Molecular imaging of fibroblast activation in systemic sclerosis using [¹⁸F]AlF-NOTA-FAPI-04 PET/CT: a prospective cohort study

Abstract

Purpose

This prospective cohort study evaluated the utility of [¹⁸F]AlF-NOTA-FAPI-04 PET/CT for whole-body assessment of fibroblast activation in systemic sclerosis (SSc), correlating organ-specific uptake with clinical indicators of severity and multi-organ dysfunction.

Methods

31 patients with SSc and 21 patients from a non-SSc cohort underwent total-body [¹⁸F]AlF-NOTA-FAPI-04 PET/CT imaging. Quantitative uptake parameters were measured in lungs, heart, kidneys, skeletal muscles, esophagus, and skin. Clinical assessments were completed within ± 14 days. Inter-organ metabolic correlations were analyzed.

Results

Abnormally elevated FAPI uptake was prevalent in the lungs (96.8%), heart (35.5%), kidneys, and skeletal muscles (12.9%). Compared with the non-SSc cohort, patients with SSc exhibited significantly elevated whole lung SUV_mean(wlSUV_mean:overall adjusted P-value(p_adj) < 0.001) and cardiac SUVmax (cSUV_max: median1.37, IQR 1.17–1.87 vs.1.01 ± 0.15, p_adj <0.001). Reduced pulmonary function showed significant negative correlations with wlSUV_mean (p_adj <0.01). SSc patients with renal impairment demonstrated markedly higher renal SUV_max(rSUV_max)and renal SUV_mean (rSUV_mean) compared to both SSc patients with preserved renal function and the non-SSc cohort(both overall p_adj < 0.001). Muscular uptake aligned with elevated creatine kinase and biopsy-proven myopathy. No significant tracer accumulation was observed in the esophagus or skin. Inter-organ correlation analysis revealed metabolic coupling across pulmonary, cardiac, and renal systems (wlSUV_mean vs. rSUV_mean: r = 0.722; cSUV_mean vs. rSUV_mean: r = 0.632).

Conclusion

[¹⁸F]AlF-NOTA-FAPI-04 PET/CT provides a noninvasive, integrative imaging platform for mapping fibroblast activation across multiple organs in SSc. It demonstrated potential for detection of fibrotic burden and inter-organ metabolic profiling. Its sensitivity in gastrointestinal and cutaneous involvement remains limited, warranting further optimization.

Graphical Abstract

Introduction

Systemic sclerosis (SSc) is a chronic autoimmune disorder characterized by widespread vascular dysfunction and progressive fibrosis affecting multiple organs, including the skin, lungs, heart, kidneys, gastrointestinal tract, and skeletal muscles. The clinical presentation of SSc varies greatly, ranging from limited cutaneous involvement to extensive systemic manifestations impacting vital organs. Central to the pathogenesis of SSc is the activation of fibroblasts, which originate primarily from resident fibroblasts, epithelial-mesenchymal transition (EMT), or pericyte differentiation. These activated fibroblasts exhibit increased proliferative and secretory activities, leading to excessive extracellular matrix (ECM) deposition, notably collagen, fibronectin, and glycosaminoglycans [1]. This pathological process significantly impairs tissue elasticity and function, contributing to substantial morbidity and mortality in affected patients.

The heterogeneity of fibroblast activation between different SSc subtypes—particularly the pronounced visceral fibrosis in diffuse cutaneous SSc (dcSSc) compared to limited cutaneous SSc (lcSSc)—highlights the necessity for systemically integrated monitoring strategies to evaluate multiorgan involvement. Molecular imaging has emerged as a powerful approach to visualize and quantify biological processes in vivo, providing essential insights into disease progression. Fibroblast activation protein (FAP), a serine protease markedly upregulated in activated fibroblasts but minimally expressed in healthy tissues, presents a promising target for molecular imaging.

Fibroblast activation protein inhibitor (FAPI) PET/CT imaging, targeting FAP, has recently shown diagnostic promise in visualizing fibroblast activation in various organs, particularly in pulmonary and cardiac fibrosis associated with SSc [2, 3]. Despite these advancements, comprehensive multi-organ profiling of fibroblast activation using FAPI PET in systemic sclerosis remains largely unexplored. This prospective study aims to validate the clinical utility of [¹⁸F]AlF-NOTA-FAPI-04 PET/CT for quantitative assessment of fibroblast activation across multiple organs, correlating tracer uptake dynamics with established clinical biomarkers and disease severity indices. We hypothesize that this novel imaging modality can effectively map the burden of fibrosis across organs, thereby facilitating earlier detection and individualized therapeutic approaches for systemic sclerosis patients.

