Abstract Body:

Introduction: Idiopathic pulmonary fibrosis (IPF) is a deadly disorder that lacks non-invasive biomarkers for assessment of disease activity, presenting a major obstacle in clinical management of this disease¹. The presence of exuberant extracellular matrix (ECM) is a hallmark of IPF. Fibronectin is an abundant ECM glycoprotein that is highly and differentially upregulated in the IPF lung, including in the fibroblastic foci, the leading edge of fibrotic activity². Due to fibronectin’s role in early fibrosis, in this study we are utilizing fluorescence and positron emission tomography (PET)-based imaging methods to target fibronectin as a biomarker of lung fibrosis via the PEGylated functional upstream domain of Streptococcus pyogenes F1 adhesin peptide (PEG-FUD)^3-5.

Methods: Tissue sections from patients with IPF were stained with Cy5-labeled PEG-FUD or mutated control, PEG-mFUD, while some were blocked with excess unlabeled FUD prior to staining. Cy5-PEG-FUD was quantified in distinct regions of fibrotic tissues identified by Masson’s trichrome-stained sequential sections. For in vivo assessment in the murine model of pulmonary fibrosis, single intratracheal dose of bleomycin (1 U/kg) or normal saline control was delivered to 10-14-week old male and female mice. Three and 11 days post-bleomycin, mice were intravenously administered ⁶⁴Cu-radiolabeled PEG-FUD or PEG-mFUD and imaged by µPET/CT up to one day later. Tissue uptake was further quantified through ex vivo biodistribution studies. Lung radiodensity (Hounsfield units of CT scans) and radiotracer uptake (%ID/g in PET scans) were determined in Inveon Research Workplace by manually drawing regions of interest. Separately, 7 and 28 days post-bleomycin, mice were administered Cy5-PEG-FUD or Cy5-PEG-mFUD control subcutaneously and their lungs imaged ex vivo 24h later using the In Vivo Imaging System (IVIS). One-way ANOVA and correlation analyses were utilized for statistical comparisons between groups of interest and to determine a relationship between µPET and µCT-based signal in lungs from mice treated with ⁶⁴Cu-PEG-FUD, respectively. 

Results: Quantification of Cy5-PEG-FUD localization in tissue samples from patients with IPF demonstrated preferential binding to fibroblastic foci, the leading edge of new fibrosis, compared to mature fibrosis (Figure A-D). Further, we found that the level of Cy5-PEG-FUD binding correlated with the fibronectin expression in these tissues (Figure E). Incubation of fibrotic human IPF tissue with excess FUD resulted in a blockade of Cy5-PEG-FUD signal confirming the probe’s specificity. In the murine model of lung fibrosis, lung injury was detected in vivo by µCT radiodensity 11, but not 3, days post-bleomycin. In contrast, significantly higher levels of ⁶⁴Cu-PEG-FUD uptake were found in bleomycin-treated mouse lungs compared to controls at both time points (Figure F-G). This finding was confirmed by ex vivo gamma counting of lung tissue at the eleven-day end-point. Additionally, a correlative relationship between µCT radiodensity and ⁶⁴Cu-PEG-FUD uptake was found eleven days post-bleomycin, during the peak of the pro-fibrotic response (Figure H). Finally, we found significantly higher uptake of Cy5-PEG-FUD in mouse lungs on 7 days post-bleomycin (pro-fibrotic phase) compared to healthy or fibrotic controls administered Cy5-PEG-FUD or Cy5-PEG-mFUD, respectively. In contrast, 28 days post-bleomycin (mature fibrosis phase), signal from bleomycin and Cy5-PEG-FUD-treated group did not differ from negative controls and was significantly lower than from mice 7 days post-bleomycin (Figure I-J).

Conclusions: We reveal that PEG-FUD preferentially targets nascent fibrosis by localizing to fibroblastic foci in human IPF tissue and to the pro-fibrotic phases of bleomycin-induced pulmonary fibrosis in mice. Additionally, in the bleomycin-injured mouse lung, we demonstrate uptake of the fibronectin-targeting ⁶⁴Cu-PEG-FUD even before fibrosis is apparent by µCT. These results demonstrate initial evidence that ⁶⁴Cu-PEG-FUD may be utilized as a novel PET imaging-based method for non-invasive assessment of IPF disease activity.

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Image/Figure Caption:

A-E. Human IPF tissue sections were stained for Masson’s trichrome (A), fibronectin (B) and with Cy5-PEG-FUD (C). Areas corresponding to nascent or mature fibrosis were outlined in the trichrome stain (A), followed by the same areas being identified and outlined in the remaining sections (B, C). Cy5 fluorescence from Cy5-PEG-FUD staining was quantified (D, E) and correlated with fibronectin integrated density (E). Circles = mature fibrosis. Triangles = nascent fibrosis.

F-J. Mice were treated with bleomycin (1 U/kg, intratracheally) or normal saline (NS) control. Eleven days later, mice were injected with a single intravenous dose of ⁶⁴Cu-PEG-FUD or ⁶⁴Cu-PEG-mFUD control followed by µPET/CT imaging 6 and 30 h later. Shown are representative µPET/CT images (F), PET time-activity curves (G) and correlation between PET and CT signal (30 h scan) using Spearman’s rank correlation method (blue dot = NS, red dot = bleo) (H). Seven or 28 days post-bleomycin, mice were administered Cy5-PEG-FUD subcutaneously. Twenty four hours later, mouse lungs were imaged ex vivo using the In Vivo Imaging System. Shown are representative images (J) and quantification of Cy5 fluorescence signal (I).

Data was analyzed via Student’s t-test (D), Pearson correlation (E, H) or One Way ANOVA with Šidak’s post-hoc test (G, I).

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