Emerging Blood Biomarkers in Systemic Sclerosis: From Single Molecules to Biomarker-Based Patient Stratification

1. Introduction

Systemic sclerosis (SSc) is a systemic autoimmune rheumatic disease characterized by immune abnormalities, microvascular injury, and fibrosis involving the skin and internal organs. The clinical phenotype of SSc varies markedly among patients, ranging from relatively mild involvement to severe multisystem disease complicated by interstitial lung disease (ILD), pulmonary arterial hypertension (PAH), cardiac dysfunction, or renal crisis. This heterogeneity represents one of the greatest challenges in clinical management and therapeutic development [1,2,3,4,5,6,7].

ILD develops in approximately half of patients with SSc and remains a leading cause of mortality. In addition, vascular complications such as PAH and digital ulcers substantially impair quality of life and prognosis. Early identification of patients at risk for progressive organ involvement is therefore critically important. However, clinical assessment alone often fails to accurately predict disease trajectories [1,2,3,4,5,6,7].

Although biomarkers obtained directly from affected organs or tissues may provide more precise information regarding local pathophysiology and organ-specific disease activity, such approaches are often invasive and difficult to apply routinely in clinical practice. In contrast, peripheral blood biomarkers can be obtained relatively easily and repeatedly, making them more practical for daily clinical assessment and longitudinal disease monitoring. Therefore, this review focuses specifically on circulating soluble biomarkers and related blood-derived molecular signatures in SSc.

Traditionally, disease-specific autoantibodies such as anti-topoisomerase I antibody and anticentromere antibody have been used for diagnosis and clinical classification. Nevertheless, autoantibodies generally reflect stable immunologic phenotypes rather than dynamic disease activity. Consequently, there has been growing interest in soluble circulating biomarkers capable of reflecting ongoing inflammation, endothelial activation, and fibrotic remodeling [8,9].

Over the last two decades, numerous cytokines, chemokines, adhesion molecules, growth factors, extracellular vesicle-associated molecules, and extracellular matrix (ECM)-related molecules have been investigated as candidate biomarkers in SSc. Initially, most studies focused on single molecules associated with disease activity or organ involvement. Subsequently, longitudinal cohort studies demonstrated prognostic significance of certain biomarkers. More recently, multidimensional approaches integrating multiple biomarkers and unsupervised clustering have emerged as promising strategies for identifying biologically distinct disease subsets.

Japanese multicenter cohort studies have made substantial contributions to this field. Early studies demonstrated associations between circulating chemokines and disease activity, while subsequent longitudinal analyses identified prognostic biomarkers for pulmonary decline and physical dysfunction. More recently, biomarker-based clustering approaches identified distinct pulmonary trajectories in early severe SSc, representing an important step toward biomarker-driven precision medicine [10,11,12,13].

This review summarizes advances in circulating soluble biomarkers in SSc, with particular emphasis on studies published since 2020 and on findings from Japanese longitudinal cohort studies.

2. Literature Search Strategy

Literature for this narrative review was primarily identified through searches of PubMed and related databases using combinations of the terms “systemic sclerosis” and “biomarker”. Particular emphasis was placed on studies published since 2020, although earlier landmark studies, especially longitudinal cohort studies from Japan, were also included when considered important for understanding the evolution of biomarker research in SSc. Priority was given to peer-reviewed clinical studies, translational investigations, systematic reviews, and recent review articles relevant to circulating blood biomarkers in SSc. This review was conducted as a narrative review and did not follow formal systematic review methodology.

3. Pathophysiological Basis of Blood Biomarkers in SSc

The pathogenesis of SSc is complex and incompletely understood but is generally considered to involve three interrelated processes: immune dysregulation, endothelial dysfunction, and fibrosis. Blood biomarkers are thought to reflect these pathogenic pathways [1,2,3,4,5,6,7,8,9,14].

3.1. Immune Dysregulation

Immune activation is an early and central event in SSc. Both innate and adaptive immune responses contribute to disease progression. Activated monocytes/macrophages, T cells, and B cells infiltrate affected tissues and produce numerous inflammatory mediators including cytokines and chemokines [3,14].

