Spatial transcriptomic profiling reveals fibroblast activation and inflammatory signatures in calcinosis cutis of autoimmune connective tissue diseases

Summary: Calcinosis cutis (CC) is a debilitating condition characterized by calcium deposition in the skin of patients with rheumatic skin diseases; however, its pathogenesis remains poorly understood. We employed spatial transcriptomics to define the molecular signatures of CC in dermatomyositis (DM) and chronic cutaneous lupus erythematosus (CCLE). Skin biopsies from patients with DM (n=3; 1 with CC, 2 without) and CCLE (n=4; 2 with CC, 2 without) were analyzed using the Xenium platform with a human skin gene panel plus 100 custom inflammatory markers. After filtering, the data were normalized, followed by unsupervised clustering and cell type annotation via the SingleR algorithm. CC samples revealed a global upregulation of pro-inflammatory genes (NLRP10, IL1R2, GSDMA) in both DM and CCLE (q<0.05), with gene set enrichment analysis demonstrating significant enrichment of calcium ion binding and ECM structural pathways in CCLE samples. In both diseases, histologically-identified CC areas showed differential expression of inflammasome components (AIM2, ZBP1) and immune cell markers (CD68, TYROBP, CXCR4) compared to adjacent non-calcified tissue. Across DM and CCLE, global comparison of fibroblasts demonstrated significant increases in markers of myofibroblast-transition (POSTN, ACTA2) and ECM regulation (ADAM12, MMP2) (q<0.05) in CC samples, with enriched pathways involved in mesenchyme development, Wnt-signaling, and Notch pathway regulation (q<0.05). Notably, SFRP2+ fibroblasts, a phenotype previously hypothesized to promote fibrosis in CCLE, were not only globally enriched in CC samples of both CCLE and DM but also spatially localized in areas of CC. Our findings suggest that various sources of persistent inflammation drive fibroblast-to-myofibroblast transition, promoting a pro-fibrotic and potentially pro-calcific microenvironment. This spatial transcriptomic approach provides novel mechanistic insights into CC pathogenesis and identifies potential therapeutic targets for this treatment-resistant condition. Aaron Bao¹, Saloni Patel¹, Jaroslaw Jedrych¹, Sewon Kang¹, Martin Alphonse¹, Jun Kang¹ 1. Johns Hopkins Medicine, Baltimore, MD, United States. Adaptive and Auto-Immunity