In Vivo Nano-imaging of Membrane Dynamics in Metastatic Tumor Cells Using Quantum Dots*

Changes in membrane morphology and membrane protein dynamics based on its fluidity are critical for cancer metastasis. However, this subject has remained unclear, because the spatial precision of previous in vivo imaging has been limited to the micrometer level and single molecule imaging is impossible. Here, we have imaged the membrane dynamics of tumor cells in mice with a spatial precision of 7–9 nm under a confocal microscope. A metastasis-promoting factor on the cell membrane, protease-activated receptor 1 (PAR1), was labeled with quantum dots conjugated with an anti-PAR1 antibody. Movements of cancer cells and PAR1 during metastasis were clearly observed in vivo. Images used to assess PAR1 dynamics were taken of representative cells for four stages of metastasis; i.e. cancer cells far from blood vessels in tumor, near the vessel, in the bloodstream, and adherent to the inner vascular surface in the normal tissues near tumor were photographed. The diffusion constant of PAR1 in static cells far from tumor blood vessels was smaller than in moving cells near the vessels and in the bloodstream. The diffusion constant of cells adhering to the inner vascular surface in the normal tissues was also very small. Cells formed membrane protrusion during migration. The PAR1 diffusion constant on these pseudopodia was greater than in other membrane regions in the same cell. Thus, the dynamics of PAR1 movement showed that membrane fluidity increases during intravasation, reaches a peak in the vessel, decreases during extravasation, and is also higher at locally formed pseudopodia.