Type I collagen-targeted PET probe for pulmonary fibrosis detection and staging in preclinical models

Abstract

Pulmonary fibrosis is scarring of the lungs that can arise from radiation injury, drug toxicity, environmental or genetic causes, and for unknown reasons [idiopathic pulmonary fibrosis (IPF)]. Overexpression of collagen is a hallmark of organ fibrosis. We describe a peptide-based positron emission tomography (PET) probe (68Ga-CBP8) that targets collagen type I. We evaluated 68Ga-CBP8 in vivo in the bleomycin-induced mouse model of pulmonary fibrosis. 68Ga-CBP8 showed high specificity for pulmonary fibrosis and high target/background ratios in diseased animals. The lung PET signal and lung 68Ga-CBP8 uptake (quantified ex vivo) correlated linearly (r2 = 0.80) with the amount of lung collagen in mice with fibrosis. We further demonstrated that the 68Ga-CBP8 probe could be used to monitor response to treatment in a second mouse model of pulmonary fibrosis associated with vascular leak. Ex vivo analysis of lung tissue from patients with IPF supported the animal findings. These studies indicate that 68Ga-CBP8 is a promising candidate for noninvasive imaging of human pulmonary fibrosis.

Conflict of interest statement

Competing interest: P.C. has equity in Collagen Medical, the company, which holds the patent rights to the peptides used in these probes. S.V. and P.W. are employees of Biogen. A patent has been filled by P.C. and P.D. regarding collagen-binding PET tracer preparation and imaging.

Copyright © 2017, American Association for the Advancement of Science.

Figures

Fig. 1. Probe 68Ga-CBP8 monitors disease progression in the bleomycin mouse model of pulmonary fibrosis

(A) Representative images of lung tissue stained with hematoxylin and eosin (H&E) and Sirius red (×10, scale bar, 300 μm) for sham and BM-treated mice at day 7 and day 14 after BM instillation; higher-magnification views are also displayed (×40, scale bar, 60 μm). (A to D) Disease progresses in a stepwise fashion as determined by histological Ashcroft scoring of lung fibrosis (B), by histological quantification of the area affected by disease (C) and by hydroxyproline (Hyp) analysis (D). (E) Ex-vivo lung uptake of 68Ga-CBP8 increases with disease progression in the BM-treated mice; 5-fold higher in BM-mice 14 days after instillation than in sham animals, 1.5 higher uptake in BM-mice 14 days after instillation compared with BM-mice 7 days after instillation. (F) Correlation of ex-vivo 68Ga-CBP8 lung uptake and Ashcroft score. (G) Correlation between ex-vivo lung uptake of 68Ga-CBP8 and lung hydroxyproline. Data were analyzed with one-way ANOVA, followed by post hoc Tukey tests with two-tailed distribution. For panels B-F, data are displayed as box plots with the dark band inside the box representing the mean, the bottom and top of the box the first and third quartiles, and the whiskers the minimum and maximum values. For all of the experiments shown in this figure: n=11 for sham, n=4 for BM-treated mice at day 7, and n=7 for BM-treated mice at day 14.

Fig. 2. 68Ga-CBP8 is specifically taken up by fibrotic but not healthy lungs (bleomycin model)

(A) Representative fused PET-CT images show specific accumulation of 68Ga-CBP8 (left images) in fibrotic but not control lungs; 68Ga-CBP12 (right images) showed no preferential uptake in the lungs of BM-treated mice compared to control mice. Greyscale image shows CT image, color scale image shows PET image from integrated data 50–80 min after probe injection. B. Hydroxyproline level was significantly higher in fibrotic BM-treated animals than in the sham-treated animals. C. PET activity values for 68Ga-CBP8 and 68Ga-CBP12 in sham and BM-treated mice (50–80 min after injection). Data are expressed as percent injected dose per cubic centimeter of tissue (%ID/cc). Data show significantly higher uptake in fibrotic lungs with 68Ga-CBP8. D. Ex vivo uptake of 68Ga-CBP8 and 68Ga-CBP12 in lungs from sham and BM-treated mice 150 min after injection expressed as %ID per lung. E. Distribution of 68Ga-CBP8 and 68Ga-CBP12, expressed as percent injected dose per gram of tissue (%ID/g), (mean ± standard error) in various organs was similar except in fibrotic lungs. (Inset) The same data as in E expressed as %ID/lung. Data were analyzed using one-way ANOVA, followed by post hoc Tukey tests with two-tailed distribution. For all of the experiments shown in this figure: n=4 for sham injected with 68Ga-CBP12, n=4 for BM-treated mice injected with 68Ga-CBP12, n=11 for sham injected with 68Ga-CBP8 and n=7 for BM-treated mice injected with 68Ga-CBP8. For panels B–D, data are displayed as box plots with the dark band inside the box representing the mean, the bottom and top of the box the first and third quartiles, and the whiskers the minimum and maximum values.

