Aim:
Integrin alpha-v-beta-6 (αvβ6) is a heterodimeric adhesion protein that is exclusively expressed on cholangiocytes during liver fibrogenesis. With the aim of developing a quantitative imaging agent for the detection of liver fibrosis progression, we developed and validated a dual-function near infrared fluorescence (NIRF)/positron emission tomography (PET) probe targeting αvβ6.
Material and methods:
A dual-labeled cyclic peptide recognizing integrin αvβ6 was designed based on a 9-mer cyclic RGD peptide, attaching a functional group with sulfo-Cy5.5 and a Ga-68-chelator for NIRF and PET imaging, resp. Binding specificity was tested in vitro on target vs. non-target expressing cell lines. We investigated the in vivo uptake, biodistribution and blood clearance in the mouse models of biliary (Mdr2 KO) and toxin (CCl4)-induced fibrosis. Whole body and organ imaging were performed using NIRF imaging at 15 min, 6 and 12h post injection (5.84 nM/mouse). For PET imaging the Ga-68-labeled construct (5 MBq/mouse) was injected and organ specific signals were quantified after 2h using a gamma-counter. Fibrosis and target expression were assessed by hydroxyproline quantification and real-time qRT-PCR.
Results:
The dual-labeled construct bound specifically and dose-dependently to αvβ6 expressing cells (highest at 1µM in 5.2-fold vs. control; p< 0.01). Both mouse fibrosis models displayed significantly elevated levels of hepatic collagen (3-fold; p< 0.0001) and Itgb6 expression compared to the non-fibrotic controls (43-fold and 4.6-fold in Mdr2 KO and CCl4 treated fibrotic mice, resp.; p< 0.05). NIRF imaging with the bimodal construct revealed a liver-specific 3.4-fold (Mdr2 KO) and a 3.2-fold (CCl4) enhanced uptake in the fibrotic livers (p< 0.01) at 6h post injection, compared to the non-fibrotic controls, with similar results in PET imaging using the Ga-68-radiolabeled bimodal cyclopeptide.
Discussion:
We designed a bimodal integrin αvβ6-specific fibrogenesis imaging agent based on a cyclic RGD peptide that can be used for NIR imaging in mice and for PET-imaging in patients. This opens the possibility of molecular imaging to quantify liver fibrogenesis in vivo and therefore to assess early treatment responses in patients treated with potential antifibrotic agents.