Macrophages play particularly important roles in SSc fibrosis. Alternatively activated macrophages produce profibrotic mediators such as transforming growth factor-beta (TGF-beta), IL-6, and CCL18. T helper cell polarization is also altered in SSc, with contributions from Th2, Th17, and T follicular helper pathways [9,14].

Recent therapeutic studies have further strengthened the concept that B cells play central pathogenic roles in SSc. The DESIRES trial demonstrated that rituximab, a monoclonal antibody targeting CD20-positive B cells, significantly improved skin sclerosis in patients with SSc and showed favorable effects on pulmonary involvement [15]. In addition, emerging evidence suggests that CD19-targeted chimeric antigen receptor (CAR)-T cell therapy may represent a novel therapeutic strategy for severe refractory SSc. Recent case series demonstrated marked improvement in skin fibrosis and interstitial lung disease following CAR-T cell therapy, further supporting the pathogenic importance of B cells in SSc [16].

Type I interferon (IFN) signaling is increasingly recognized as another major immunologic pathway in SSc. IFN-inducible gene signatures have been identified in subsets of patients and are associated with inflammatory phenotypes and progressive disease [17]. Because these inflammatory pathways generate soluble mediators detectable in serum, circulating cytokines and chemokines may reflect ongoing disease activity [9,17].

3.2. Endothelial Dysfunction and Vasculopathy

Vascular injury is a hallmark of SSc and often precedes overt fibrosis. Endothelial cell activation and apoptosis lead to impaired angiogenesis, vascular remodeling, and tissue ischemia [3,14]. Activated endothelial cells express adhesion molecules such as intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1 (VCAM-1), and selectins, facilitating leukocyte recruitment into tissues. Platelet activation further amplifies vascular inflammation through release of chemokines and growth factors [11,12].

Although angiogenic mediators such as vascular endothelial growth factor (VEGF) are elevated in SSc, effective angiogenesis is paradoxically impaired, suggesting dysregulated vascular repair mechanisms. Recent clinical studies evaluating IL-8, VEGF, basic fibroblast growth factor, and IFN-alpha further support the concept that angiogenic and inflammatory mediator profiles reflect vascular involvement and disease course in SSc [18].

Endoglin is a TGF-beta co-receptor expressed on endothelial cells and involved in vascular remodeling. A systematic review highlighted soluble endoglin as a candidate endothelial biomarker in SSc, particularly in relation to PAH and vascular complications [19].

3.3. Fibrosis and Extracellular Matrix Remodeling

Fibrosis results from persistent activation of fibroblasts and myofibroblasts. In addition to resident fibroblasts, mesenchymal transition of endothelial cells, pericytes, epithelial cells, and other cellular sources have been proposed to contribute to myofibroblast accumulation in fibrotic diseases, including SSc [20]. Activated fibroblasts produce excessive collagen and ECM proteins, leading to progressive tissue stiffness and organ dysfunction [3,14,20].

TGF-beta signaling represents a central profibrotic pathway in SSc. Additional mediators including IL-6, connective tissue growth factor, endothelin-1, and periostin also contribute to fibroblast activation [9,14,20]. ECM turnover generates measurable circulating biomarkers including collagen neoepitopes, matrix metalloproteinases (MMPs), and matricellular proteins. These molecules may directly reflect ongoing fibrotic activity [20,21].

4. Cytokines and Chemokines

4.1. IL-6 and Related Inflammatory Mediators

IL-6 is among the most extensively studied cytokines in SSc and is strongly implicated in inflammation and fibrosis. Elevated serum IL-6 levels are associated with diffuse cutaneous disease, higher modified Rodnan skin score (mRSS), inflammatory phenotypes, progressive skin fibrosis, and worse pulmonary outcomes [9,21]. IL-6 promotes fibroblast activation through JAK/STAT signaling and enhances collagen synthesis. It also contributes to B-cell activation and Th17 differentiation [9,14].

Emerging inflammatory biomarkers are also being investigated in SSc. Recently, soluble oncostatin M receptor was proposed as a potential diagnostic biomarker, suggesting possible involvement of oncostatin M signaling pathways in SSc-related inflammation and fibrosis [22].

