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ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix

Abstract

The extracellular matrix (ECM) is a master regulator of cellular phenotype and behavior. It has a crucial role in both normal tissue homeostasis and disease pathology. Here we present a fast and efficient approach to enhance the study of ECM composition and structure. Termed in situ decellularization of tissues (ISDoT), it allows whole organs to be decellularized, leaving native ECM architecture intact. These three-dimensional decellularized tissues can be studied using high-resolution fluorescence and second harmonic imaging, and can be used for quantitative proteomic interrogation of the ECM. Our method is superior to other methods tested in its ability to preserve the structural integrity of the ECM, facilitate high-resolution imaging and quantitatively detect ECM proteins. In particular, we performed high-resolution sub-micron imaging of matrix topography in normal tissue and over the course of primary tumor development and progression to metastasis in mice, providing the first detailed imaging of the metastatic niche. These data show that cancer-driven ECM remodeling is organ specific, and that it is accompanied by comprehensive changes in ECM composition and topological structure. We also describe differing patterns of basement-membrane organization surrounding different types of blood vessels in healthy and diseased tissues. The ISDoT procedure allows for the study of native ECM structure under normal and pathological conditions in unprecedented detail.

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Figure 1: Surgical modification of vascular flow for decellularization.
Figure 2: Integrity validation and analysis of the structure of ISDoT tissues.
Figure 3: The 3D structure of the primary breast cancer niche.
Figure 4: Mass spectrometry analysis of ISDoT ECM-enriched tissues.
Figure 5: Visualization of metastatic lymph node remodeling.
Figure 6: Visualization of metastatic lung remodeling.

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Acknowledgements

We thank I. Novak and N. Meyn Christensen (Centre for Advanced Bioimaging (CAB), University of Copenhagen) for imaging assistance. We thank J.R. Brewer (University of Southern Denmark) for providing access to his custom-built two-photon microscope. We thank J. Koch for help with decellularization procedures. We thank E. Sahai (Francis Crick Institute) for providing the MATLAB code. We thank J. Couchman, K.B. Jensen (both from Biotech Research & Innovation Centre, University of Copenhagen) and E. Sahai for their critical reading of the manuscript. We thank R. Linding (BRIC, University of Copenhagen) for providing access to mass spectrometry facilities. This work was supported by the Danish Cancer Society (R56-A3342, R124-A7862) (A.E.M.-G. and E.R.H.); the Novo Nordisk Foundation (Hallas Møller Stipend; to C.D.M. and J.T.E.); the European Research Council (ERC-2015-CoG-682881-MATRICAN; to A.E.M.-G. and E.R.H.); the Ragnar Söderberg Foundation Sweden (N19/15; C.D.M.); Cancerfonden Sweden (CAN 2016/283); the Innovation Foundation Denmark (1311-00010B; to T.R.C.); the National Health and Medical Research Council (NHMRC) Australia (APP1129766; T.R.C.); and the Danish Council for Independent Research YDUN grant (1084181001; F.V.A.).

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Authors

Contributions

A.E.M.-G. conceived the project, and the project was developed together with C.D.M., T.R.C. and J.T.E. Together, A.E.M.-G., C.D.M., T.R.C. and J.T.E. designed all experiments. A.E.M.-G. designed the surgical decellularization experiments. C.D.M. performed all imaging, assisted by A.E.M.-G., and T.R.C. performed and analyzed all mass spectrometry data. E.R.H. performed the clustering analysis. T.R.C. performed fiber orientation, diameter, gap and periodicity analyses, and was assisted by C.D.M. E.R.H., F.A.V. and A.E.M.-G. performed ECM staining. A.E.M.-G., C.D.M., T.R.C. and J.T.E. wrote and edited the manuscript.

Corresponding author

Correspondence to Janine T Erler.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–10 and Supplementary Tables 1–3 (PDF 26132 kb)

Fully narrated surgical procedure for lung decellularization and decellularization set-up. (MP4 105511 kb)

Z-stack of a healthy lung freshly resected (AVI 240 kb)

Z-stack of a healthy decellularized lung (AVI 6776 kb)

Z-stack of a healthy lymph node freshly resected. (AVI 4121 kb)

Z-stack of a healthy decellularized lymph node. (AVI 2678 kb)

Z-stack of a healthy decellularized peripheral nerve bundle stained for collagen-4. (AVI 1144 kb)

Z-stack of a decellularized 4T1 breast tumour immunostained for Nidogen-1. (AVI 3989 kb)

3D representation of a decellularized 4T1 satellite lymph nodes immunostained for Collagen type IV. (AVI 11435 kb)

Z-stacks of decellularized healthy (left panel) and decellularized B16F10 tongue melanoma satellite (right panel) lymph nodes immunostained for Collagen type IV. (AVI 6705 kb)

Z-stacks of decellularized healthy (left panel) and decellularized B16F10 tongue melanoma satellite (right panel) lymph nodes immunostained for Nidogen 1 The Z-stacks descend from cortex into the medulla. (AVI 11607 kb)

Z-stack of a decellularized B16F10 tongue melanoma satellite lymph node. (AVI 2737 kb)

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Mayorca-Guiliani, A., Madsen, C., Cox, T. et al. ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix. Nat Med 23, 890–898 (2017). https://doi.org/10.1038/nm.4352

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