Fibroblast Activating Protein: Skimming the Surface of Molecular Imaging to Assess Fibrotic Disease Activity

Abstract

Noninvasive studies to assist with the diagnosis of pulmonary fibrosis and individual prognostication represent a “holy grail” for idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases (ILDs). Multiple studies have investigated the ability of physiologic measurements, molecular profiling, circulating cellular markers (i.e., monocytes), and imaging biomarkers to predict disease progression. However, there remains no clinically used disease activity marker for single–time point assessment to assist with treatment decisions. In this issue of the Journal, Yang and colleagues (pp. 160–172) evaluate “fibroblast activating protein” (FAP) as a molecular marker of disease activity with the potential to be leveraged for prognostic purposes (1). FAP is a transmembrane serine protease that was initially found to be expressed by cancer-associated stromal cells and subsequently shown to be expressed on other cell types, including macrophages (2). While associated with fibrosis, FAP has been shown to have antifibrotic actions linked to its role in the proteolytic degradation and processing of collagen. Indeed, FAP-deficient mice have increased collagen accumulation in lungs after radiation- or bleomycin-induced injury (3). However, FAP is also reported to enhance α2-antiplasmin function, which would be expected to stabilize fibrinogen and fibronectin and contribute to lung fibrosis (4, 5). FAP expression is increased by the profibrotic cytokine TGF-β (transforming growth factor-β), and expression is increased in murine models of lung fibrosis. Within the lungs of patients with IPF, FAP was upregulated within fibroblastic foci, but not in the adjacent alveolar epithelium or in normal lung tissues (6). However, increased expression may not be detrimental; a recent study showed that depletion of FAP+ cells or genetic deletion of FAP exacerbated fibrosis in a murine model of repetitive bleomycin injury and had no impact on fibrosis induced by adenoviral overexpression of TGF-β (7). Regardless of its role in pathogenesis, recent studies have used FAP to direct delivery of approved and emerging antifibrotic drugs, supporting its potential as an indicator of active wound repair and fibrosis (8, 9). Here Yang and colleagues (1) evaluate FAP expression using human lung fibroblasts, a murine model of pulmonary fibrosis, single-cell sequencing, and a radiolabeled FAP inhibitor administered to patients with ILD. This study has several major findings. Fibroblast expression of FAP is induced by TGF-β. Similar findings have been shown in fibroblasts from several sources and disease states (2, 6). In vivo, increased FAP expression was detected 1 week after bleomycin-induced lung injury. FAP expression was increased in fibrotic human lung tissue, with half of explanted IPF samples and almost all of silicosis lung tissue samples showing elevations, demonstrating that increased FAP expression is not specific for IPF. Single-cell transcriptomic analysis indicated that FAP was highly expressed in mesenchymal cells and that there was moderate correlation with other mesenchymal cell markers associated with fibrosis (α-SMA [α-smooth muscle actin], fibronectin, and collagen I). In a small group of healthy volunteers and a larger group of subjects with ILD (IPF and non-IPF), 68Ga-FAPI-04 positron emission technology (PET) uptake was increased in the lungs of subjects with ILD compared with healthy volunteers. Differences in total standardized uptake value (SUVtotal) but not mean standardized uptake value were seen between the IPF and non-IPF ILD groups, with the IPF group having a higher SUVtotal. As SUVtotal, a measurement defined by the authors, captures the extent of probe uptake above a threshold, this finding hints at underlying biologic heterogeneity among ILD subtypes. SUVtotal correlated moderately with subsequent changes in FVC and Dl CO; however, the follow-up times for pulmonary function testing were nonuniform, ranging from 4 to 24 months. A major strength of this study lies in the application of molecular imaging to establish a link between FAP expression as a biologic indicator of fibroblast activation and a noninvasive indicator of disease activity. PET probes targeting type I collagen, CCR2 (C-C chemokine receptor 2), αvβ6 integrin, and CXCR4 (C-X-C motif chemokine receptor 4) have been recently applied to patients with IPF to detect disease or to assess treatment effects (10–13). 68Ga-FAPI-04 PET/computed tomography has been performed in systemic sclerosis–associated ILD, and the degree of probe uptake was associated with disease progression (14). This study by Yang and colleagues (1) illustrates several important applications of molecular imaging to ILD. First, it can be used to obtain molecular information otherwise not available and thus advance our understanding of molecular heterogeneity within individuals and ILD subtypes. Second, imaging of molecular pathways involved in fibrogenesis could provide a window into disease activity at a single time point. This study also highlights important considerations in using molecular imaging. PET biomarkers that incorporate both magnitude of uptake and amount of lung involved may better predict disease progression for diffuse lung diseases compared with traditional PET measurements such as mean standardized uptake value. Several important gaps remain in establishing FAP as a reliable marker of disease activity. This study does not definitively link FAP expression with profibrotic fibroblast activity. Indeed, although FAP correlated with ACTA2 (actin alpha 2, smooth muscle), COL1A1 (collagen type I alpha 1 chain), and FN1 (fibronectin 1) in single-cell analysis, transcriptional expression does not always reflect protein concentration. Given its role in collagen turnover, FAP may have a functional role in controlling the wound-repair response to prevent the excessive collagen accumulation that characterizes fibrosis. Thus, studies will need to determine the ability of molecular imaging for FAP to distinguish between physiologic and fibrotic repair. More definitive studies are needed to determine if 68Ga-FAPI-04 PET can reliably predict disease progression in ILD and whether FAP activity can predict responsiveness to antifibrotic therapies. Although the premise of differentiating “active” from “stable” disease by noninvasive FAP assessment as a decision tool to guide pharmacotherapy is appealing, fibrosis progression is not linear and for some individuals may occur in a stepwise manner. In the liver, FAP expression in hepatic stellate cells correlates with fibrosis severity (15), and circulating FAP is increased in patients with increased liver stiffness, supporting a potential role for FAP in the risk stratification of patients for liver fibrosis (16). In the lung, one modality may not provide the sensitivity and specificity needed for risk prediction or guidance of therapy in an individual patient. Nevertheless, the combination of accessible serum or genetic markers with physiologic measurements and molecular imaging may improve our ability to risk stratify individuals with ILD.