Single-cell spatial transcriptomics of formalin-fixed, paraffin-embedded biopsies reveals colitis-associated cell networks.

Imaging-based, single-cell, spatial transcriptomics (iSCST) of FFPE tissue enables comprehensive analysis of archived specimens while preserving spatial context, critical to an understanding of ulcerative colitis (UC) pathology. Here, we deployed a robust framework for applying iSCST to clinical FFPE mucosal biopsies from patients with UC or immune checkpoint inhibitor-induced colitis, as well as patients serving as healthy controls. iSCST using custom Xenium gene panels enabled precise detection of diverse cell subsets and disease-specific genes. We mapped transcriptionally distinct fibroblast subsets within mucosal niches, including inflammation-associated fibroblasts (IAFs), and identified colitis-specific neighborhoods formed by IAFs, monocytes, and neutrophils. Transcriptional signatures and spatial neighborhoods uncovered through iSCST were associated with vedolizumab (VDZ) response, with nonresponders exhibiting either an innate IAF-monocyte-neutrophil signature or adaptive gut-associated lymphoid tissue signature, while responders showed enrichment of an epithelial cellular neighborhood. These signatures were validated in an internal and an external dataset, supporting the existence of 2 distinct archetypes of treatment resistance to VDZ in UC. This iSCST framework provides a powerful approach for analyzing FFPE tissues, offering insights into colitis-associated cellular networks and identifying biomarkers to enhance patient risk stratification in routine clinical workflows.