1. What are the current scientific issues in this field?
The current lack of a non-invasive technique to assess active fibrosis in interstitial lung disease (ILD) results in delayed disease diagnosis or “blind” prescribing of anti-fibrotic drugs.
2. How does this research contribute to this field?
Preclinical and clinical data from this study show that FAP is a highly specific biomarker of active fibrosis in ILD and that FAP-targeted PET imaging can be used to predict disease progression.
Rationale
Persistent activation of lung fibroblasts and the resulting oversynthesis of extracellular matrix are deleterious events in patients with ILD. Lung biopsy is the primary assessment technique for fibrotic status in ILD, but is also an important risk factor for triggering exacerbations.
Fibroblast activation protein (FAP) is a well-known surface biomarker of activated fibroblasts, but its expression pattern and diagnostic significance in ILD are unclear.
Aim of the study
This study aimed to comprehensively investigate whether FAP expression intensity can be used as a quantitative estimate/measure of the number of activated fibroblasts in ILD lungs.
Research Methods
For the first time, multiple methods were used to detect the expression of FAP in human primary lung fibroblasts and lung tissue samples, including qPCR, western blot, immunofluorescence staining, and immunohistochemistry of whole sections. Deep learning and analysis of single-cell sequencing data.
In addition, FAP-labeled PET/CT was performed in patients with different types of ILD (baseline data are shown in the appendix at the end of the article), and the relationship between FAP tracer uptake and pulmonary function parameters was analyzed.
Key findings
For the first time, FAP expression was significantly upregulated in the early stages of lung fibroblast activation events in response to low doses of profibrotic cytokines.
Single-cell sequencing data further indicated that almost all FAP-positive cells in ILD lungs were collagen-producing fibroblasts. Immunohistochemical analysis confirmed that FAP expression levels were closely related to the abundance of fibroblast foci in lung biopsy sections of patients with ILDs.
The study found that total intake of FAPI PET tracer (FAPI SUV total) was significantly associated with decreased lung function in patients with ILD.
Study Conclusions
The findings strongly support that in vitro and in vivo assays of FAP can assess the pro-fibrotic effects of ILDchemical activity, which may aid in early diagnosis and selection of appropriate therapeutic windows.
Figure 1. TGF-β regulates FAP expression in primary human lung fibroblasts.
(A) Human primary lung fibroblasts (HPLF) from healthy donors were treated with TGF-β (10 ng/ml) at the indicated times (24, 48 and 72 hours). Corresponding negative controls were set at each time point, and then the expressions of FAP, fibronectin and α-SMA were analyzed by Western blot results.
(B) Meanwhile, immunofluorescence staining was used to detect the expression of FAP at different time points after TGF-β (10ng/ml) treatment. Red indicates FAP immunoreactive signal and blue indicates DAPI staining.
(C) In a TGF-β dose-dependent experiment, 5 concentration gradients (0.5, 1, 2, 5, 10 ng/ml) of TGF-β and a negative control (0 ng/ml) were used /ml TGF-β) group was treated for 48h. Western blot was used to analyze the expression of FAP, fibronectin, collagen type I and α-SMA.
(D) Expression of FAP in lung tissue of mice at different time points in bleomycin model.
Figure 2. Expression of FAP protein and mRNA in transplanted lung tissue of IPF and non-IPF patients.
(A-B) Western blot using frozen lung tissue from healthy donors (n=6) and patients with IPF (n=8) or non-IPF ILD (silicosis, n=12) Assessing FAP expression, **P
(C) qPCR analysis of FAP mRNA expression. β-actin was used as an internal reference. The relative expression of FAP in one healthy donor (1#) was designated as the sample control and set to 1. The fold expression in other individuals was calculated by comparison with controls. The dotted line is the median, **P
Figure 3. Single-cell sequencing from public databases shows FAP-specific expression by fibroblasts.
(A) UMAP showing single-cell population data from control versus IPF in the GSE135893 study.
(B) FAP expression levels in each cell population using single-cell RNAseq data from study GSE135893.
(C) UMAP showing FAP-expressing cells from healthy donors and IPF patients from study GSE135893.
(D) Expression correlation analysis between FAP and ACTA2, FN1, COL1A1 (x-axis and y-axis represent log2 normalized expression levels).
Figure 4. FAP protein expression in lung biopsies from IPF and non-IPF patients. Immunohistochemical staining for FAP was performed on lung biopsies from IPF (n=7) and non-IPF ILD (n=26).
(A) Representative high magnification immunohistochemical images of FAP expression in donors and IPF patients. Scale bars, 200 μm.
(B) Representative immunohistochemical images of ILD patients with different grades of fibroblast lesions (+, ++, ++). The upper row represents the raw immunohistochemical panorama image of the whole section image (WSI), and the lower row represents the positive signal extraction (blue) from the AI analysis. Fibroblast lesion grade (C) and percentage of FAP-positive area within WSI (D) in donor, IPF, and non-IPF ILD patients. The dotted line is the median, **P
(E) Correlation between percentage of FAP-positive area and fibroblast lesion grade in patients including IPF and non-IPF (n=33), r=0.6056; P=0.0002, The data are shown in the correlation graph.
Figure 5. FAP-targeted PET/CT images of ILD patients
78 patients, 20 with IPF, 63 with non-IPF ILD, and 8 healthy volunteers, underwent 68Ga-FAPI-04PET-CT examination.
(A) From left to right, shows the patient’s PET whole body uptake 68Ga-FAPI-04, lung tomographic 68Ga-FAPI-04 uptake, and whole lung PET 3D reconstruction 68Ga-FAPI- 04 Intake.
(B) Mean SUV uptake of 68Ga-FAPI-04 in all ILD patients and healthy volunteer patients. The dotted line is the median, ****P
(C) Quantitative analysis of total PET tracer uptake of FAP targets (FAPI SUVtotal) in patients with different subtypes of ILD and healthy volunteers, dashed line is the median, **** P
(D) The correlation analysis of KL-6 and 68Ga-FAPI-04 uptake (SUVmean and SUVtotal) in ILD (including IPF and non-IPF patients) was plotted. P
Figure 6.68 Relationship of Ga-FAPI-04 uptake and lung function.
(A) Timeline of PET/CT and Pulmonary Function (PFT): The first PFT is performed no more than one month after the PET/CT scan. The time interval between two PFT measurements was approximately 4-20 months.
(B) Correlation analysis between 68Ga-FAPI-04 (SUVtotal and SUVmean) uptake and PFT (baseline DLCO and FVC).
(C)68Ga-FAPI-04 (SUVtotal and SUVmean) uptake and correlation analysis of PFT changes (DLCO and FVC baseline changes). Plot linear regression (R) and linear regression lines. P
Appendix Clinical Characteristics of Patients Participating in the Study
Excerpt
Tian Xin Lun
Professor, Chief Physician of Department of Respiratory and Critical Care Medicine, Peking Union Medical College Hospital, Master Supervisor.
This article is reproduced from the public account “Shuaifuyuan Forum” (ID: shuaifuyuanluntan)
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Analysis of fibroblast activation protein (FAP) expression in patients with interstitial lung disease
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