Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging

Abstract

Intravital multiphoton microscopy has provided powerful mechanistic insights into health and disease and has become a common instrument in the modern biological laboratory. The requisite high numerical aperture and exogenous contrast agents that enable multiphoton microscopy, however, limit the ability to investigate substantial tissue volumes or to probe dynamic changes repeatedly over prolonged periods. Here we introduce optical frequency domain imaging (OFDI) as an intravital microscopy that circumvents the technical limitations of multiphoton microscopy and, as a result, provides unprecedented access to previously unexplored, crucial aspects of tissue biology. Using unique OFDI-based approaches and entirely intrinsic mechanisms of contrast, we present rapid and repeated measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy, thereby demonstrating the potential of OFDI to facilitate the exploration of physiological and pathological processes and the evaluation of treatment strategies.

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Figure 1: Principles of in vivo multiparametric OFDI.
Figure 2: Comparison of multiphoton and OFDI angiography.
Figure 3: Contrast-free lymphangiography using OFDI.
Figure 4: Imaging tissue viability.
Figure 5: Multiparametric response of directed anticancer therapy characterized by OFDI.

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Acknowledgements

We thank J. Baish for insightful input regarding fractal analysis. We also thank J. Kahn and S. Roberge for preparation of animal models, P. Huang for animal care and colony maintenance, and E. di Tomaso and C. Smith for histological preparations. We thank Genentech for supplying the MDA-MB-361HK mammary carcinoma cells and J.B. Little of the Harvard School for Public Health for HSTS26T. This research was funded in part by US National Institutes of Health grants P01-CA080124, R01-CA085140, R01-CA115767, R01 CA126642, R33-CA125560, K25-CA127465, K99-CA137167, and R01-CA096915. R.M.L. is supported in part by US Department of Defense Breast Cancer Research Program fellowship W81XWH-06-1-0436.

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Authors

Contributions

B.J.V. developed OFDI technology. B.J.V. and R.M.L. designed and performed most of the experiments, developed methodology, headed all data analysis and wrote the manuscript. J.A.T. contributed to vascular tracing of OFDI data. T.P.P. performed lymphangiography experiments and contributed to data analysis and manuscript preparation. L.A.B. performed VEGFR-2 blockade in vivo experiments. T.S. developed and performed fractal characterization and contributed to manuscript preparation. L.L.M. contributed to vascular data analysis. G.J.T. contributed to OFDI technology development. D.F. contributed to experimental design and manuscript preparation. R.K.J. and B.E.B. contributed to the design of experiments, preparation of the manuscript and supervised the project.

Corresponding authors

Correspondence to Rakesh K Jain or Brett E Bouma.

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Competing interests

R.K.J. is a consultant for AstraZeneca, Dyax and Millenium and has received research grants from AstraZeneca and Dyax. He has received honoraria for lectures from Roche and Pfizer. He is on the scientific advisory boards for Enlight and SynDevRx. B.J.V., G.J.T. and B.E.B. are inventors of the optical frequency domain imaging apparatus and methods used in the research described by this manuscript.

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Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Methods (PDF 6251 kb)

Supplementary Video 1

Time-lapse video of an MCaIV tumor implanted in the dorsal skinfold chamber. Changes in tumor microvasculature are shown starting 8 h before to 40 h after DC101 treatment. Tumor volume and mean intratumoral vessel diameter are tracked at 2 h intervals for 48 h. (MOV 2611 kb)

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Vakoc, B., Lanning, R., Tyrrell, J. et al. Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging. Nat Med 15, 1219–1223 (2009). https://doi.org/10.1038/nm.1971

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