Subcellular imaging tracks cancer cells in mice

Visualization of microscopic cancer is essential for understanding and controlling cancer cell dormancy, growth, spread, and colonization of distant sites. One of our most useful imaging methods involves tagging biomarkers of cancer with fluorescent dyes, then exciting those molecules and imaging the fluorescence. Several approaches involving tumor cell labeling have been developed for visualizing tumor cells in vivo. The Escherichia coli β-galactosidase (lacZ) gene has been used to detect micrometastases. However, lacZ detection requires extensive histological preparation and the sacrifice of tissue or of the animal. Luciferase has also been used to label tumor cells, but its weak and unstable signal and low resolution preclude imagingindividual tumor cells in vivo. Therefore, other techniques are required for real-time imaging and study of tumor cells in viable fresh tissue or living animals.

To externally image and follow the natural course or impediment of tumor progression and metastasis, a strong signal and high resolution are necessary. We chose to use green fluorescent protein (GFP) and red fluorescent protein (RFP) to satisfy these conditions because of their intrinsic brightness. The first use of GFP to image cancer cells in vivo was pioneered by Takashi Chishima and others in our laboratory.¹ We have developed the use of GFP and other fluorescent proteins to successfully follow individual tumor cells in vivo.²

Using cancer cells that express GFP in the nucleus and RFP in the cytoplasm with a highly sensitive whole-mouse macroimaging/microimaging system, the Olympus OV100, we developed real-time dynamic subcellular imaging of cancer cell trafficking in live mice. We imaged the cytoplasmic and nuclear dynamics of tumor cells moving through and out of blood vessels in live mice as well as cancer cell viability.^3–6.

We saw individual cancer cells traveling in vessels of various sizes in the live mice and noted some behaviors. Cancer cells either crawled along the vessel wall or moved with the blood flow. Cancer cells also deformed both their cytoplasm and nuclei to fit and move in small vessels. Finally, cancer cells often formed aggregates.

We saw aggregates collide with other aggregates, both those moving with the blood tissue and some already attached to the vessel wall. Some aggregates became larger by repeated collisions. The cellular adhesion within the aggregates is not strong, and some cells escaped into the bloodstream. The double labeling of the nucleus and cytoplasm allowed us to distinguish the individual cells and nuclei in the aggregates.

We also noted that the cancer cells frequently contacted other cancer cells or vessel walls. Cancer cells left the blood vessels by first extending their cytoplasmic processes through the wall. Then the nuclei followed along the extension, undergoing varying degrees of deformation to fit within the extended cytoplasmic protrusion. The whole cell eventually emerged. Cancer cells that initially left remained in the proximity of the blood vessels and then surrounded and grew on the vessel surface.^3–6

This method offers a way to watch cancer cells move and grow. Our understanding of the complex steps of metastasis will significantly increase with our new ability to continuously image cancer cells at the subcellular level in a live animal. In addition, new drugs can be developed to target these newly visible steps of metastasis.

This study was supported in part by National Cancer Institute grants CA099258, CA103563, and CA101600.