The clinical importance of IL-6 was reinforced by clinical trials of tocilizumab, an anti-IL-6 receptor antibody. Biomarker analyses from the focuSSced trial suggested that IL-6-related inflammatory pathways are linked to progressive pulmonary fibrosis [21,22,23]. Tocilizumab also appeared to stabilize lung function in early SSc-ILD, further supporting the relevance of inflammatory biomarkers in identifying treatment-responsive phenotypes [23,24].

4.2. CCL2 (MCP-1)

CCL2 is a monocyte chemoattractant strongly implicated in SSc fibrosis. CCL2 recruits monocytes and macrophages into tissues and may directly promote fibroblast activation [9,10,14]. Serum CCL2 levels are elevated in SSc and are associated with ILD severity and skin fibrosis. Earlier Japanese cohort studies demonstrated that serum CCL2 levels correlated with disease activity and longitudinal pulmonary dysfunction [10,11]. Because macrophage-driven fibrosis is central to SSc pathogenesis, CCL2 remains one of the most biologically plausible profibrotic biomarkers [9,10,11].

4.3. CXCL8 (IL-8)

CXCL8 is a neutrophil-attracting chemokine associated with inflammatory and vascular activation [10,11,18]. Japanese longitudinal cohort studies demonstrated elevated CXCL8 levels in early SSc and identified baseline CXCL8 levels as predictors of subsequent physical dysfunction [11]. These findings suggested that soluble inflammatory mediators may predict future disease progression rather than merely reflect current activity [11].

4.4. IFN-Related Chemokines and Type I Interferon Signaling

CXCL9 and CXCL10 are IFN-inducible chemokines associated with Th1-type inflammation and activation of type I IFN pathways. Elevated serum levels of these chemokines have been observed particularly in early inflammatory SSc and are associated with inflammatory disease activity and diffuse cutaneous involvement [10,11,17]. Accumulating evidence increasingly supports the importance of type I IFN signaling in the pathogenesis of SSc. IFN-inducible gene signatures and serum IFN scores have been associated with inflammatory phenotypes, progressive fibrosis, and biologically distinct disease subsets [17].

These findings suggest that IFN-related biomarkers may eventually contribute to molecular classification and biomarker-based precision medicine approaches in SSc. Furthermore, IFN-related pathways may represent potential therapeutic targets in inflammatory SSc phenotypes [17].

4.5. CXCL4

CXCL4 has emerged as one of the most promising biomarkers in SSc. Originally identified as a platelet-derived chemokine, CXCL4 is strongly associated with fibrosis and vascular disease [25,26,27]. High CXCL4 levels correlate with ILD progression, PAH, digital ulcers, and mortality. Experimental and translational studies suggest that CXCL4 may contribute to endothelial dysfunction, innate immune activation, and fibroblast activation [25,26,27]. Because CXCL4 appears closely linked to severe fibrotic phenotypes, it may represent both a biomarker and pathogenic mediator [25,26,27].

5. Adhesion Molecules and Endothelial Biomarkers

5.1. ICAM-1 and Selectins

Adhesion molecules play critical roles in leukocyte recruitment and endothelial activation [12]. A multicenter Japanese study demonstrated elevated serum ICAM-1, E-selectin, and P-selectin levels in patients with early SSc. Importantly, baseline ICAM-1 levels predicted subsequent decline in pulmonary function [12]. P-selectin levels were also associated with future disability progression, suggesting relationships between platelet/endothelial activation and systemic disease severity [12].

5.2. VEGF, Endostatin, and Angiogenic Mediators

VEGF is elevated in SSc despite defective angiogenesis. This paradox suggests dysregulated vascular repair mechanisms. VEGF levels have been associated with digital ulcers, PAH, and abnormal nailfold capillary findings, although its clinical utility as an isolated biomarker remains uncertain [18].

Recent meta-analytic evidence further supports the importance of angiogenesis-related biomarkers in SSc. Circulating endostatin, an endogenous anti-angiogenic glycoprotein, is significantly elevated in patients with SSc, particularly in those with digital ulcers and PAH. These findings suggest that endostatin may reflect impaired vascular repair and progressive vasculopathy in SSc [28].

