Real-time visualization of the function of bone-resorbing cells within
animals
Developed a functional small molecular probe for in vivo imaging

Researchers of Osaka University have discovered a way to visualize sites where boneresorbing cells (osteoclasts) were in the process of resorbing bone in living mice. This
real-time visualization of changes in osteoclast localization and activity allowed thesuccessful measurement of bone resorption intensity. Since this enables simple andquick access to information on the activity of osteoclasts, this discovery will contribute tothe early diagnosis of affected areas and the development of new therapeutic drugs.

Osteoclasts are bone-resorbing cells that may cause osteoporosis and rheumatoid
arthritis if there is an excess in bone resorption. So far, information on osteoclast
localization could be gathered by using fluorescent proteins, but this method did not allow
an examination of osteoclast activity. A group of researchers at the Immunology Frontier
Research Center (IFReC), Osaka University, led by Kazuya Kikuchi, professor at theGraduate School of Engineering, and Masaru Ishii, professor at the Graduate School ofMedicine, Osaka University, produced fluorescent probes that visualize those sites
where osteoclasts are in the process of resorbing bone and developed their own imaging
device. They thereby conducted a successful in vivo evaluation of osteoclast function.

The research team developed a mechanism by which small molecular probes (SMPs)
are selectively delivered to the location of target cells. Up until then, application of SMPs
to in vivo imaging was particularly challenging, as delivery to target tissues proved
difficult. The researchers managed to optimize molecular delivery so that the molecularprobes only had to be injected into the mice for imaging. The SMPs were equipped witha switch function that is only triggered at those areas where bone is being resorbed. This
enabled the selective visualization of osteoclast activity. In combination with fluorescentproteins that label target cells, the researchers succeeded in real time visualization ofchanges in cell localization and activity as well as the measurement of bone resorption
intensity.

This research will have great impact in the field of in vivo imaging as it established an invivo imaging method that allows simple and quick measurement in detecting osteoclasts
that are in the process of resorbing bone. This will also benefit early diagnosis and thescreening of new therapeutic drugs. The study carries broader social and academic
relevance as it was conducted as interdisciplinary research, spanning such areas asmolecular design based on physical chemistry principles, the creation of functional
fluorescent probes by synthetic organic chemistry, and the clarification of intravitalmechanisms using immunological knowledge and technology. It therefore serves as an
example of research that achieved positive results starting from basic research that led
to application in medical research.

This research was featured in the electronic version of Nature Chemical Biology on June7, 2016.

Figure 1: In vivo excitation spectra of pHocas-3 and tdTomato

Figure 2: Photostable pH-activatable Probe**************************************
Article Information:
Article title: Real-time intravital imaging of pH variation associated with osteoclast activity.
Journal: Nature Chemical Biology
Authors: Hiroki Maeda, Toshiyuki Kowada, Junichi Kikuta, Masayuki Furuya, Mai
Shirazaki, Shin Mizukami, Masaru Ishii, and Kazuya Kikuchi.
DOI: 10.1038/nchembio.2096
Funded by: Ministry of Education, Culture, Sports, Science and Technology, Japan(MEXT), Japan Agency for Medical Research and Development (AMED)