvist
  6. Institute of Biomedicine/Pathology, University of Turku, FI-20014, Turku, Finland

    • Lorand Kis
    • , Claes Lindh
    • , Sara Corvigno
    • , Johan Hartman
    • , Artur Mezheyeuski
    • , Carina Strell
    • , Joseph W. Carlson
    • , Hanna Dahlstrand
    •  & Arne Östman
  7. Department of Oncology-Pathology, Karolinska Institutet, SE-17176, Stockholm, Sweden

    • Lorand Kis
    • , Claes Lindh
    • , Johan Hartman
    •  & Joseph W. Carlson
  8. Department of Pathology and Cytology, Karolinska University Hospital, SE-17176, Stockholm, Sweden

    • Przemysław Mitura
  9. Department of Urology and Oncological Urology, Medical University in Lublin, ul. Jaczewskiego 8, 20-954, Lublin, Poland

    • Andrzej Stepulak
  10. Department of Biochemistry and Molecular Biology, Medical University in Lublin, ul. Chodzki 1, 20-093, Lublin, Poland

    • Patrick Micke
    •  & Hanna Dahlstrand
  11. Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185, Uppsala, Sweden

    • Hanna Dahlstrand
  12. Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Karolinska University Hospital, SE-14186, Stockholm, Sweden

    • Carlos Fernández Moro
  13. Department of Clinical Pathology/Cytology, Karolinska University Hospital, SE-14186, Stockholm, Sweden

    • Carlos Fernández Moro
  14. Department of Oncology-Pathology, Karolinska University Hospital, SE-17176, Stockholm, Sweden

    • Peter Wiklund
    •  & Ayako Miyakawa
  15. Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-17177, Stockholm, Sweden

    • Peter Wiklund
    •  & Ayako Miyakawa
  16. Department of Urology, Karolinska University Hospital, SE-17176, Stockholm, Sweden

    • Karl Deisseroth
  17. Howard Hughes Medical Institute, W080 Clark Center, Stanford University, 318 Campus Drive West, Stanford, CA, 94305, USA

    • Karl Deisseroth
  18. Department of Bioengineering, W080 Clark Center, Stanford University, 318 Campus Drive West, Stanford, CA, 94305, USA

    • Karl Deisseroth
  19. Department of Psychiatry and Behavioral Sciences, W080 Clark Center, Stanford University, 318 Campus Drive West, Stanford, CA, 94305, USA

    • Cecilia Sahlgren


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N.T., S.K., A.Mi. and P.U. designed the study. N.T., S.K., D.K., L.L. and K.M. performed the experiments. N.T., S.K. and R.T. performed 3D image processing. R.T. and K.D. developed the custom-built light-sheet microscope system. C.Sa., P.K., L.K., C.L., P.M., A.S., S.C., J.H., P.M., A.Me., C.St., J.W.C., C.F.M., H.D. and A.Mi. provided human tumour samples. P.W., M.O., A.Ö. and K.D. provided conceptual advice. N.T. and P.U. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Per Uhlén.

Electronic supplementary material

  1. Supplementary Information

    Supplementary figures, tables and video legends.

  2. Life Sciences Reporting Summary

    Life Sciences Reporting Summary

  3. Supplementary Video 1

    Three-dimensional volume reconstruction of hTumour 1immunostained for E-cadherin.

  4. Supplementary Video 2

    Three-dimensional volume reconstruction of hTumour 3 immunostained for N-cadherin.

  5. Supplementary Video 3

    Three-dimensional volume reconstruction of hTumour 6 immunostained for CD34.

  6. Supplementary Video 4

    Three-dimensional volume reconstruction of the CD34 signal.

  7. Supplementary Video 5

    Single-cell 3D volume reconstruction of hTumour 7 immunostained for Vimentin.

  8. Matlab script 1

    Generation of centroids list (point cloud) from Hmaxima images.

  9. Matlab script 2

    Calculation of mean intensity value of each dots area.

  10. Matlab script 3

    Generation of binary images from XYZ coordinates.

  11. Supplementary Table 1

    Clinicopathological characteristics of 50 human urothelial FFPE samples.

  12. Supplementary Table 2

    Clinicopathological characteristics of 16 human ovarian cancer FFPE samples.