5.3. Endothelin-1 and Endoglin

Endothelin-1 is a potent vasoconstrictor implicated in vascular remodeling and fibrosis [2]. Elevated endothelin-1 levels have been associated with PAH, digital ulcers, and pulmonary fibrosis. A recent systematic review and meta-analysis further confirmed significantly elevated circulating endothelin-1 levels in patients with SSc compared with healthy controls, supporting its role as a biomarker of vascular dysfunction and fibrosis [29].

Endoglin integrates vascular and profibrotic signaling pathways and may represent an important mechanistic biomarker. Elevated soluble endoglin levels have been associated with PAH and vascular complications in SSc [19].

6. Fibrosis and Extracellular Matrix Biomarkers

6.1. KL-6 and SP-D

KL-6 and SP-D are widely used biomarkers for SSc-associated ILD. KL-6 reflects alveolar epithelial injury and correlates with high-resolution computed tomography abnormalities and pulmonary function impairment. Longitudinal cohort studies further demonstrated that elevated KL-6 levels were associated with subsequent decline in DLCO and progressive pulmonary dysfunction, supporting its prognostic utility in SSc-ILD [30].

Candidate serum biomarker studies have shown that KL-6, SP-D, CCL18, and related pneumoproteins are associated with ILD severity and progression, although their performance differs across cohorts and disease stages [31,32].

6.2. CCL18

CCL18 is produced mainly by alternatively activated macrophages and is strongly associated with pulmonary fibrosis [32,33]. Several studies identified elevated CCL18 levels as predictors of progressive ILD and mortality in SSc [32,33]. Because macrophage activation plays central roles in fibrosis, CCL18 may represent one of the most robust fibrosis-related biomarkers [32,33].

6.3. Periostin, IGFBP7, COMP, and Collagen Turnover Markers

Periostin is a matricellular protein induced by IL-4, IL-13, and TGF-beta signaling and has emerged as a candidate biomarker reflecting cutaneous and pulmonary fibrosis in SSc. Biomarker analyses from therapeutic trials further support its role as a fibrosis-related biomarker [21]. Serum periostin levels were positively correlated with mRSS, particularly in diffuse cutaneous SSc, and were associated with progressive skin sclerosis during longitudinal follow-up [34].

Insulin-like growth factor-binding protein (IGFBP) 7 has also emerged as a candidate fibrosis-related biomarker in SSc. Elevated serum IGFBP7 levels were associated with diffuse cutaneous disease, increased skin thickness, and interstitial lung disease, suggesting potential involvement in fibroblast activation and tissue remodeling [35].

Cartilage oligomeric matrix protein (COMP) and collagen neoepitopes such as Pro-C3 reflect ECM remodeling and collagen synthesis. Recent studies demonstrated associations between these markers and progressive cutaneous and pulmonary fibrosis, suggesting that they may eventually allow direct monitoring of active fibrogenesis[21].

6.4. Matrix Metalloproteinases

MMPs regulate ECM degradation and remodeling. Altered levels of MMP-7 and MMP-12 have been associated with ILD severity and progressive fibrosis [31,36]. Recent studies suggest that combined MMP/TIMP signatures may improve diagnostic accuracy for connective tissue disease-associated ILD. In particular, MMP-7, MMP-9, MMP-10, and MMP-12 were significantly elevated in SSc-ILD and may contribute to earlier identification of pulmonary involvement [36].

7. Biomarkers for Organ Involvement

Although many biomarkers discussed in the previous sections overlap across inflammatory, vascular, and fibrotic pathways, their relative clinical relevance may differ according to specific organ involvement. Therefore, this section summarizes biomarker profiles from the perspective of major organ complications in SSc.

7.1. Skin Fibrosis

Skin fibrosis remains a central clinical manifestation and therapeutic target in SSc. mRSS is widely used for assessment of severity, longitudinal progression, and therapeutic response of skin fibrosis. Several circulating biomarkers have been associated with the extent and progression of cutaneous involvement.

Elevated serum IL-6 levels have been associated with diffuse cutaneous involvement, higher mRSS, and inflammatory phenotypes [9,21]. CCL2 has also been linked to skin fibrosis and profibrotic macrophage activation [10,11].

Periostin has emerged as a promising biomarker reflecting cutaneous fibrosis. Serum periostin levels correlated positively with mRSS and progressive skin fibrosis, particularly in diffuse cutaneous SSc [34]. Similarly, IGFBP7 was associated with diffuse cutaneous disease and increased skin thickness [35].