Methods

Patients

This prospective observational study was conducted between May 2024 and February 2025. 31 patients (5 males and 26 females; age: 54.14 ± 13.82 years, range 23–79 years) diagnosed with SSc according to the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria were prospectively recruited following written informed consent. A non-SSc disease-control cohort consisted of 21 patients (4 males, 17 females; age 45.9 ± 12.1 years, range 24–66) with thyroid-associated orbitopathy due to autoimmune thyroid disease (Graves’ disease, n = 18; Graves–Hashimoto overlap, n = 3), derived from a dedicated TED registry (ChiCTR2400083394). At PET/CT, all were euthyroid after treatment; blood pressure and renal function were within normal limits. No participant had systemic autoimmune disease other than autoimmune thyroid disease, nor a history of malignancy, cardiovascular disease, or pulmonary disease. Additionally, they were age- and sex-matched to the study cohort. All participants provided written informed consent. The study was approved by the institutional ethics committee (Approval No. M2023856) and registered with ClinicalTrials.gov (ChiCTR2500098083).

Clinical assessment

Participants underwent comprehensive clinical assessments within ± 14-day window relative to PET/CT imaging. Evaluations included the modified Rodnan Skin Score (mRSS), pulmonary function tests (FVC%, FEV1%, DLCO%), serum creatinine, Glomerular Filtration Rate (eGFR), 24-hour urinary protein quantification, High-Resolution Computed Tomography (HRCT), ‌electrocardiogram(ECG) and echocardiography. Pulmonary function tests were completely performed in 26 patients (Two patients were diagnosed with respiratory failure through arterial blood gas analysis, and three patients failed to complete pulmonary function tests due to inability to cooperate).Patients with suspected organ involvement underwent additional selective diagnostic tests, including cardiac speckle imaging (n = 21), cardiac magnetic resonance (CMR)(n = 15), muscle MRI (n = 2), electromyography (EMG) (n = 2), and muscle biopsies (n = 2).

The clinical evaluation results were independently reviewed by two experienced rheumatologists to determine organ involvement according to predefined criteria: (1) Pulmonary dysfunction was defined as either forced expiratory volume in 1 s (FEV1%) < 80% of predicted value or resting room-air PaO₂ <60 mmHg; (2) Cardiac involvement required documented abnormalities on ECG, echocardiography, speckle tracking echocardiography, or CMR, excluding alternative etiologies; (3) Renal impairment was confirmed by eGFR < 60 mL/min/1.73 m² or 24-hour urinary protein > 150 mg; (4) Musculoskeletal involvement mandated both clinical myositis and elevated serum creatine kinase; (5) Gastrointestinal involvement was defined by typical symptoms, while skin involvement was assessed using the modified Rodnan Skin Score (mRSS) [4].All discrepancies were resolved through interdisciplinary consensus.

[¹⁸F]AlF-NOTA-FAPI-04 PET/CT

All patients underwent whole-body PET/CT using a PET/CT scanner (Biograph 64,Siemens, Germany) approximately 55 ± 16 min post-injection of 212 ± 40 MBq of [¹⁸F]AlF-NOTA-FAPI-04. Low-dose CT (120 kV, 30–50 mAs) was used for attenuation correction, followed by PET acquisition from vertex to feet (2–3 min/bed, matrix size: 168 × 168, slice thickness: 3 mm). After that, maintaining the patient position, a deep inspiratory HRCT scan was performed using 64 × 1.25-mm detectors, with a pitch of 0.53 and 1.25-mm collimation (120 kVp and 100 mAs). Images were reconstructed using OSEM and analyzed with MedEx PET/CT image and information system (Beijing Medix Technology Co., Ltd) and United Imaging artificial intelligence (uAI) platforms.

Organ-oriented image analysis

All [¹⁸F]AlF-NOTA-FAPI-04 PET/CT scans were independently reviewed by two board-certified nuclear medicine physicians who were blinded to the clinical data. Discrepancies were resolved by consensus. Whole-body images were segmented according to commonly affected organ systems in SSc, including the skin, lungs, heart, kidneys, gastrointestinal tract, and skeletal muscles. FAPI-avid lesions were defined as areas of localized or diffuse radiotracer uptake exceeding the surrounding background, excluding physiological uptake and non-SSc-related abnormalities (Fig. 1).

Fig. 1.

