Angewandte
Chemie
DOI: 10.1002/anie.201310979
Tumor Diagnosis
In Vivo Imaging of Mouse Tumors by a Lipidated Cathepsin S
Substrate**
Hai-Yu Hu, Divya Vats, Matej Vizovisek, Lovro Kramer, Catherine Germanier, K. Ulrich Wendt,
Markus Rudin, Boris Turk,* Oliver Plettenburg,* and Carsten Schultz*
Abstract: The synthesis and evaluation of two cathepsin S-
specific probes is described. For long-term retention of the
probe at the target site and a high signal-to-noise ratio, we
introduced a lipidation approach via the simple attachment of
palmitoic acid to the reporter. After cathepsin S-specific
cleavage in cultured cells and in a grafted tumor mouse
model, fluorescence increased owing to dequenching and we
observed an intracellular accumulation of the fluorescence in
the target tissue. The lipidated probe provided a prolonged and
strongly fluorescent signal in tumors when compared to the
very similar non-lipidated probe, demonstrating that non-
invasive tumor identification is feasable. The homing principle
by probe lipidation might also work for selective administra-
tion of cytotoxic compounds to specifically reduce tumor mass.
cells. They are required for promoting tumor growth, tissue
[
4]
invasion, and the altered metabolism of tumor cells.
A
number of probes targeting proteases have been developed in
the past and several are on the way to the clinic, among them
[
5]
several substrates. A common problem with these probes is
a lack of local accumulation of the signal generated, often
referred to as “homing”. Therefore, we hypothesized that
probes with specific cancerous tissue accumulation would
enhance the signal-to-noise ratio after being cleaved by the
enzyme.
Among the proteases that have a major role in cancer are
cysteine cathepsins, in particular cathepsins B, L, and S, as
judged on the basis of pharmacological and genetic
[
6]
approaches. In cancer, the primary source of these cathe-
psins are tumor and tumor-associated cells, and in particular
[
4,7]
I
t is the ultimate goal in cancer diagnostics to locate tumors
macrophages.
mechanistic role of cathepsin S in cancer using in vitro and
A number of groups have studied the
[1]
in humans early and with minimal invasion. In the past
decade, optical molecular imaging technologies have become
a powerful addition to both tumor detection and therapeutic
treatment evaluation owing to its high selectivity, good spatial
[
7b]
in vivo models.
Genetic ablation of cathepsin S in the
pancreatic islet model revealed that the enzyme plays a role in
tumor invasion, resistance to apoptosis, and also in tumor
[
2]
[7b]
resolution, and non-invasive mode of action. However, the
limitation of this method over techniques such as MRI and
CT is that fluorescent signals are attenuated by body mass,
skin, and hair, mostly through light absorption and scattering.
Additionally, high autofluorescence in animal tissue reduces
angiogenesis.
Moreover, Kwok et al. have shown that
cathepsin S is expressed on the surface of carcinoma cells
[
8]
and stays associated with the cell membrane.
Based on the reverse design principle, we equipped
a highly specific non-peptidic substrate for cathepsin S with
an NIR fluorophore and a suitable quencher and used
a lipidation approach to target the fluorescent product of
the enzymatic reaction to the region of enzymatic activity,
that is, the tumor (Scheme 1). Reverse design for protease
substrates is based on the idea that a non-peptidic high
[
3]
the signal-to-noise ratio. One way of mitigating these
problems is the use of tumor-specific enzyme substrates in
combination with dyes emitting in the near infrared (NIR).
Proteases are typical enzymes secreted into the tumor
microenvironment by both tumor and tumor-associated
[
+]
[
*] Dr. H.-Y. Hu, Priv.-Doz. Dr. C. Schultz
Prof. B. Turk
Cell Biology & Biophysics Unit
Faculty of Chemistry and Chemical Technology
University of Ljubljana (Slovenia)
European Molecular Biology Laboratory
Meyerhofstrasse 1, 69117 Heidelberg (Germany)
E-mail: schultz@embl.de
[+]
Dr. H.-Y. Hu, Dr. K. U. Wendt, Dr. O. Plettenburg
Sanofi Deutschland GmbH, Diabetes Division, R&D
Industriepark Park Hçchst, 65926 Frankfurt (Germany)
E-mail: Oliver.Plettenburg@sanofi.com
[
+]
Dr. D. Vats, C. Germanier, Prof. M. Rudin
Institute for Biomedical Engineering
ETH Zꢀrich and University of Zꢀrich (Switzerland)
+
[
] These authors contributed equally.
[
+]
M. Vizovisek, L. Kramer, Prof. B. Turk
Jozef Stefan Institute
[
**] All authors acknowledge funding by LIVIMODE, a collaborative
project funded by the EU 7FP. The work was partially supported by
grant P1-0140 from the Slovene Research Agency to B.T. We would
like to thank R. Vidmar (JSI) for mass spectrometry analysis of probe
cleavage.
Department of Biochemistry and Molecular Biology
Jamova 39, 1000 Ljubljana (Slovenia)
E-mail: Boris.Turk@ijs.si
[
+]
M. Vizovisek, L. Kramer
International Postgraduate School Jozef Stefan
Ljubljana (Slovenia)
[
+]
M. Vizovisek, Prof. B. Turk
Center of Excellence CIPKEBIP
Ljubljana (Slovenia)
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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