Intravital imaging of metastatic behavior through a mammary imaging window

Abstract

We report a technique to evaluate the same tumor microenvironment over multiple intravital imaging sessions in living mice. We optically marked individual tumor cells expressing photoswitchable proteins in an orthotopic mammary carcinoma and followed them for extended periods through a mammary imaging window. We found that two distinct microenvironments in the same orthotopic mammary tumor affected differently the invasion and intravasation of tumor cells.

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Figure 1: The MIW allows for long-term, high-resolution imaging of the orthotopic tumors.
Figure 2: Photoswitching through the MIW is a tool for studying orthotopic tumor microenvironments.

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Acknowledgements

This work was supported by US Department of Defense (BC061403 to D.K.), US National Institutes of Health (U54GM064346 to J.v.R.; CA100324 to J.C., J.E.S. and J.W.; U54CA126511 to J.C. and B.G.; and GM070358 and GM073913 to V.V.V.). We thank the staff of the Analytical Imaging Facility and D. Entenberg for help with microscopy, the immunohistochemistry facility for help with histology, M. Rottenkolber for help in fabrication of the imaging box, J. Pollard (Albert Einstein College of Medicine) for providing the F4/80 antibody, S. Garofalo for technical assistance, and members of the Condeelis, Segall, Cox and Verkhusha laboratories for discussions.

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Correspondence to John Condeelis or Jeffrey E Segall or Jacco van Rheenen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Methods (PDF 778 kb)

Supplementary Movie 1

Movie recorded through the ocular, at low magnification (10×). After focusing the field and checking for the flowing vessels in the green epifluorescence channel, the photoswitched area can be recognized in the red channel. (MOV 803 kb)

Supplementary Movie 2

120-minute timelapse of a 250 × 250 µm area photoswitched inside the tumor. Notice the cells which move towards the fast flowing vessel. Non-photoswitched cells (green), photoswitched cells (red) and matrix (blue). (MOV 2261 kb)

Supplementary Movie 3

Z-stack of collagen (blue) and Dendra2-MTLn3 tumor cells (green and red) visualized in the live animal using a multiphoton microscope. The stepsize is 5µm. (MOV 1644 kb)

Supplementary Movie 4

10-minute timelapse of flowing vessels labeled using AlexaFluo647-10K (red, moving shadows are red blood cells) and Dendra2-MTLn3 tumor cells (green). Other channels were omitted for better contrast. (MOV 708 kb)

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Kedrin, D., Gligorijevic, B., Wyckoff, J. et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat Methods 5, 1019–1021 (2008). https://doi.org/10.1038/nmeth.1269

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