Radiolabeled GPVI-Fc for PET Imaging of Multiple Extracellular Matrix Fibers: A New Look into Pulmonary Fibrosis Progression

Abstract

Invariably fatal and with a particularly fast progression, pulmonary fibrosis (PF) is currently devoid of curative treatment options. Routine clinical diagnosis relies on breathing tests and visualizing the changes in lung structure by CT, but anatomic information is often not sufficient to identify early signs of progressive PF. For more efficient diagnosis, additional imaging techniques were investigated in combination with CT, such as ¹⁸F-FDG PET, although with limited success because of lack of disease specificity. Therefore, novel molecular targets enabling specific diagnosis are investigated, in particular for molecular imaging techniques. Methods: In this study, we used a ⁶⁴Cu-radiolabeled platelet glycoprotein VI fusion protein (⁶⁴Cu-GPVI-Fc) targeting extracellular matrix (ECM) fibers as a PET tracer to observe longitudinal ECM remodeling in a bleomycin-induced PF mouse model. Results: ⁶⁴Cu-GPVI-Fc showed significant uptake in fibrotic lungs, matching histology results. Contrary to ¹⁸F-FDG PET measurements, ⁶⁴Cu-GPVI-Fc uptake was linked entirely to the fibrotic activity of tissue and not was susceptible to inflammation. Conclusion: Our study highlights ⁶⁴Cu-GPVI-Fc as a specific tracer for ECM remodeling in PF, with clear therapy-monitoring and clinical translation potential.

Keywords: PET; bleomycin; pulmonary fibrosis.

Graphical abstract

FIGURE 1.

Experimental workflow for longitudinal PF imaging. Day 0: intratracheal deposition of bleomycin (BLM) or saline. Days 3, 7, 14, 21, and 28 after bleomycin deposition: PET and MRI scans after radiotracer injection. After in vivo imaging: euthanasia, biodistribution, microscopy imaging.

FIGURE 2.

Representative images of ⁶⁴Cu-GPVI-Fc accumulation 48 h after radiotracer injection in animals that received intratracheal saline (control) or bleomycin 14 or 21 d after deposition. (A) Maximum-intensity-projection PET images fused with single-slice MR images (n = 4–9). (B) Corresponding quantification of in vivo tracer uptake in lungs. (C) Corresponding quantification of ex vivo tracer uptake in lungs. *P ≤ 0.05. **P ≤ 0.01. ****P ≤ 0.0001. Ctrl = control.

FIGURE 3.

(A) Hematoxylin and eosin and Masson trichrome staining in control animals and animals on days 3, 7, 14, 21, and 28 after bleomycin deposition. (B) Histologic scoring of inflammation and modified Ashcroft score (n = 4–5) in mouse lungs compared with controls. (C) Fluorescence microscopy for colocalization of GPVI-Fc with extracellular matrix fibers of collagen I–III and with fibronectin and fibrinogen. Nuclei stain added for cell localization. *P ≤ 0.05. ****P ≤ 0.0001. Col = collagen; Ctrl = control; ECM = extracellular matrix; H&E = hematoxylin and eosin; MT = Masson trichrome; Nuc = nuclei.

FIGURE 4.

Uptake specificity of ⁶⁴Cu-GPVI-Fc. (A) Representative maximum-intensity-projection PET images fused with single-slice MR images 14 d after bleomycin deposition in control animals (n = 9), bleomycin-induced PF animals injected with ⁶⁴Cu-GPVI-Fc (n = 9), and bleomycin-induced PF animals injected with denatured ⁶⁴Cu-GPVI-Fc (n = 4). (B) Corresponding quantification of in vivo tracer uptake in lungs. (C) Corresponding quantification of ex vivo tracer uptake in lungs. ****P ≤ 0.0001. -BLM = no bleomycin (control); +BLM = bleomycin-induced; denat. = denaturated.

FIGURE 5.

¹⁸F-FDG PET imaging of mice with bleomycin-induced PF. (A) Representative single-slice PET images fused with single-slice MR images 14 and 21 d after bleomycin deposition and in control animals (n = 4–5). (B) Mean in vivo tracer uptake in the right lung lobe. (C) Ex vivo tracer uptake in same lobe. *P ≤ 0.05. **P ≤ 0.01. ***P ≤ 0.001.