Brain metastasis frequently occurs in individuals with cancer and is often fatal. We used multiphoton laser scanning microscopy to image the single steps of metastasis formation in real time. Thus, it was possible to track the fate of individual metastasizing cancer cells in vivo in relation to blood vessels deep in the mouse brain over minutes to months. The essential steps in this model were arrest at vascular branch points, early extravasation, persistent close contacts to microvessels and perivascular growth by vessel cooption (melanoma) or early angiogenesis (lung cancer). Inefficient steps differed between the tumor types. Long-term dormancy was only observed for single perivascular cancer cells, some of which moved continuously. Vascular endothelial growth factor-A (VEGF-A) inhibition induced long-term dormancy of lung cancer micrometastases by preventing angiogenic growth to macrometastases. The ability to image the establishment of brain metastases in vivo provides new insights into their evolution and response to therapies.
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F.W. and J.H. received funding for this work from the German Cancer Foundation (Deutsche Kebshilfe), and F.W. received a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft; WI1930/4-1). J.H. received additional funding from the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung) and German Federal Ministry of Economics and Technology (Bundesministerium für Wirtschaft und Technologie) (16IN0675; 13N10171; 13N9268). Y.K. received a research grant from the Hypatia Foundation. We wish to thank C. Eggert for her excellent technical assistance, A. Ullrich, W. Hiddemann, R. Jain and U. Koedel for helpful discussions, K. Ogston for editing the manuscript and G. Mitteregger for her supervision of the animal facility. The PC14-PE6 human lung cancer cell line was kindly provided by I.J. Fidler, MD Anderson Cancer Center.
Initial arrest of cancer cells in the brain microvasculature. Passive movement of PC14-PE6 lung carcinoma cells (red) in relation to perfused brain microvessels (green, FITC dextran) imaged immediately after internal carotid artery injection for 3 min. Single images were acquired every 0.5 s, 50 μm deep in the brain parenchyma. Cells are arrested in capillaries of the same diameter, primarily at vascular bifurcations; n = 4 mice.
Cancer cell arrest stays dynamic for hours. When followed over 1 h after internal carotid injection, it becomes evident that cells continuously give up and reenter a resting position; permanent arrest is rare. This could explain why neither areas with permanent absence of blood flow in vivo, nor brain infarcts in histological examinations have been detected. Bifurcations of capillaries of the same diameter are still the preferred sites for arrest. Images were acquired every 30 s for 60 minafter injection of PC14-PE6 lung carcinoma cells (n = 4 animals; here: 20–50 μm depth).
Transmigration of the vascular wall. A melanoma cell (red, arrows) has partly extravasated through a hole in the wall (detected by additional GFP-positivity of endothelial cells in this animal, not shown) of blood vessel number 1. Note the extensive movement of the extravasated part, extending and retracting lamellipodia-like protrusions (arrowheads). The extravasated part is located in close proximity to a deeper larger vessel, which has no disruption of the endothelial lining (number 2). This video demonstrates the activity of the extravasation process, which always took longer than 30 min (n = 5 extravasating A2058 melanoma cells in n = 4 animals observed). Images were acquired every 45 s for 60 min, 24 h after injection (20–100 μm depth).