Overview of organ-specific imaging and clinical parameters assessed in systemic sclerosis using [¹⁸F]AlF-NOTA-FAPI-04 PET/CT. Key quantitative imaging biomarkers and reference functional tests are depicted for the lungs, heart, kidneys, muscles, esophagus, and skin. eSUV_max: esophageal SUV_max; cSUV_max: cardiac SUV_max; cSUV_mean: cardiac SUV_mean; cFAV: cardiac FAPI active volume; cTL_FAPI: cardiac Total Lesion FAPI; mSUV_max: muscle SUV_max; sSUV_max: Skin SUV_max; wlFAV: whole lung FAPI active volume; wlTL_FAPI: whole lung Total Lesion FAPI; wlSUV_mean : whole lung SUV_mean; wlSUV_max: whole lung SUV_max; rSUV_max: renal SUV_max; rSUV_mean: renal SUV_mean; LAA: left atrial area; LVEF: left ventricular ejection fraction; LVEDV: left ventricular end-diastolic volume; LVEDM: Left Ventricular End-Diastolic Mass; CMR: Cardiovascular Magnetic Resonance;FEV1: Forced Expiratory Volume in 1 s; PFT: pulmonary function test; ABG: Arterial Blood Gas Analysis;24 h UP: 24-Hour Urine Protein

For pulmonary Assessment, Semi-automated segmentation was performed for regions exhibiting elevated pulmonary uptake. Quantitative parameters, including SUV_max, SUV_mean, FAPI-active volume (FAV), and total lesion FAPI (TL_FAPI), were derived using a volume of interest (VOI) threshold set at 41% of the SUV_max. Additionally, automated whole-lung segmentation using the uAI Research Portal provided global parameters such as whole-lung SUV_mean (wlSUV_mean), SUV_max (wlSUV_max), FAV (wlFAV), and TL_FAPI (wlTL_FAPI). These parameters were compared between patients with preserved versus impaired pulmonary function; when analyzing differences in wlSUV_mean, the non-SSc cohort served as the reference.

For cardiac assessment, the cardiac SUV_max (cSUV_max) was first compared between SSc patients and non-SSc cohort; subsequently, myocardial involvement was evaluated based on visually localized or heterogeneous myocardial uptake.These findings were correlated with cardiac function metrics obtained from CMR, including left ventricular ejection fraction (LVEF), left atrial area (LAA), left ventricular end-diastolic volume (LVEDV), and left ventricular mass (LVEDM). Comparative analysis was conducted between patients with and without myocardial FAPI uptake. Semiautomatic segmentation of myocardial areas exhibiting elevated FAPI uptake was performed using a threshold of 41% SUV_max. This enabled the extraction of the following myocardial FAPI uptake parameters: cSUV_max, cardiac SUV_mean (cSUV_mean), cardiac FAPI active volume (cFAV), and cardiac Total Lesion FAPI (cTL_FAPI).

For renal assessment, the renal uptake was quantified by calculating SUV_max and SUV_mean at the upper, mid, and lower poles of each kidney, carefully avoiding the collecting systems [5]. The averaged values from both kidneys (designated as rSUV_max and rSUV_mean) served as the reference metrics. Renal FAPI parameters were compared among three groups: SSc patients with preserved renal function, SSc patients with renal impairment, and the non-SSc cohort.

For skeletal muscle assessment, the muscular FAPI uptake (mSUV_max) was evaluated in conjunction with clinical parameters, including serum creatine kinase levels, EMG, muscle MRI, and biopsy results when available.

For gastrointestinal assessment, the maximum esophageal FAPI uptake was quantified as eSUV_max, and compared between SSc patients and non-SSc cohort. For cutaneous assessment, SUV_max was measured in bilateral facial dermatomes, averaged, and labeled as skin SUV_max(sSUV_max). Statistical comparisons of sSUV_max were then performed among SSc patients stratified by facial mRSS scores and the non-SSc cohort.

Statistical analysis

Continuous variables were characterized using either the mean ± standard deviation or the median (interquartile range), contingent upon their adherence to a normal distribution. The comparison of continuous variables between groups was conducted using the Student’s t-test or the Mann-Whitney U test, as appropriate. Multivariate correlation analysis of radiotracer uptake parameters across visceral organ systems was conducted utilizing Spearman’s rank correlation coefficients. All statistical tests were two-tailed with a significance level of α = 0.05. P-values were adjusted (p_adj)for multiple testing using the Benjamini-Hochberg method to control the false discovery rate. The analyses were performed using SPSS version 29.

