Clarification of molecular mechanism of lymphatic metastasis and development of new therapy for cancer metastasis

To clarify molecular mechanism of lymphatic metastasis and apply the mechanism to development of new therapy for cancer metastasis, we aim at the improvement of a quantitative in vivo molecular imaging method from various aspects like a fluorescent material, the living body operation technique, the imaging device, and the image analysis, etc. In this year, we mainly got two results. One is for the "development of fluorescent nano-particle using silica-coating technology ". Another is for the "visualization of angiogenesis mechanism".

Quantum dot (QD), which is bright and photostable nano-particles, is expected to be a good tool for in vivo imaging. Previously, we succeeded in imaging a DDS process and metastatic cancer cells in mice and lymph vessel networks in pig stomach with QDs. However, the oxidation of QDs in tissues leads to elevated levels of cadmium toxicity and decreases in their photostability. We prepared QD/SiO2 core-shell nanoparticles that consist of a single QD and a uniform silica shell. We varied the shell thickness and evaluated the photostability of the nanoparticles with high-accuracy single-particle imaging measurements, demonstrating that the photostability was severalfold greater than QDs alone. Moreover, in vivo fluorescence imaging showed that subcutaneously injected QD/SiO2 specifically labeled the lymph node. The silica layer also enhanced the photostability of QD/SiO2 in SLN tissues.

Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis. However, as vascular imaging at the molecular level is impossible, the detailed in vivo dynamics of VEGF and its receptor (VEGF-R) remain unknown. To understand the molecular distribution of VEGF and the VEGF-R, we prepared ischemic mice with a new surgical method and induced angiogenesis in the gastrocnemius muscle. Then, we made a VEGF-conjugated QD and performed immunostaining of VEGF-R-expressing cells with the fluorescent probe, demonstrating the high-affinity of the probe for VEGF-R. To observe the physiological molecular distribution of VEGF-R, we performed in vivo single particle imaging of gastrocnemius in the ischemic leg with the VEGF-conjugated QD. The results suggested that only a 3-fold difference of VEGF-receptor distribution is involved in the formation of branched vasculature in angiogenesis, although previous ex vivo data showed 10 to 20-fold difference in its distribution, indicating that a method inducing a several-fold local increase of VEGF-R concentration may be effective in generating site-specific angiogenesis in ischemic disease. This new in vivo imaging of ischemic mice could make useful contributions to understanding the mechanisms of angiogenesis and to developing a VEGF-R-related drug.

Publications

Hamada Y, Gonda K, Takeda M, Sato A, Watanabe M, Yambe T, Satomi S, Ohuchi N. In vivo imaging of the molecular distribution of the VEGF receptor during angiogenesis in a mouse model of ischemia. Blood 118: e93-e100 (2011).
Kobayashi Y, Nozawa T, Nakagawa T, Gonda K, Takeda M, Ohuchi N. Fabrication and fluorescence properties of multilayered core-shell particles composed of quantum dot, gadolinium compound, and silica. J Mater. Sci. 47: 1852-1859 (2012).
Ueno H, Ishikawa T, Bui KH, Gonda K, Ishikawa T, Yamaguchi T. Mouse respiratory cilia with the asymmetric axonemal structure on sparsely distributed ciliary cells can generate overall directional flow. Nanomedicine in press.