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Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes


A long-term goal of cancer diagnosis is to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted 'activatable' fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH–activatable probes based on the boron-dipyrromethene fluorophore were synthesized and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex vivo and in vivo imaging of human epidermal growth factor receptor type 2–positive lung cancer cells in mice was performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific, cell surface–targeting molecules that result in cellular internalization.

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Figure 1: Mechanism of a cancer imaging strategy using target-specific activatable probes.
Figure 2: Development of a series of fluorescence probes for various acidic environments.
Figure 3: In vitro imaging of NIH3T3 HER2+ cells with Ph-BDP–, DiMeN-BDP–, EtMeN-BDP– or DiEtN-BDP–labeled trastuzumab.
Figure 4: The activatable probe can produce a fluorescence signal only from HER2-positive tumors and not from HER2-negative tumors, with minimal background from normal tissue.
Figure 5: The activatable probe can produce a fluorescence signal only from living HER2-positive cells and tumors and not from dead cells or fixed tumors.


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This study was supported in part by the Precursory Research for Embryonic Sciences and Technology from the Japan Science and Technology Agency, by research grants 19021010 and 19205021 to Y.U. and by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research to Y.H., Y.K., T.B., P.L.C. and H.K.

Author information

Authors and Affiliations



Y.U. and H.K. planned the projects, developed the probes, performed the in vivo experiments and wrote and edited the manuscript. D.A. and M.K. developed the probes, performed the in vivo experiments and wrote and edited the manuscript. Y.H., Y.K., T.B., T.W. and A.H. performed the in vivo experiments and wrote and edited the manuscript. T.N. and P.L.C. planned the projects and wrote and edited the manuscript.

Corresponding authors

Correspondence to Yasuteru Urano or Hisataka Kobayashi.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–4, Supplementary Tables 1–3 and Supplementary Methods (PDF 563 kb)

Supplementary Video 1

Reversible activation by altering the pH. (AVI 4293 kb)

Supplementary Video 2

Fluorescence-guided Laparoscope (MOV 12792 kb)

Supplementary Video 3

Fluorescence-guided Laparoscope (MOV 5792 kb)

Supplementary Video 4

Fluorescence-guided Laparoscope (MOV 8635 kb)

Supplementary Video 5

pH-activatable rhodamine (MOV 2877 kb)

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Urano, Y., Asanuma, D., Hama, Y. et al. Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes. Nat Med 15, 104–109 (2009).

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