Spatially informed phenotyping by cyclic-in-situ-hybridisation identifies novel fibroblast populations and their pathogenic niches in systemic sclerosis

Yi-Nan Li¹, Tim Filla², Andrea-Hermina Györfi³, Minrui Liang⁴, Veda Devakumar², Alexandru Micu², Hongtao Chai², Christina Bergmann⁵, Ann-Christin Pecher⁶, Jörg Henes⁶, Pia Moinzadeh⁷, Suzan Al-Gburi⁷, Thomas Krieg⁸, Alexander Kreuter⁹, Jiucun Wang¹⁰, Georg Schett⁵, Bernhard Homey¹¹, Sascha Dietrich¹², Jörg H W Distler¹³, Alexandru-Emil Matei¹⁴

Affiliations

¹ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany. Electronic address: yi-nan.li@med.uni-duesseldorf.de.
² Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
³ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, and Fraunhofer Cluster of Excellence for Immune Mediated Diseases CIMD, Frankfurt am Main, Germany.
⁴ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Department of Rheumatology, Huashan Hospital, Fudan University, Shanghai, P. R. China.
⁵ Department of Internal Medicine 3, Rheumatology and Clinical Immunology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and University Hospital Erlangen, Erlangen, Germany; Deutsches Zentrum Immuntherapie (DZI), FAU Erlangen-Nürnberg and University Hospital Erlangen, Erlangen, Germany.
⁶ Department of Internal Medicine II, Hematology, Oncology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, Tübingen, Germany.
⁷ Department of Dermatology, University Hospital Cologne, Cologne, Germany.
⁸ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Translational Matrix Biology, Faculty of Medicine, University Hospital Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 50931, Cologne, Germany.
⁹ Department of Dermatology, Venereology and Allergology, Helios St. Elisabeth Klinik Oberhausen, Duisburg, Germany.
¹⁰ Department of Rheumatology, Huashan Hospital, Fudan University, Shanghai, P. R. China; State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, P. R. China; Research Unit of Dissecting the Population Genetics and Developing New Technologies for Treatment and Prevention of Skin Phenotypes and Dermatological Diseases (2019RU058), Chinese Academy of Medical Sciences, Shanghai, P. R. China.
¹¹ Department of Dermatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
¹² Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
¹³ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, and Fraunhofer Cluster of Excellence for Immune Mediated Diseases CIMD, Frankfurt am Main, Germany. Electronic address: joerg.distler@med.uni-duesseldorf.de.
¹⁴ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, and Fraunhofer Cluster of Excellence for Immune Mediated Diseases CIMD, Frankfurt am Main, Germany. Electronic address: Alexandru-Emil.Matei@med.uni-duesseldorf.de.

PMID: 40603206
DOI: 10.1016/j.ard.2025.06.002

Free article

Spatially informed phenotyping by cyclic-in-situ-hybridisation identifies novel fibroblast populations and their pathogenic niches in systemic sclerosis

Yi-Nan Li et al. Ann Rheum Dis. 2025.

Free article

. 2025 Jul 1:S0003-4967(25)01042-8.

doi: 10.1016/j.ard.2025.06.002. Online ahead of print.

