Background/Purpose: SSc frequently results in serious gastrointestinal (GI) complications that contribute to increased morbidity and mortality. Understanding the molecular changes across different disease stages is crucial for elucidating the pathophysiology. Our study employs high-definition spatial transcriptomics to decipher the molecular and cellular landscape of the GI, assessing distinct alterations in the lamina propria associated with early and late disease stages.

Methods: We studied gastroduodenal biopsies from SSc patients enrolled in the ReSScue trial (n=10, 2013 ACR/EULAR classification criteria). Patients presented moderate to severe lower GI symptoms assessed by the UCLA GIT score. Formalin-fixed paraffin-embedded (FFPE) samples were processed for spatial transcriptomics using the Visium HD Spatial Gene Expression Reagent kit v1. Libraries were sequenced on the Illumina NovaSeq X platform and alignment was performed with Space Ranger 3.1.3. The data was analyzed using the packages Seurat v5 and Banksy for cell typing. Speckle was used for compositional analysis between SSc patients with early (symptom onset < 3.5 years) and late stage disease (symptom onset > 13.5 years). Differentially expressed genes (DEG) were identified between early and late patients using MAST (adjusted p-value < 0.05, |log2 FC| ≥ 0.2) and the clusterProfiler package was used for pathway over-representation analysis (ORA) and gene set enrichment analysis (GSEA) with the Gene Ontology (GO) database.

Results: We included 5 SSc patients with early and 5 with late stage, with mean disease durations of 1.9 years and 18.6 years, respectively. The two groups did not differ in other clinical parameters (Fig. 1). Six tissue domains were annotated: Epithelium (villi), crypts, lamina propria, Brunner’s glands, muscularis mucosae and isolated lymphoid follicles (ILF; Fig. 2A-B). Compositional analysis between early and late disease showed no significant differences in the tissue regions (Fig. 2C). DEG of the lamina propria revealed 61 significantly upregulated genes and 2,355 significantly downregulated genes in early compared to late disease (Fig. 3A). ORA showed upregulated pathways related to pathogen defense (GBP3, GBP5, LYZ, CHGA) in early compared to late disease (Fig. 3B). In contrast, several pathways associated with extracellular matrix (ECM) organization, including ADAM19 and various collagen types, were significantly downregulated in early compared to late disease (Fig. 3C). Also, both ORA and GSEA showed downregulation in early SSc of immune response pathways, including JAK-STAT signaling and interferon responses (e.g., JAK1, STAT2, IRF8, IRF4), and pathways related to actin cytoskeleton organization and angiogenesis (e.g. CAPZA2, GSN, VEGFA, VEGFB; Fig. 3C).

Conclusion: This is a first report investigating the molecular landscape in GI tissue of SSc patients using high-definition spatial transcriptomics across different disease stages. Our findings could allude to the importance of exogenous agents in early disease stages, with an upregulation of pathogen defense pathways in early SSc. In addition, our results show an upregulation of fibrosis pathways and ongoing inflammatory processes in late SSc.

Fig. 1: Characteristics and clinical data for SSc patients with early and late disease.

mRSS = modified Rodnan skin score, ILD = Interstitial lung disease. * symptoms assessed by the UCLA GIT score.

Fig. 2: Spatial transcriptomic analysis reveals 6 distinct tissue regions in the duodenum of SSc patients. A) UMAP of all 10 samples (908,423 8×8µm bins) and representative spatial plots of 1 sample show 6 distinct tissue regions. B) A dotplot of the 6 identified tissue regions shows the top 5 expressed marker genes in each cluster, which were used for manual annotation. C) Compositional analysis shows no significant differences in the proportions of the tissue regions between early and late SSc patients.

Fig. 3: DEG of the lamina propria show downregulated genes and pathways related to immune response and ECM in early compared to late patients. A) Volcano plot of 61 significantly upregulated and 2,355 significantly downregulated genes in early compared to late SSc, highlighting genes related to the pathways identified by GO. Upregulated genes are marked in yellow. B) ORA with the GO database shows upregulated pathways related to pathogen defense in early SSc (red boxes). C) ORA with the GO database shows downregulated pathways related to immune response (red boxes), ECM organization (green boxes) and actin cytoskeleton organization (blue boxes) in early compared to late SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deciphering-the-molecular-landscape-of-the-small-intestine-in-early-and-late-ssc-using-high-definition-spatial-transcriptomics/