Stable, high-resolution intravital imaging of the lung has become possible through the utilization of vacuum-stabilized imaging windows. However, this technique is extremely invasive and limited to only hours in duration. Here we describe a minimally invasive, permanently implantable window for high-resolution intravital imaging of the murine lung that allows the mouse to survive surgery, recover from anesthesia, and breathe independently. Compared to vacuum-stabilized windows, this window produces the same high-quality images without vacuum-induced artifacts; it is also less invasive, which allows imaging of the same lung tissue over a period of weeks. We further adapt the technique of microcartography for reliable relocalization of the same cells longitudinally. Using commonly employed experimental, as well as more clinically relevant, spontaneous metastasis models, we visualize all stages of metastatic seeding, including: tumor cell arrival; extravasation; growth and progression to micrometastases; as well as tumor microenvironment of metastasis function, the hallmark of hematogenous dissemination of tumor cells.
This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work: Einstein's Integrated Imaging Program, Montefiore's Ruth L. Kirschstein T32 Training Grant of Surgeons for the Study of the Tumor Microenvironment (CA200561), NIH grants CA100324, CA216248, P30CA013330, and SIG #1S10OD019961-01. We thank M. Rottenkolber, R. Ibagon and A. Leggiadro of the Einstein Machine Shop for their skilled craftsmanship and design insight; and we thank C. Rodriguez-Tirado, B. Canella and U. Steidl's lab for help with evaluating blood counts.
Integrated supplementary information
WHRIL bearing mice are able to perform tasks of daily living (grooming, feeding, nesting, stretching, etc.) without impediment.
Left: raw, unprocessed video showing single cell resolution imaging. Right: Same video with red (blood) channel averaged over time to improve definition of the vascular boundaries. Time between frames = 1 sec, FOV = 170 μm. Cyan = CFP labeled myeloid cells, Red = vasculature labeled by 155kD TMR-dextran.
Time lapse video of raw data shown in Figure 4A showing arrival of a tumor cell to join other cells trapped in the lung vasculature.
Time between frames = 1.35 min. Time stamp = hh:mm:ss. FOV = 117x67 μm. Green = tumor cell, red = vasculature, cyan = macrophages.
Time lapse video of blood averaged data shown in Figure 4B showing extravasation of an experimentally metastasized tumor cell from inside the lung vasculature into an alveolus.
Left: Maximum intensity projection of 3 slices (9 μm) within the lung vasculature. Right: 3D reconstruction of the entire data set (8 slices, 24 μm) FOV = 60 μm. Time between frames = 64 sec. Time stamp = hh:mm:ss.
Raw unprocessed data corresponding to Figure 4B showing a single optical section of the lung vasculature. Time between frames = 64 sec. Green = GFP tumor cells, Cyan = CFP macrophages, Red = Ve-Cad labeled endothelia and 155kD TMR dextran. Time stamp = hh:mm:ss.
Video of data presented in Figure 5A showing one cell of a group of spontaneously metastasized tumor cells appearing to undergo division in the lung.
Time between frames = 1.42 min. FOV = 75 μm. Green = GFP labeled tumor cells, Red = 155kD TMR dextran, Blue = SHG from collagen I fibers. Time stamp =mm:ss
Yellow arrow indicates location of transient vascular leakage seen as red dextran appearing in the extravascular space.