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In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts

Nature Methods volume 6, pages 143–145 (2009)Cite this article

Abstract

Here we integrated multiphoton laser scanning microscopy and the registration of second harmonic generation images of collagen fibers to overcome difficulties in tracking stromal cell-matrix interactions for several days in live mice. We show that the matrix-modifying hormone relaxin increased tumor-associated fibroblast (TAF) interaction with collagen fibers by stimulating β1-integrin activity, which is necessary for fiber remodeling by matrix metalloproteinases.

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Figure 1: Buckling of a collagen fiber in a relaxin-treated tumor.
Figure 2: Quantitative analysis of collagen fiber length and area of colocalization between stromal cells and fibers.
Figure 3: Flow cytometry analysis of HSTS26T tumor–associated fibroblasts.

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Acknowledgements

This work was supported by US National Cancer Institute grants R01-CA98706 (Y.B.), R01-CA85140 and P01-CA80124 (R.K.J.), and fellowship Swiss National Funding for young scientists 107362, Fond Decker and Fond de Perfectionement du Centre Hospitalier Universitaire Vaudois (J.Y.P.). We thank J. Kahn for technical assistance.

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Author notes

  1. Jean Y Perentes & Trevor D McKee

    Present address: Present addresses: Department of Thoracic and Vascular Surgery, Centre Hospitalier Universitaire Vaudois, rue du Bugnon 46, 1010 Lausanne, Switzerland (J.Y.P.) and Department of Signaling Biology, Ontario Cancer Institute, 610 University Avenue, Toronto M5G 2M9, Canada (T.D.M.).,

  2. Jean Y Perentes and Trevor D McKee: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School, 100 Blossom Street, Boston, 02114, Massachusetts, USA

    Jean Y Perentes, Trevor D McKee, Carsten D Ley, Hannah Mathiew, Michelle Dawson, Timothy P Padera, Lance L Munn, Rakesh K Jain & Yves Boucher

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Contributions

J.Y.P. designed and performed experiments, analyzed data and wrote the paper. T.D.M. conceived the project, designed and performed experiments, analyzed data, and wrote the paper. C.D.L. performed imaging and immunostaining experiments. H.M. performed imaging and analyzed data. M.D. performed flow cytometry analysis. T.P.P. analyzed data and wrote the paper. L.L.M. analyzed data and wrote the paper. R.K.J. coordinated the project and wrote the paper. Y.B. coordinated the project, designed experiments, analyzed data and wrote the paper.

Corresponding authors

Correspondence to Rakesh K Jain or Yves Boucher.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-4, Supplementary Table 1, Supplementary Methods (PDF 4000 kb)

Supplementary Video 1

Region of interest in which 8 cells are tracked over the 4 day time course. Some cells exhibit projections with no directed movement, while others move a distance of one cell body or greater over the 4 day time-course. Cells of interest are drawn as outlines on the image in yellow, to indicate the extent of each individual cell over time. (MOV 566 kb)

Supplementary Video 2

Collagen fiber interacting with a migrating stromal cell. For each time point, the SHG, GFP and the merged GFP-SHG maximum intensity projection of the acquired volumes are shown on four consecutive days. The bottom panel is a schematic drawing. The collagen fiber is dragged by the migrating stromal cell. Bar = 10 μm. (MOV 267 kb)

Supplementary Video 3

Buckling rearrangement of a collagen fiber by stromal cells in a relaxin treated tumor. For each time point, the SHG, GFP and the merged GFP-SHG maximum intensity projections of the acquired volumes are shown. The bottom panel is a schematic drawing. Relaxin treatment seems to cause a long lasting stromal cell interaction with the collagen fiber eventually leading to buckling of the latter. Bar = 20 μm. (MOV 676 kb)

Supplementary Video 4

Gap formation in a collagen fiber induced by relaxin. For each time point, the SHG, GFP and the merged GFP-SHG maximum intensity projection of the acquired volumes are shown. The bottom panel is a schematic drawing. There is an increased interaction of stromal cells with collagen fibers between day 1 and 2 and fiber thinning and gap formation at later time points. Bar = 20 μm. (MOV 807 kb)

Supplementary Video 5

Global rearrangement of collagen fiber bundles. For each time point, the SHG and the merged GFP and SHG maximum intensity projection of the acquired volumes is represented. From day 2, a group of fibers associated to stromal cells seems to be pushed towards the center of the images. Bar = 25 μm. (MOV 420 kb)

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Perentes, J., McKee, T., Ley, C. et al. In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts. Nat Methods 6, 143–145 (2009). https://doi.org/10.1038/nmeth.1295

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