1867: Deciphering the Molecular Landscape of the Small Intestine in Early and Late SSc Using High-Definition Spatial Transcriptomics

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.