ECM turnover markers including COMP and Pro-C3 may reflect active fibrogenesis in skin tissues and could potentially serve as biomarkers of ongoing cutaneous fibrosis [21].

7.2. Interstitial Lung Disease

ILD is the most important determinant of mortality in many SSc cohorts [5,6]. Multiple biomarkers have been associated with ILD severity or progression, including KL-6, SP-D, CCL18, IL-6, CCL2, CXCL4, ICAM-1, periostin, MMPs, and extracellular vesicle-associated biomarkers [21,30,31,32,33,34,35,36,37,38,43]. However, single biomarkers alone often show limited predictive accuracy [31,32,37]. Consequently, integrated biomarker approaches may provide superior prediction of pulmonary trajectories [13,31,37].

Recent studies have also highlighted extracellular vesicles (EVs) as emerging biomarkers in SSc-ILD. Increased circulating EV subpopulations, particularly ICAM1-positive EVs, were associated with progressive fibrosing ILD and independently predicted pulmonary progression during longitudinal follow-up [38].

7.3. Pulmonary Hypertension

Biomarkers associated with PAH include BNP/NT-proBNP, endoglin, endothelin-1, endostatin, VEGF-related molecules, and EV-associated signatures. NT-proBNP remains the most clinically established biomarker for PAH screening and prognosis and has been incorporated into screening algorithms such as the DETECT algorithm for SSc-PAH [39]. In addition, endostatin and endothelin-1 may reflect vascular remodeling and impaired angiogenesis [2,19,28,29]. Recent longitudinal studies have further identified GDF-15 and PSP-D as promising candidate biomarkers for future development of SSc-associated PAH [40].

7.4. Cardiac Involvement

Cardiac involvement in SSc ranges from subclinical myocardial fibrosis to severe arrhythmias and heart failure. Candidate biomarkers include troponins, natriuretic peptides, inflammatory cytokines, and fibrosis markers [41,42]. Because early detection of cardiac involvement remains difficult, additional biomarker development is needed.

8. Japanese Longitudinal Cohort Studies in SSc Biomarker Research

Japanese multicenter longitudinal cohort studies have provided important insights into circulating biomarkers associated with disease progression and organ involvement in SSc [10,11,12,13]. Early studies demonstrated elevated serum levels of chemokines including CCL2, CXCL8, CXCL9, and CXCL10 in patients with SSc [10,11]. Subsequent longitudinal analyses demonstrated that baseline CXCL8 levels predicted future physical dysfunction in early SSc, establishing the prognostic significance of soluble inflammatory biomarkers [11].

Further studies investigated endothelial biomarkers including ICAM-1 and selectins. Baseline ICAM-1 levels predicted subsequent pulmonary decline, linking endothelial activation to progressive ILD [12]. In a recent multicenter Japanese cohort study, patients with early severe SSc were classified into three biomarker-defined clusters based on serum chemokine and adhesion molecule profiles using k-means clustering analysis [13].

Cluster 1 was characterized by elevated sICAM-1 and sE-selectin levels, whereas Cluster 2 showed elevated CCL2, CXCL8, and sP-selectin levels. Cluster 3 showed no distinctive biomarker pattern and served as the reference group. Importantly, these clusters demonstrated distinct pulmonary function trajectories. Cluster 1 showed impaired pulmonary function at baseline with only minimal further decline during follow-up, whereas Cluster 2 initially showed relatively preserved pulmonary function but demonstrated progressive decline over time. In contrast, pulmonary function remained relatively stable throughout the disease course in Cluster 3 [13].

These findings suggest that serum biomarker profiles may reflect biologically distinct inflammatory and vascular disease processes associated with different patterns of pulmonary progression in early severe SSc. This progression from single biomarkers to integrated clustering approaches represents a major conceptual advance in SSc biomarker research [10,11,12,13].

Because SSc is biologically and clinically heterogeneous, single biomarkers alone may not sufficiently capture complex disease phenotypes or differences across ethnic populations. Integrated multi-biomarker clustering approaches may therefore provide more robust stratification of patients by simultaneously reflecting multiple pathogenic pathways, including inflammation, endothelial dysfunction, and fibrosis. Such approaches may also improve longitudinal prediction of organ progression beyond conventional single-marker analyses. Major Japanese longitudinal cohort studies and their principal biomarker findings are summarized in Table 1.

