Contrast Media Mol Imaging. 2013 Jul-Aug;8(4):350-60. doi: 10.1002/cmmi.1529.

Optical imaging detection of microscopic mammary cancer in ErbB-2 transgenic mice through the DA364 probe binding αv β3 integrins.

Conti L, Lanzardo S, Iezzi M, Montone M, Bolli E, Brioschi C, Maiocchi A, Forni G, Cavallo F.

Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Turin, Via Nizza 52, 10126 Torino.

 

Abstract

Despite spontaneous tumor growth in genetically engineered mice being one of the most recognized tools for the in vivo evaluation of novel diagnostic and therapeutic anticancer compounds, monitoring early stage lesions in live animals is a goal that has yet to be achieved. A large number of targets for the molecular imaging of various diseases have been identified and many imaging technologies, including optical techniques are emerging. One of the most commonly exploited targets in tumor imaging is αVβ3 integrin. The aim of this study was to set up an optical imaging (OI) method for the early detection of autochthonous mammary cancer in female BALB/c mice transgenic for the rat-ErbB-2 oncogene (BALB-neuT). We show that DA364, a near-infrared fluorescence arginine–glycine–aspartic acid cyclic probe, was taken up by neoplastic mammary glands and that its uptake increased with cancer progression. By contrast, the nonaccumulation of DA364 in the healthy mammary glands of virgin and lactating wild-type mice suggests that the probe specifically targets breast cancers. Comparisons of OI with whole-mount and histological findings showed that DA364 allows the noninvasive visualization of atypical hyperplasia and microscopic foci of in situ carcinoma 2 months before mammary lesions become detectable by palpation. Moreover, DA364 was successfully used to monitor the outcome of anticancer vaccination. Therefore, DA364 can be considered a promising early detection tool in near-infrared noninvasive OI for the early diagnosis of breast cancer.

Copyright © 2013 John Wiley & Sons, Ltd.

PMID: 23613438

 

Supplement

One of the most commonly exploited targets for tumor imaging is αVβ3 integrin, which is the receptor for a variety of extracellular matrix proteins that contain an arginine–glycine–aspartic acid (RGD) sequence and is a marker of angiogenesis, tumor development and metastasis in several types of cancer (1), including breast cancer (2). To develop a tool for αVβ3 integrin OI visualization, we synthesized DA364, a cyclic Cy5.5 conjugated-RGD peptide with high affinity for αVβ3 integrin (3). The efficiency of this probe to in vivo image tumor has been demonstrated in several transplantable mouse model of glioma and glioblastoma tumor (3); however, in order to approach its clinical translation, we chose an ErbB-2-driven mammary carcinogenesis mouse model that recapitulates the features of human ErbB-2 positive tumors: the BALB-neuT mice (4). As epithelial tumors are clearly visible in OI and considering that diffuse optical tomography (DOT) has been tested in a clinical setting, these mice are a good model for setting up noninvasive early breast cancer diagnostic tools.

In cancer-prone BALB-neuT mice, autochthonous mammary cancers develop in all 10 mammary glands in an asynchronous manner starting from 4 weeks of age, but this random onset makes the prediction of which glands will develop the first palpable tumors practically impossible.

The inspection of tumor onset performed by calliper permits the identification of the first tumor, classified as in situ carcinoma by hystopathological analysis, around week 20 (Figure 1 A and 1 C). When DA364 is intravenously injected in BALBneuT mice it allows the visualization of preneoplastic lesions 2 months earlier than palpation, at 12 weeks of age, when widespread atypical hyperplasia and microscopic foci of in situ carcinomas are present in only a few mammary glands (Figure 1 B and C). Federica Cavallo-fig1Figure 1. Visualization of mammary tumors in BALBneuT mice by palpation (A) and OI (B).

In (C) the kinetics of the onset of mammary lesions, evaluated by palpation (black bars) or OI (red bars), are expressed as the mean number of lesions ± SEM per mouse, in 60 individually evaluated mammary glands. *** P < 0.001; ** P < 0.01, Student’s t-test.

The anticipatory diagnostic ability of DA364 OI can be exploited to monitor tumor progression and response to treatment in other experimental mouse models in which αVβ3 integrin is over-expressed. This possibility represents a marked improvement in the detection of autochthonous tumors in transgenic animals, and is a valuable alternative to the use of reporter genes which, in fact, may alter the results of immune-based anticancer therapies as they encode for foreign antigens that can induce an immune response in the transgenic animal.

The ability of DA364-based NIR OI to detect very small mammary pre-neoplastic lesions identified by us (Figure 2) makes the possibility of its translation to human breast cancer diagnosis a very challenging issue, opening the doors to other kinds of diagnostic applications in human pathology, such as the detection of superficial tumors and the visualization of neoplastic lesions during fluorescence guided surgery.

 

References:

1.         Costouros, N. G., F. E. Diehn, and S. K. Libutti. 2002. Molecular imaging of tumor angiogenesis. J Cell Biochem Suppl 39:72-78.

2.         Chen, X., R. Park, M. Tohme, A. H. Shahinian, J. R. Bading, and P. S. Conti. 2004. MicroPET and autoradiographic imaging of breast cancer alpha v-integrin expression using 18F- and 64Cu-labeled RGD peptide. Bioconjug Chem 15:41-49.

3.         Lanzardo, S., L. Conti, C. Brioschi, M. P. Bartolomeo, D. Arosio, L. Belvisi, L. Manzoni, A. Maiocchi, F. Maisano, and G. Forni. 2011. A new optical imaging probe targeting alphaVbeta3 integrin in glioblastoma xenografts. Contrast Media Mol Imaging 6:449-458.

4.         Boggio, K., G. Nicoletti, E. Di Carlo, F. Cavallo, L. Landuzzi, C. Melani, M. Giovarelli, I. Rossi, P. Nanni, C. De Giovanni, P. Bouchard, S. Wolf, A. Modesti, P. Musiani, P. L. Lollini, M. P. Colombo, and G. Forni. 1998. Interleukin 12-mediated prevention of spontaneous mammary adenocarcinomas in two lines of Her-2/neu transgenic mice. J Exp Med 188:589-596.

 

Contact:

Federica Cavallo

Professor, Immunology

University of Turin

Molecular Biotechnology Center

Via Nizza, 52

10126, Torino, Italy

Phone: +39 011 670 6454/6457/6458

Fax: +39 011 236 5417

e-Mail: federica.cavallo@unito.it

Federica Cavallo-fig2

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