Results

Clinical characteristics and findings

The clinical characteristics of the 31 SSc patients included in this study were summarized in Table 1. Baseline organ-specific [¹⁸F]AlF-NOTA-FAPI-04 PET/CT uptake parameters for both the SSc group and non-SSc cohort were provided in Supplementary Tables 1 and 2 in Online Resource 1.[¹⁸F]AlF-NOTA-FAPI-04 PET/CT findings were categorized into three types: (1) abnormal focal or diffuse uptake not attributable to causes other than SSc; (2) no abnormal radiotracer uptake was observed on PET, but concurrent CT demonstrated localized abnormalities; (3) absence of focal high uptake or CT abnormalities. Focal increased FAPI uptake was most frequently observed in the lungs (96.77%, 30/31), heart (35.48%, 11/31), and skeletal muscles (12.90%, 4/31). Isolated pulmonary involvement was identified in 18 patients. 8 patients (25.81%) exhibited simultaneous involvement of heart and lungs, one patient (3.23%) showed concurrent lung and skeletal muscle involvement, three patients (9.68%) displayed involvement in all three organs. Abnormal visceral FAPI uptake was absent in only a single patient. SSc patients with impaired renal function demonstrated significantly higher renal FAPI uptake values (SUV_max and SUV_mean) compared to SSc patients with preserved renal function. Despite being the most frequently involved digestive organ in SSc, the esophagus demonstrated no detectable uptake enhancement, although luminal dilation was observed in the majority of SSc patients. Facial skin fell into the third category with no significant FAPI uptake or CT abnormalities observed.

Table 1.

Clinical characteristics of the patients

Characteristics	SSc patients
Num.(n)	31
Age(y)	54.14 ± 13.82
Gender(Female) Gender(Male)	26/31(83.87%) 5/31(16.13%)
Disease duration(y)	7.68 ± 6.05
mRSS SCORE	8.72 ± 5.74
Disease Subtypes
Localized cutaneous type	6/31(19.35%)
Diffuse cutaneous type	11/31(35.48%)
Overlap syndrome	13/31(41.94%)
No scleroderma type	1/31(3.23%)
Lung involvement
Num. of patients with Interstitial fibrosis in CT	30/31(96.77%)
Num. of patients with Reduced FEV1%	11/26(42.31%)
Num. of patients with Reduced FVC%	9/26(34.62%)
Num. of patients with Reduced DLCO%	24/25(96.00%)
Num. of patients with respiratory failure	2/31(6.45%)
Num. of patients with Elevated PASP	11/31(35.48%)
Cardiac involvement
Num. of patients with elevated BNP	9/31(29.03%)
Num. of patients with reduced LVEF in UCG or CMR	4/31(12.90%)
Num. of patients with abnormal ECG	13/31(41.94%)
Num. of patients with reduced GLS in Cardiac speckle imaging	6/21(28.57%)
Num. of patients with abnormal CMR	9/15(60.00%)
Renal involvement
Num. of patients with decreased eGFR(mL/min/1.73m2)	2/31(6.45%)
Num. of patients with elevated urine protein quantification	10/31(32.26%)
Skeletal muscle involved
Patients with muscle symptoms	4/31(12.90%)
Patients with elevated serum CK or CK-MB	4/31(12.90%)
Patients with positive muscle MR findings	2/2(100.00%)
Gastrointestinal involvement
Patients with gastrointestinal symptoms	25/31(80.65%)
Patients with gastroesophageal reflux or Barret esophagus indicated by gastroscopy	13/17(76.47%)
Treatment
glucocorticoid	18(58.06%)
CTXa	10(32.26%)
MMFb	17(54.84%)
Nintedanibc	2(6.45%)

MMF：Mycophenolate mofetil CTX：cyclophosphamide a:8 patients received a combination of glucocorticoids and CTX b:6 patients received a combination of hormone and MMF c:1 patient received a combination of Nintedanib and MMF and 1 patient received a combination of Nintedanib and CTX

Table 2.

Comparison of pulmonary FAPI uptake parameters between SSc patients with normal pulmonary function and declined pulmonary function

Variables	Normal pulmonary function (n = 15)	Declined pulmonary function (n = 9)	non-SSc cohort (n = 21)	P_value	Padj value
wlTL-FAPI (g)	192.84 (45.72–502.80)	604.73 (416.29–898.66)	NA	< 0.05	0.10
wlFAV(mL)	104.73 ± 90.46	220.51 ± 157.82	NA	0.07	0.10
wlSUVmax	3.28 ± 1.35	3.14 ± 0.76	NA	0.75	0.75
wlSUVmean	0.72 (0.65–0.82)	0.94 (0.83–1.16)	0.55 ± 0.11	< 0.001	< 0.001

Intra Organ-Oriented FAPI activity

Pulmonary involvement

Pulmonary interstitial fibrosis, the most common cause of mortality in SSc patients, was identified through increased FAPI uptake in 30 out of 31 patients, aligning visually with HRCT findings(Fig. 2 and Supplementary Table 3). After excluding four patients with respiratory muscle involvement (Table 2),Significant differences in wlSUV_mean were observed across three groups (Overall p_adj <0.001,Table 2): SSc patients with normal pulmonary function (median 0.72, IQR 0.65–0.82), those with impaired function (0.94, 0.83–1.16), and non-SSc cohort (0.55 ± 0.11). Pairwise comparisons were detailed in Fig. 3A.

Fig. 2.