Authors

Affiliations

¹ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany. Electronic address: yi-nan.li@med.uni-duesseldorf.de.
² Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
³ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, and Fraunhofer Cluster of Excellence for Immune Mediated Diseases CIMD, Frankfurt am Main, Germany.
⁴ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Department of Rheumatology, Huashan Hospital, Fudan University, Shanghai, P. R. China.
⁵ Department of Internal Medicine 3, Rheumatology and Clinical Immunology, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and University Hospital Erlangen, Erlangen, Germany; Deutsches Zentrum Immuntherapie (DZI), FAU Erlangen-Nürnberg and University Hospital Erlangen, Erlangen, Germany.
⁶ Department of Internal Medicine II, Hematology, Oncology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, Tübingen, Germany.
⁷ Department of Dermatology, University Hospital Cologne, Cologne, Germany.
⁸ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; Translational Matrix Biology, Faculty of Medicine, University Hospital Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 50931, Cologne, Germany.
⁹ Department of Dermatology, Venereology and Allergology, Helios St. Elisabeth Klinik Oberhausen, Duisburg, Germany.
¹⁰ Department of Rheumatology, Huashan Hospital, Fudan University, Shanghai, P. R. China; State Key Laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, and Human Phenome Institute, Fudan University, Shanghai, P. R. China; Research Unit of Dissecting the Population Genetics and Developing New Technologies for Treatment and Prevention of Skin Phenotypes and Dermatological Diseases (2019RU058), Chinese Academy of Medical Sciences, Shanghai, P. R. China.
¹¹ Department of Dermatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
¹² Department of Hematology, Oncology and Clinical Immunology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany.
¹³ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, and Fraunhofer Cluster of Excellence for Immune Mediated Diseases CIMD, Frankfurt am Main, Germany. Electronic address: joerg.distler@med.uni-duesseldorf.de.
¹⁴ Department of Rheumatology, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Hiller Research Center, University Hospital Düsseldorf, Heinrich Heine University, Düsseldorf, Germany; Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, and Fraunhofer Cluster of Excellence for Immune Mediated Diseases CIMD, Frankfurt am Main, Germany. Electronic address: Alexandru-Emil.Matei@med.uni-duesseldorf.de.

PMID: 40603206
DOI: 10.1016/j.ard.2025.06.002

Abstract

Objectives: Spatially nonresolved transcriptomic data identified several functionally distinct populations of fibroblasts in health and disease. However, in-depth transcriptional profiling in situ at the single-cell resolution has not been possible so far. We thus aimed to profile these populations by single-cell spatial transcriptomics using cyclic in situ hybridisation (cISH).

Methods: We studied fibroblast subpopulations in the skin of systemic sclerosis (SSc) patients and heathy individuals using cISH as a novel approach for transcriptional phenotyping with subcellular resolution. Clustering was performed using Building Aggregates with a Neighbourhood Kernel and Spatial Yardstick (BANKSY) as a novel approach for spatially informed transcriptional phenotyping. The findings were further validated by integration with single-cell RNA sequencing in distinct SSc cohorts.

Results: BANKSY-based spatially informed clustering identified 9 fibroblast (FB) subpopulations, with SFRP2+ reticular dermis (RetD) FB and CCL19+ nonperivascular (nonPV) FBs as novel subpopulations that reside in specific cellular niches and display unique gene expression profiles. SFRP2+ RetD FBs and CCL19+ nonPV FBs as well as COL8A1+ FBs display altered frequencies in SSc skin and play specific, disease-promoting roles for extracellular matrix release and leukocyte recruitment as revealed by their transcriptional profile, their cellular interactions, and ligand-receptor analyses. The frequencies of COL8A1+ FBs and their interactions with monocytic cells and B cells are associated with the progression of skin fibrosis in SSc.

Conclusions: Our cISH-based, spatially resolved transcriptomic approach identified novel fibroblast subpopulations deregulated in SSc skin with specific pathogenic roles. COL8A1+ FBs and their immune interactions may also have potential as biomarkers for future progression of skin fibrosis.

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Conflict of interest statement

Competing interests AHG received lecture fees from Boehringer Ingelheim. JHWD has consultancy relationships with Active Biotech, Anamar, ARXX, AstraZeneca, Bayer Pharma, Boehringer Ingelheim, Callidatas, Celgene, Galapagos, GSK, Inventiva, Janssen, Kyverna, Novartis, Pfizer, Quell Therapeutics and UCB; has received research funding from Anamar, ARXX, BMS, Bayer Pharma, Boehringer Ingelheim, Cantargia, Celgene, CSL Behring, Exo Therapeutics, Galapagos, GSK, Incyte, Inventiva, Kiniksa, Kyverna, Lassen Therapeutics, Mestag, Sanofi-Aventis, RedX, UCB and ZenasBio; and is CEO of 4D Science and scientific lead of FibroCure.

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Spatially informed phenotyping by cyclic-in-situ-hybridisation identifies novel fibroblast populations and their pathogenic niches in systemic sclerosis

Affiliations

Spatially informed phenotyping by cyclic-in-situ-hybridisation identifies novel fibroblast populations and their pathogenic niches in systemic sclerosis

Authors

Affiliations

Abstract

Conflict of interest statement

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