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

ABSTRACT 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. Access through your institution Buy or subscribe This is a preview of subscription content, access via

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS HIGH RESOLUTION MAPPING OF THE TUMOR MICROENVIRONMENT USING INTEGRATED

SINGLE-CELL, SPATIAL AND IN SITU ANALYSIS Article Open access 19 December 2023 A MULTI-MODAL SINGLE-CELL AND SPATIAL EXPRESSION MAP OF METASTATIC BREAST CANCER BIOPSIES ACROSS

CLINICOPATHOLOGICAL FEATURES Article Open access 30 October 2024 SPATIALLY RESTRICTED DRIVERS AND TRANSITIONAL CELL POPULATIONS COOPERATE WITH THE MICROENVIRONMENT IN UNTREATED AND

CHEMO-RESISTANT PANCREATIC CANCER Article Open access 22 August 2022 CHANGE HISTORY * _ 02 JANUARY 2018 In this Article originally published, owing to a technical error, author affiliations

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Outpatient Test. JAMA_ 259, 1194–1198 (1988). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS 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 * Nobuyuki Tanaka and Shigeaki

Kanatani contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177, Stockholm, Sweden Nobuyuki Tanaka, 

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

Kazuhiro Matsumoto & Mototsugu Oya * Department of Biological Sciences, Columbia University, New York, NY, 10027, USA Raju Tomer * Turku Centre for Biotechnology, University of Turku and

Faculty of Science and Engineering, Åbo Akademi University, FI-20521, Turku, Finland Cecilia Sahlgren * Institute for Complex Molecular Systems, Eindhoven University of Technology,

Eindhoven, MB, 5600, The Netherlands Cecilia Sahlgren * Institute of Biomedicine/Pathology, University of Turku, FI-20014, Turku, Finland Pauliina Kronqvist * Department of

Oncology-Pathology, Karolinska Institutet, SE-17176, Stockholm, Sweden Lorand Kis, Claes Lindh, Sara Corvigno, Johan Hartman, Artur Mezheyeuski, Carina Strell, Joseph W. Carlson, Hanna

Dahlstrand & Arne Östman * Department of Pathology and Cytology, Karolinska University Hospital, SE-17176, Stockholm, Sweden Lorand Kis, Claes Lindh, Johan Hartman & Joseph W.

Carlson * Department of Urology and Oncological Urology, Medical University in Lublin, ul. Jaczewskiego 8, 20-954, Lublin, Poland Przemysław Mitura * Department of Biochemistry and Molecular

Biology, Medical University in Lublin, ul. Chodzki 1, 20-093, Lublin, Poland Andrzej Stepulak * Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185, Uppsala,

Sweden Patrick Micke & Hanna Dahlstrand * Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Karolinska University Hospital, SE-14186, Stockholm, Sweden

Carlos Fernández Moro * Department of Clinical Pathology/Cytology, Karolinska University Hospital, SE-14186, Stockholm, Sweden Carlos Fernández Moro * Department of Oncology-Pathology,

Karolinska University Hospital, SE-17176, Stockholm, Sweden Hanna Dahlstrand * Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-17177, Stockholm, Sweden Peter Wiklund 

& Ayako Miyakawa * Department of Urology, Karolinska University Hospital, SE-17176, Stockholm, Sweden Peter Wiklund & Ayako Miyakawa * Howard Hughes Medical Institute, W080 Clark

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

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

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also search for this author inPubMed Google Scholar CONTRIBUTIONS 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. CORRESPONDING AUTHOR Correspondence to Per

Uhlén. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE: Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional affiliations. A correction to this article is available online at https://doi.org/10.1038/s41551-017-0162-1. 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. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Tanaka, N., Kanatani, S., Tomer, R. _et al._ Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. _Nat Biomed Eng_ 1, 796–806 (2017).

https://doi.org/10.1038/s41551-017-0139-0 Download citation * Received: 11 January 2017 * Accepted: 31 August 2017 * Published: 02 October 2017 * Issue Date: October 2017 * DOI:

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