Representative case of a SSc patient with interstitial lung disease. A: Diffuse bilateral pulmonary increased radiotracer uptake on PET; B: Visually identified high-uptake regions with Semi-automated ROI delineation; D: Corresponding interstitial lung abnormalities on co-registered CT

Fig. 3.

[¹⁸F]AlF-NOTA-FAPI-04 PET/CT image of a representative SSc patient with cardiac involvement‌ (whole-body MIP, PET, fused images, and cardiac magnetic resonance). ‌A-C:‌ Diffuse left ventricular myocardial radiotracer uptake on PET, accentuated in the apical region; ‌D:‌ Multifocal subendocardial late gadolinium enhancement (LGE) involving all left ventricular walls on CMR

Cardiac involvement

Compared with non-SSc cohort, SSc patients exhibited significantly elevated myocardial FAPI uptake(cSUV_max: median1.37, IQR 1.17–1.87 vs.1.01 ± 0.15,p_adj<0.001)(Fig. 3C).Eleven patients exhibited localized or heterogeneous FAPI uptake in the myocardium, with their clinical conditions detailed in Supplementary Tables 4–5(Online Resource 1). Among these, 10 out of 11 patients demonstrated abnormal findings on echocardiography and/or cardiac MRI. Myocardial fibrosis was concurrently identified by both [¹⁸F]AlF-NOTA-FAPI-04 PET/CT and CMR in seven patients (Patients 3, 5, 9, 12, 16, 24, 31) (Fig. 4). 3 patients (Patient 2,17,27) presented with abnormal electrocardiograms and echocardiograms but did not undergo further CMR evaluations. 1 patient (Patient 19) demonstrated no abnormalities during routine cardiac examinations. Furthermore, delayed myocardial enhancement and elevated left ventricular myocardial Native T1 values were observed on CMR in two additional patients, however, no abnormal radiotracer uptake was identified on [¹⁸F]AlF-NOTA-FAPI-04 PET/CT.

Fig. 4.

[¹⁸F]AlF-NOTA-FAPI-04 PET/CT image of a representative SSc patient from the renal function preserved group and renal function impaired group. Patient 21 (A, B,C) demonstrated both eGFR and 24-hour urinary protein quantification within normal limits (110 mL/min/1.73 m² and 115.9 mg/24h, respectively). Patient 5 (D, E, F) maintained an eGFR within the normal range (88 mL/min/1.73 m²), however, demonstrated markedly elevated urinary protein quantification (676 mg/24h)

We further compared cardiac function parameters between patients with myocardial FAPI-avid uptake and those with non-avid uptake. No statistically significant differences were observed in cardiac function parameters between the two patient groups (all p_adj >0.05; Table 3).

Table 3.

Comparison of cardiac function parameters between the SSc patients presenting and absenting myocardial FAPI uptake

Variables	FAPI-non-avid group (n = 20)	FAPI-avid group (n = 11)	P Value	Padj Value
LAA(cm²)	16.29 (15.10–19.37.10.37)	23.89 (17.66–25.05)	< 0.05	0.12
RAA(cm²)	15.84 ± 2.88	17.04 ± 3.46	0.34	0.62
LVEDD(cm)	5.15 (4.60–5.50)	5.50 (5.15–6.30)	0.07	0.18
AAD(cm)	3.40 (2.80–3.80)	3.40 (3.10–3.80)	0.59	0.76
MPAD(cm)	2.70 (2.20–2.78)	2.50 (2.20–2.90)	0.88	0.88
LVEF	0.60 (0.56–0.65)	0.57 (0.48–0.60)	0.05	0.16
LVEDV(mL)	112.20 (99.00–131.00.00.00)	117.00 (115.50–153.50.50.50)	< 0.05	0.13
LVEDM(g)	71.00 (67.70–79.30)	85.00 (74.00–103.00.00.00)	< 0.05	0.13
LVCO(L/min)	5.05 (4.18–5.88)	5.20 (4.95–6.10)	0.53	0.76
RVEF(%)	0.45 (0.42–0.59)	0.46 (0.44–0.55)	0.71	0.85
BNP(pg/mL)	91.45 (60.70–119.00)	93.00 (40.90–625.00)	0.79	0.85
CK(U/L)	48.00 (31.75–79.25)	65.00 (38.00–147.50.00.50)	0.35	0.62
CK MB(U/L)	9.00 (7.00–14.75.00.75)	15.00 (8.00–23.50.00.50)	0.38	0.62

