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Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity


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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  • 02 January 2018

    In this Article originally published, owing to a technical error, author affiliations were incorrectly assigned in the HTML version; the PDF was correct. These errors have now been corrected.


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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

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.

Correspondence to Per Uhlén.

Supplementary information

Supplementary Information

Supplementary figures, tables and video legends.

Life Sciences Reporting Summary

Supplementary Video 1

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

Supplementary Video 2

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

Supplementary Video 3

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

Supplementary Video 4

Three-dimensional volume reconstruction of the CD34 signal.

Supplementary Video 5

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

Matlab script 1

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

Matlab script 2

Calculation of mean intensity value of each dots area.

Matlab script 3

Generation of binary images from XYZ coordinates.

Supplementary Table 1

Clinicopathological characteristics of 50 human urothelial FFPE samples.

Supplementary Table 2

Clinicopathological characteristics of 16 human ovarian cancer FFPE samples.

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Further reading

Fig. 1: Assessment of embedding tissues in paraffin before 3D imaging.
Fig. 2: Whole-tissue 3D imaging of mouse bladder tumours.
Fig. 3: Whole-tissue 3D imaging of human FFPE tumours.
Fig. 4: Single-cell analysis of a human FFPE tumour.
Fig. 5: Re-embedding of cleared human FFPE tumours.
Fig. 6: Diagnostic assessment of clinical UC FFPE samples using the DIPCO pipeline.
Fig. 7: Diagnostic assessment of clinical ovarian cancer FFPE samples using the DIPCO pipeline.