Fig. 3. 68Ga-CBP8 detects and stages disease in a mouse model of pulmonary fibrosis with enhanced vascular permeability

(A) Treatment scheme for the LDBVL group, which was treated with a low dose of BM (0.1 U/kg) and the vascular leak agent FTY720. (B to F) Mice were treated with a very low dose of BM (LDB), the vascular leak agent FTY720 (FTY), or both, administered together (LDBVL). The LDBVL, but not LDB or FTY, group exhibited a fibrotic response as determined by histological Ashcroft scoring of lung fibrosis (B), by Sirius red staining (% total area affected by disease) (C) and by hydroxyproline (Hyp) analysis (D). Data show significantly higher uptake of 68Ga-CBP8 in lungs of LDBVL animals compared to FTY or LDB animals as quantified by PET data (E) and by ex vivo uptake (F). (G and H) Correlations between hydroxyproline levels and %ID/cc (G) and %ID/lung (H). Data were analyzed using one-way ANOVA, followed by post hoc Tukey tests with two-tailed distribution. For all of the experiments shown in this figure: n=6 for the FTY group, n=4 for the LDB group, n=9 for the LDBVL. For panels B–F, data are displayed as box plots with the dark band inside the box representing the mean, the bottom and top of the box the first and third quartiles, and the whiskers the minimum and maximum values.

Fig. 4. 68Ga-CBP8 PET allows monitoring of the response to anti-fibrotic therapy in mice

(A) Representative images of lung tissue stained with hematoxylin and eosin (H&E) and Sirius red (×10, scale bar, 300 μm) for mice treated with 3G9 only (n=5), for mice treated with a low dose of BM and FTY720 (LDBVL, n=9), for LDBVL mice receiving 3G9, LDBVL+3G9 (n=11) and for LDBVL mice receiving 1E6, LDBVL+1E6 (n=11); higher-magnification views are also displayed (×40, scale bar, 60 μm). (B) Representative fused PET-CT images show specific accumulation of 68Ga-CBP8 in the lungs of mice from the LDBVL and LDBVL+1E6 groups and but low PET signal in the lungs of the control 3G9 or treated LDBVL+3G9 groups. Greyscale image shows CT image, color scale image shows PET image from integrated data 50–80 min after probe injection. (C) Hydroxyproline content was significantly higher in animals from the LDBVL and LDBVL+1E6 groups than in animals from the 3G9 and LDBVL+3G9 groups showing that the treatment of LDBVL animals with 3G9 is preventive against fibrosis. The treatment of LDBVL animals with 1E6 has no effect on fibrosis. (D) and (E) Similar effects were seen for quantitative PET data (D) and ex vivo uptake in the lungs. Data were analyzed using one-way ANOVA, followed by post hoc Tukey tests with two-tailed distribution. For panels C–E, data are displayed as box plots with the dark band inside the box representing the mean, the bottom and top of the box the first and third quartiles, and the whiskers the minimum and maximum values. For all of the experiments shown in this figure: n=5 for the 3G9 group, n=9 for the LDBVL group, n=11 for the LDBVL+3G9 and n=11 for the LDBVL+1E6.

Fig. 5. Probe 68Ga-CBP8 signal reflects changes in collagen concentration in human IPF lung tissues

A. Uptake of 68Ga-CBP8 in human lung samples taken from the upper, middle, and lower lobes of explanted lung of IPF patients. The uptake is defined as %ID/g +/− standard error. Data were analyzed using one-way ANOVA, followed by post hoc Tukey tests with two-tailed distribution. For patient 1, n=5 (upper lobe), n=5 (middle lobe) and n=6 (lower lobe). For patient 2, n=8 (upper lobe), n=6 (middle lobe) and n=6 (lower lobe). For patient 3, n=5 (upper lobe), n=5 (middle lobe) and n=9 (lower lobe). B. Representative images of Sirius red staining of collagen showing early stage of IPF (left, lung section taken from the upper lobe of patient 2) characterized by thickening of the alveolar septa and knot-like formation and late stage of fibrosis (right, lung section taken from the lower lobe of patient 2) characterized by a denser deposition of collagen. C. Correlation of 68Ga-CBP8 uptake in lung tissue samples from IPF patients with histological staining of collagen as measured by quantification of % collagen staining (Sirius red) in the area affected by disease (r2=0.94, p<0.0001).