Renal fibrosis

Renal FAPI uptake was compared among three groups: SSc patients with preserved renal function (n = 21), SSc patients with renal impairment (n = 10), and non-SSc controls (n = 21).After confirming comparable mediastinal blood pool SUV_mean between SSc patients and non-SSc cohort (0.98 ± 0.17 vs. 1.00 ± 0.28, p = 0.96), compared to the kidneys of SSc patients with preserved renal function and non-SSc cohort, the rSUV_max and rSUV_mean were significantly elevated in SSc patients with renal impairment, but no significant difference was observed between SSc patients with preserved renal function and the non-SSc cohort for either parameter (SSc with normal renal function - SSc with with renal impairment – non SSc cohort, rSUV_max: median 1.42, IQR 1.17–1.56 vs. 2.44,1.68–2.97 vs. 1.30 ± 0.23, Overall p_adj<0.001; rSUV_mean: median 1.20, IQR 1.02–1.30 vs. 2.02,1.48–2.32 vs. 1.13 ± 0.18, Overall p_adj<0.001; pairwise comparisons were shown in Fig. 3: D-E).These findings supporting the potential role of [¹⁸F]AlF-NOTA-FAPI-04 PET/CT as an early indicator of renal fibrosis in SSc(Fig. 5, Supplementary Table 6 in Online Resource 1).

Fig. 5.

Representative PET/CT imaging of an SSc patient with myopathic involvement‌ (PET, co-registered CT, and fused images). ‌Multifocal and diffuse increased FAPI uptake in skeletal muscles on PET imaging (A,B,E,H), with no correlative structural abnormalities on co-registered CT(C,D,F,G,I,J). The biopsy of the focal hypermetabolic nodule in the left adductor magnus muscle suggested localized inflammation or myopathic changes

Skeletal muscle involvement

Elevated FAPI uptake in skeletal muscle tissue was observed in four patients, consistent with clinical manifestations of muscle weakness and elevated muscle enzyme levels (CK or CK-MB(Supplementary Table 7 in Online Resource 1). MRI and EMG confirmed inflammatory changes in two of these patients, with biopsies showing fibrotic and inflammatory myopathy consistent with [¹⁸F]AlF-NOTA-FAPI-04 PET/CT results.

A notable case was a 53-year-old man presenting with acute limb myalgia, weakness, and markedly elevated CK (8392 U/L) and CK-MB (233 U/L). [¹⁸F]AlF-NOTA-FAPI-04 PET/CT revealed multifocal muscular uptake, and biopsy confirmed severe fiber atrophy with significant CD4 + T cell/macrophage infiltration, indicative of systemic sclerosis-associated myopathy (SScAM) (Fig. 6).

Fig. 6.

Quantitative comparison of organ-specific FAPI uptake parameters between SSc patients and the non-SSc cohort. Al statistically signifcant differences were summarized in the figure. Panels display p_adj (Benjamini–Hochberg FDR, 5%) for all tested contrasts. Statistical significance is indicated as follows:*p_adj<0.05,**p_adj<0.01,***p_adj<0.001,****p_adj<0.0001. PF=pulmonary function, RF-=renal function

Gastrointestinal fibrosis

80.6% (25/31) of patients reported gastrointestinal symptoms. However, compared to the esophagus in the non-SSc cohort, the esophagus in patients with SSc did not exhibit a significant increase in radioactive uptake, a noticeable dilation of the esophagus was observed(eSUV_max: 1.87 ± 0.83 vs.1.54 ± 0.29, p_adj =0.366; width: 2.20 cm ± 0.85 cm vs. 1.25 cm ± 0.31 cm, p_adj<0.001) (Fig. 3F).

Skin fibrosis

No significant differences in sSUV_max were found among SSc patients stratified by mRSS severity (mRSS 0–1: 1.07 ± 0.29; mRSS 2–3: 1.14 ± 0.36) and non-SSc cohort (0.95 ± 0.24) (overall p_adj=0.17). Pairwise comparisons were presented in Fig. 3B, indicating limited utility for assessing skin fibrosis.

Inter-organ-oriented FAPI activity

To evaluate inter-organ metabolic associations, key molecular imaging parameters were extracted from the heart, lungs, and kidneys. These included cSUV_max, cSUV_mean, cFAV, cTL_FAPI, wlTL_FAPI, wlFAV, wlSUV_max, wlSUV_mean, rSUV_max and rSUV_mean. Pairwise Pearson correlation coefficients were computed among all extracted variables to generate a comprehensive correlation matrix, providing a quantitative assessment of linear associations and potential metabolic coupling between organs.

Based on Pearson correlation analysis, multiple metabolic parameters across different organs exhibited moderate to strong associations. Within the heart, strong intra-organ correlations were observed:

cFAV and cTL_FAPI showed the strongest correlation in the entire dataset (r = 0.991); cSUV_max was also strongly correlated with both cTL_FAPI (r = 0.962) and cFAV (r = 0.945); A moderate correlation was found between cSUV_max and cSUV_mean (r = 0.722). Beyond intra-cardiac coherence, the analysis revealed moderate positive inter-organ correlations between the lungs and kidneys, suggesting potential systemic metabolic coupling: wlSUV_mean correlated with rSUV_max at r = 0.681; The correlation between wlSUV_mean and rSUV_mean was even stronger at r = 0.722.In addition, cSUV_mean was moderately correlated with rSUV_mean (r = 0.632), indicating a possible metabolic linkage along the cardiac–renal axis (Fig. 7).

