Imaging nuclear - cytoplasm dynamics of cancer cells in the intravascular niche of live mice.

We have previously shown that cancer cells can form an intravascular niche where they can proliferate and undergo apoptosis as well as traffic and extravasate. In the present study, green fluorescent protein (GFP) was expressed in the cytoplasm of HT-1080 human fibrosarcoma cells, and red fluorescent protein (mCherry), linked to histone H2B, was expressed in the nucleus to further investigate intravascular cancer cell nuclear-cytoplasmic dynamics. Nuclear mCherry expression enabled visualization of nuclear dynamics, whereas simultaneous cytoplasmic GFP expression enabled visualization of nuclear-cytoplasmic ratios as well as simultaneous cell and nuclear deformation. Cancer cells were injected in the epigastric cranialis vein in an abdominal flap of nude mice to enable subcellular in vivo imaging. The cell cycle position of individual living cells was readily-visualized by the nuclear-cytoplasmic ratio and nuclear morphology. Real-time induction of apoptosis was observed by nuclear size changes and progressive nuclear fragmentation. Intra- and extra-vascular mitotic cells were visualized by imaging. One hour after cell injection, round and elongated cancer cells were observed in the vessels. Three hours after injection, invadopodia-like structures of the cancer cells were observed. Five hours after injection, dual-color cancer cells began to divide within the vessel. By 10 h, some intravascular cancer cells underwent apoptosis. Deformed new blood vessels in the tumor were observed 10 days later. Extravascular cancer cells were imaged dividing in the tumor at day 14 after injection. The subcellular in vivo imaging approach described in the present report provides new visual targets for trafficking and proliferating intravascular cancer cells as well as extravasating and invading cancer cells.