ORIGINAL PAPER Journal of Pathology J Pathol 2010; 222: 52–63 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/path.2745 High-resolution in vivo imaging of breast cancer by targeting the pro-invasive integrin αvβ6 Antonio Saha, 1 David Ellison, 2 Gareth J Thomas, 1 Sabarinath Vallath, 1 Stephen J Mather, 2 Ian R Hart 1 and John F Marshall 1 * 1 Centre for Tumour Biology, Queen Mary University of London, Barts and London School of Medicine & Dentistry, Institute of Cancer and CR-UK Clinical Centre, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK 2 Centre for Molecular Oncology and Imaging, Queen Mary University of London, Barts and London School of Medicine & Dentistry, Institute of Cancer and CR-UK Clinical Centre, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK *Correspondence to: John F Marshall, Centre for Tumour Biology, Institute of Cancer and CR-UK Clinical Research Centre, John Vane Science Centre, Barts and London School of Medicine & Dentistry, Charterhouse Square, London EC1M 6BQ, UK e-mail: j.f.marshall@qmul.ac.uk Abstract The integrin αvβ6 is expressed only on epithelia and then usually only during processes of tissue remodelling including cancer, where its high expression correlates with reduced survival. Thus, αvβ6 represents an important target for imaging and therapy of cancer and new molecular-specific targeting agents are required. We have developed A20FMDV2, a peptide derived from the VP1 coat protein of foot-and-mouth-disease virus that binds specifically and stably to αvβ6. Using a newly generated pair of isogenic human cell lines that differ only in αvβ6 expression, it was shown, using biodistribution and SPECT imaging, that indium-111-labelled A20FMDV2 locates specifically to αvβ6-expressing tissues in vivo, achieving at least seven-times higher retention in αvβ6-positive than in αvβ6-negative tumours. In further studies with MCF10.DCIS.COM and MCF10A.CA1a breast carcinoma cell lines, which express αvβ6 endogenously, the radiopeptide achieved similar levels of tumour retention and permitted excellent discriminatory imaging of tumours. Thus, A20FMDV2 can be used for molecular-specific targeting of αvβ6 for imaging in vivo the often more aggressive, αvβ6-positive cancers. In the future, A20FMDV2 could serve also to deliver therapy to these same cancers. Copyright  2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: integrin; αvβ6; imaging; SPECT; PET Received 24 March 2010; Revised 20 May 2010; Accepted 29 May 2010 No conflicts of interest were declared. Introduction Integrins are heterodimeric glycoproteins composed of non-covalently linked α and β subunits [1]. Inte- grins dynamically translate extracellular matrix cues into intracellular responses, including mechanical, bio- chemical, and genetic signals, thereby modulating cell proliferation, survival, migration, and invasion [2]; thus, they are key determinants of the survival and spread of cancer. One of these integrins, αvβ6, is expressed only on epithelia and then usually only during processes of tissue remodelling; these include wound healing, chronic inflammation, and cancer [3,4]. We, and others, have shown clearly in experimen- tal studies that αvβ6 imparts a pro-invasive and thus more aggressive phenotype when overexpressed on cancer cells [5–10]. This behaviour appears to reflect the clinical situation in humans, since survival from cancer of the colon, cervix or from non-small cell lung cancer is reduced significantly if the cancers express high levels of αvβ6 [11–13]. Thus, since αvβ6 is weak or absent in normal tissue but its presence can drive tumour invasion and shorten survival, this integrin represents a major new target for molecular- specific targeting for many cancers. To this end, we are developing peptides for αvβ6-specific imaging and therapy. Integrins bind to their extracellular ligands partly through the recognition of specific amino acid se- quences; eight different integrins, including αvβ6, recognize the RGD (Arg-Gly-Asp) motif [14]. We identified a 20-mer peptide, A20FMDV2 (NAVPNL- RGDLQVLAQKVART), derived from the VP1 coat- protein of foot-and-mouth-disease virus, that exhibited exquisite specificity for αvβ6 over other RGD-directed integrins [15]. This specificity was dependent on the formation of a specific secondary structure when it bound to αvβ6, comprising a hairpin shape with RGD at the apex followed c-terminally by an α-helix [15]. Importantly, upon binding, A20FMDV2 formed a highly stable association with αvβ6 [16]. As a pre- lude to determining whether our peptide could be used as a therapeutic targeting agent, we first wished to establish that it could be used as a molecular- specific imaging agent. In this study, we have used Copyright  2010 Pathological Society of Great Britain and Ireland. J Pathol 2010; 222: 52–63 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com