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.
