In vivo subcellular resolution optical imaging in the lung reveals early metastatic proliferation and motility

Abstract

To better understand breast cancer metastatic cell seeding, we have employed multiphoton microscopy and a vacuum stabilized window which eliminates the need for complex registration software, video rate microscopy or specialized gating electronics to observe the initial steps of tumor cell seeding within the living, breathing lung. We observe that upon arrival to the lung, tumor cells are found exclusively in capillary vessels, completely fill their volume and display an initial high level of protrusive activity that dramatically reduces over time. Further, we observe a concomitant increase in positional stability during this same period. We employ several techniques accessible to most imaging labs for optimizing signal to noise and resolution which enable us to report the first direct observation, with subcellular resolution, of the arrival, proliferation, and motility of metastatic tumor cells within the lung.

Keywords: Intravital imaging; cancer; macrophage; metastasis; multiphoton; vacuum window.

Figure 1.

Lung Imaging Window: The Lung Imaging Window utilizes vacuum to attach the lung tissue to the coverglass, thereby immobilizing the tissue, and fixtures the window within a stage plate, thereby immobilizing the window. This new design prevents motion artifacts and eliminates the need for either timing image acquisition with breathing/ventilation or complex image registration algorithms. (A) Computer Aided Design (CAD) drawing of Lung Imaging Window. (B) CAD drawing of fixturing stage plate. The center hole which serves as an imaging aperture through which the objective lens images the lung tissue, has a tightly toleranced step which accepts the imaging window. (C–E) Cartoons showing the steps of the lung window surgery. (C) An incision in the neck through the skin and salivary gland allows access to the trachea for insertion of a catheter which is connected to a ventilator. (D) With the catheter in place, the skin, muscle and ribs are removed exposing the left lung. (E) The vacuum window is attached to the stage plate and connected to the vacuum line. The mouse is inverted and positioned so the exposed lung tissue is covers the window. The vacuum is then turned on and imaging may be performed through the imaging aperture.

Figure 2.

Stable imaging with sub-cellular resolution of the intact, breathing lung. Still images from a 140 min long time lapse movie showing a micro-metastasis in the lung 5 d after single tumor cells were introduced into the mouse via tail vein injection. In one cell, the unlabeled nucleus (arrows) appears as a shadow within the GFP labeled cytoplasm and reveals the cell progressing through the different stages of mitosis. The telophase stage is marked by the separation of pinching and separation of the cytoplasm of the 2 daughter cells (arrow heads). The red channel (blood labeled with 155kD Rhodamine-dextran) has been averaged over the duration of the movie and overlaid with the green (GFP tumor cells) and cyan (CFP macrophages) channels. Frames were acquired every 2.5 min with 3 frame averages per acquisition.

Figure 3.

Tumor cells lodge in capillaries and completely fill their lumen immediately after tail vein injection. (A) Stills taken from a time lapse movie showing a tumor cell lodged in a capillary immediately after tail vein injection. Dextran signal was quantified within 80μm² circles in 3 locations: 1 – on top of the tumor cell, 2 – in the extravascular space and 3 – in the lumen of a flowing vessel. Cyan = macrophages, Green = Tumor cells, Red = Dextran. (B) Quantification of the time course of the dextran signals within the 3 locations indicated in A). (C) Analysis of the dextran exclusion observed in movies of 5 cells within the lungs of 3 different mice. No statistical difference was observed between the dextran levels measured within the tumor cell volume and the extravascular space while the level of dextran within the flowing vessels averaged 20 times greater. Error bars: SEM, n=5 from 3 mice, *** = p<.0001. (D) Histogram of the diameter of vessels containing tumor cells immediately after tail vein injection. All of the vessels (50 cells in 5 mice) were under the single cell diameter of ∼20 μm

Figure 4.

Quantification of motility and protrusions. Immediately after tail vein injection, tumor cells lodge in the vasculature of the lung, exhibit large protrusions and continue to slowly move down the length of the capillary. These protrusions decrease with time. (A) Outlines of cell boundary over the length of the movies overlaid on a single image. (B) Top: Quantification of mean cell area shows no statistically significant difference between 0, 12 and 24 hrs. Bottom: Quantification of the standard deviation of the area difference between each time point and the starting frame. Larger standard deviations in this differential area are indicative of larger protrusions and decrease significantly by 12 and 24 hrs. Error Bars = SEM. (C) Analysis of the cell average instantaneous velocity (speed) and its jitter (as measured by the standard deviation) show a significant reduction between 0 and 24 hrs indicating a stabilization in cell position. No difference is observed between 0 and 12 hrs and between 12 and 24 hrs. Top: Quantification of the cell centroid average instantaneous velocity. Bottom: Quantification of the standard deviation of the cell centroid speed. Error Bars = SEM.