Fig. 7.

Organ-level distribution and inter-organ connectivity of FAPI uptake along the heart-lung-kidney axis

Discussion

Our research indicates that [¹⁸F]AlF-NOTA-FAPI-04 PET/CT imaging provides a comprehensive assessment of organ involvement, notably in the lungs, heart, and skeletal muscles, aligning effectively with clinical findings and standard diagnostic methods. Elevated uptake on [¹⁸F]AlF-NOTA-FAPI-04 PET/CT imaging appears promising as an early indicator of kidney injury in patients with SSc. However, limitations exist, notably in detecting digestive tract involvement, where [¹⁸F]AlF-NOTA-FAPI-04 PET/CT imaging may result in false negatives. Concurrent CT imaging partially mitigates this limitation, and false negatives in detecting skin involvement highlight a need for further imaging refinements or complementary diagnostic approaches.

Pulmonary involvement, the leading cause of SSc mortality, mainly presents as ILD and pulmonary hypertension. Metabolic imaging show promise for SSc-ILD assessment [6–8], with ¹⁸F-FDG PET/CT SUV_min plus ILD-GAP index proven as survival predictor [9]. Prior studies linked FAP expression in fibrotic lesions to imaging findings and disease progression [3, 10, 11]. We innovatively used automated wlSUV_mean as global fibrosis indicator, effectively distinguishing SSc patients by pulmonary function and showing higher values versus non-SSc controls, suggesting fibrosis biomarker potential. This reliable, patient-independent method is particularly valuable for critically ill patients unable to undergo standard tests.

Cardiac involvement significantly impacts the prognosis of SSc patients, yet current diagnostic approaches primarily detect advanced-stage lesions [12, 13]. In contrast to the limitations of FDG-PET [14], FAPI PET/CT demonstrates superior specificity owing to its negligible physiological myocardial uptake [2]. Our study revealed that 90.9% (10/11) of FAPI-positive patients showed abnormalities in conventional examinations, confirming its potential for evaluating cardiac involvement in SSc. The discrepancies between [¹⁸F]AlF-NOTA-FAPI-04 PET/CT and CMR likely stem from its specific detection of active fibroblast activation rather than established fibrosis, consistent with Treutlein et al.‘s findings [2]. No significant differences in cardiac function parameters were observed between SSc patients with FAPI-positive and FAPI-negative myocardial involvement, suggesting that fibroblast activation precedes functional decline by a significant margin. FAPI-PET sensitively captures this window of myocardial involvement in SSc, providing critical imaging evidence for early clinical intervention.

Skeletal muscle involvement, though relatively rare, significantly contributes to morbidity and mortality among SSc patients. Conventional diagnostic methods include serum creatine kinase measurements, electromyography, and muscle MRI [15, 16]. Zhang et al. demonstrated that FAPI PET serves as a visual assessment tool for disease activity in idiopathic inflammatory myopathy (IIM) [17].In our study, patients exhibited increased skeletal muscle uptake on [¹⁸F]AlF-NOTA-FAPI-04 PET/CT, correlating with elevated serum muscle enzymes and clinical evidence of muscle involvement, highlighting the clinical value of [¹⁸F]AlF-NOTA-FAPI-04 PET/CT as a sensitive, real-time imaging modality for detecting and guiding muscle biopsy in patients with skeletal involvement.

Using localized FAPI uptake not attributable to alternative etiologies as a positive criterion for SSc involvement in muscles and viscera, we found involvement of two organs in 9 patients and three organs in 3 patients, which suggests that [¹⁸F]AlF-NOTA-FAPI-04 PET/CT may have potential in evaluating the simultaneous involvement of multiple organs.

Renal involvement, another critical aspect of SSc, showed promising results with diffuse renal uptake identified via [¹⁸F]AlF-NOTA-FAPI-04 PET/CT imaging. This uptake potentially represents early stages of renal fibrosis, aligning with previous studies exploring renal fibrosis in contexts other than systemic sclerosis [5, 18]. Although the sample size was limited, the results suggest [¹⁸F]AlF-NOTA-FAPI-04 PET/CT’s potential in detecting subclinical renal involvement, warranting further validation in larger, prospective cohorts to establish definitive clinical thresholds.