9. Biomarker-Based Precision Medicine

Recent advances in machine learning and systems biology have accelerated development of biomarker-based precision medicine approaches [13,45]. Because SSc is biologically heterogeneous, patients with similar clinical phenotypes may have fundamentally different pathogenic pathways [1,13]. Molecular profiling may therefore allow identification of biologically distinct patient subsets with different prognoses and therapeutic responsiveness.

Integrative clustering analyses combining clinical and proteomic data have further strengthened the concept of biologically distinct SSc subsets. Dans-Caballero et al. identified two clinically and molecularly distinct clusters characterized by different patterns of organ involvement, autoantibody profiles, and circulating proteomic signatures associated with fibrosis, endothelial dysfunction, and inflammation [44]. Importantly, serum obtained from patients in the severe cluster induced profibrotic and inflammatory gene expression in dermal fibroblasts in vitro, supporting the biological relevance of molecular stratification approaches and suggesting that circulating molecular profiles may actively contribute to disease progression [44].

Transcriptome-integrated biomarker studies have identified soluble CD13 as a potential novel circulating biomarker in SSc. Elevated soluble CD13 levels were associated with inflammatory and immune-related transcriptomic signatures in peripheral blood, suggesting possible utility for molecular stratification and precision medicine approaches [45].

Extracellular vesicle-associated microRNA signatures may further improve molecular stratification in SSc. A recent study identified a four-miRNA extracellular vesicle signature associated with SSc-ILD and progressive profibrotic pathways [46].

Potential applications of integrated biomarker strategies include prediction of ILD progression, identification of inflammatory versus fibrotic phenotypes, therapeutic response prediction, and enrichment of clinical trial populations. Integration of circulating biomarkers with transcriptomics, proteomics, imaging, and clinical data may further improve disease stratification and individualized therapeutic approaches.

Representative circulating biomarkers in SSc, including their biological functions and associations with organ involvement and fibrosis, are summarized in Table 2. Potential clinical applications of circulating biomarkers and integrated biomarker-based strategies in SSc are summarized in Table 3.　

10. Future Perspectives

Future biomarker research in SSc will likely focus on longitudinal multiomics integration, standardized assays, and AI-driven prediction models. Single-cell RNA sequencing, proteomics, metabolomics, spatial transcriptomics, and EV-associated molecular profiling may reveal novel pathogenic pathways and therapeutic targets.

Despite substantial advances, prediction of progressive SSc-ILD remains challenging. A recent systematic review and meta-analysis demonstrated robust associations of KL-6, SP-D, and IL-8 with SSc-ILD, but also highlighted the limited validation and prognostic consistency of many other proposed biomarkers [47]. A scoping review emphasized that few biomarkers consistently predict ILD progression, although KL-6 currently appears among the most reproducible prognostic biomarkers [48]. Future studies should determine whether composite biomarker models integrating multiple pathogenic pathways outperform individual biomarkers in predicting organ progression and therapeutic response.

Another important future direction is biomarker-guided therapy selection. For example, inflammatory biomarker signatures may identify patients more likely to respond to IL-6 blockade or JAK inhibition, whereas fibrosis-dominant signatures may identify patients requiring antifibrotic therapy [21,22,23,24]. Ultimately, biomarker-driven precision medicine may allow individualized management strategies based on underlying disease biology rather than conventional clinical phenotypes alone.

11. Conclusions

Substantial progress has been made in identifying circulating blood biomarkers associated with inflammation, endothelial dysfunction, and fibrosis in SSc [8,9,13]. Recent advances have shifted the field from isolated biomarker analyses toward multidimensional biomarker profiling and biologically based patient stratification [13,43,44,45,46]. Findings from Japanese longitudinal cohort studies have also supported multidimensional biomarker stratification approaches in SSc [10,11,12,13].

Although additional validation and standardization remain necessary, integrated biomarker-based strategies may ultimately facilitate precision medicine approaches and improve prognostication, patient stratification, and therapeutic decision-making in SSc.