The gastrointestinal tract is the most frequently affected visceral organ in SSc, though non-invasive evaluation studies remain limited [19, 20].Our study demonstrated low detection rates of esophageal lesions by FAPI imaging. While the Sjögren hypothesis identifies fibrosis as a key pathological feature [21], autopsy studies reveal that esophageal involvement in SSc primarily manifests as smooth muscle atrophy rather than significant fibrosis [22]. Additionally, perioral skin involvement and sicca symptoms may also contribute to dysphagia. We hypothesize that negative esophageal FAPI-PET findings may result from: (1) missing the active fibrosis window; (2) fibrosis not being the predominant pathological alteration; (3) partial volume effects due to thinned esophageal walls [23].

Our study revealed unexpectedly low [¹⁸F]AlF-NOTA-FAPI-04 PET/CT detection of skin involvement in SSc despite characteristic clinical manifestations. The pathogenesis involves immune/vascular/fibrotic dysregulation [24–26], with aberrant fibroblast activation driving collagen deposition (skin hardening) and atrophy via pro-fibrotic secretion and Endothelial-to-Mesenchymal Transition(EndMT). Potential explanations include: (1) Temporal mismatch with fibroblast activity—immune/fibroblast dysregulation normalizes over time in early diffuse SSc [26], reducing late-stage detectability; and(2) Partial volume effects, as atrophic facial skin may falls below PET resolution.

Ethically, TED patients comprised the non-SSc cohort. Both disorders share fibroblast activation, myofibroblast differentiation, and TGF-β-mediated ECM deposition, but differ mechanistically. TED involves thyrotropin receptor(TSHR)/insulin-like growth factor-1 receptor(IGF-1R) autoantibodies driving orbital hyaluronic acid/adipogenesis [28–30], while SSc features systemic collagen I/III overproduction from vascular/immune triggers [31]. Thus, TED is antibody-mediated orbital disease versus SSc’s multifactorial systemic fibrosis. The TED cohort’s lack of skin/visceral involvement and rigorous screening ensured validity, matching prior FAPI-PET designs [3, 32].

This study demonstrated metabolic coupling pulmonary-renal (r = 0.72) and cardio-renal (r = 0.63) axis correlations. This cross-organ synchrony confirms FAPI-PET’s dual utility for assessing both single-organ and multiorgan pathophysiology, potentially mediated by systemic Fibro-activation [27]. Although SSc is systemic, organ involvement is heterogeneous. FAPI-PET quantifies inter-organ fibroblast activation covariation, enabling endophenotyping, hypothesis generation on cross-talk, and potentially more stable integrative endpoints than single-organ readouts.

This study has several limitations that warrant acknowledgment. First, the cross-sectional design lacks longitudinal follow-up data, and pathological confirmation was unattainable due to ethical and practical constraints. Second, the determination of FAPI-avid uptake was based solely on visual assessment. Third, although the inclusion of 31 SSc patients represents the largest FAPI PET cohort to date, the inherent rarity of SSc and substantial subtype heterogeneity resulted in limited subgroup sample sizes. Furthermore, potential influences of disease duration and treatment heterogeneity on FAPI uptake were not accounted for, necessitating cautious interpretation and avoidance of overgeneralization of the findings. Additionally, the use of a non-healthy control group may introduce confounding variables. Future studies should employ large-scale prospective longitudinal designs with standardized documentation of treatment regimens and appropriately timed [¹⁸F]AlF-NOTA-FAPI-04 PET/CT follow-up scans to analyze the correlation between dynamic changes in FAPI uptake and clinical outcomes, thereby validating and optimizing the prognostic value of [¹⁸F]AlF-NOTA-FAPI-04 PET/CT imaging in systemic sclerosis.

Conclusion

Our preliminary study suggests that [¹⁸F]AlF-NOTA-FAPI-04 PET/CT has potential value for the one-stop evaluation of multi-organ involvement. It revealed significant uptake patterns in the lungs, heart, kidneys, and skeletal muscle of systemic sclerosis patients. These findings suggest its utility for fibrotic burden quantification and inter-organ metabolic characterization, offering valuable perspectives for monitoring disease progression in future research.

Author contributions

Material preparation, data collection and analysis were performed by Meixin Zhao, Hui Wei, Meng Wang and Hui Li. Statistical analysis was performed by Xinwei Li and Zeyang Wang. The first draft of the manuscript was written by Meixin Zhao, Xiang Li, and Annan Zhang and Song Xue. Marcus Hacker, Weifang Zhang, Rong Mu and Xiang Li has co-contributed to the study conception and design. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82271836), China Medical Health Development Foundation, and Key Clinical Projects of Peking University Third Hospital (No. BYSY2022071, BYSYZD2023016 and BYSYZD2024005).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

“The authors affirm that human research participants provided informed consent for publication of the images.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Peking University Third Hospital (01-04-2024/M2023856).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.