bioRxiv : the preprint server for biology · 2024
Single-cell spatial transcriptomics of fixed, paraffin-embedded biopsies reveals colitis-associated cell networks.
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Mennillo Elvira, Lotstein Madison L, Lee Gyehyun, Johri Vrinda, Ekstrand Christina, Tsui Jessica, Hou Julian, Leet Donna E, He Jun Yan, Mahadevan Uma, Eckalbar Walter, Oh David Y, Fragiadakis Gabriela K, Kattah Michael G, Combes Alexis J
Аннотация
BACKGROUND & AIMS: Imaging-based, single-cell spatial transcriptomics (iSCST) using formalin-fixed, paraffin-embedded (FFPE) tissue could transform translational research by retaining all tissue cell subsets and spatial locations while enabling the analysis of archived specimens. We aimed to develop a robust framework for applying iSCST to archived clinical FFPE mucosal biopsies from patients with inflammatory bowel disease (IBD).
METHODS: We performed a comprehensive benchmarking comparison of three iSCST platforms capable of analyzing FFPE specimens. We analyzed FFPE mucosal biopsies (n=57) up to 5 years old from non-IBD controls (HC; n=9) and patients with ulcerative colitis (UC;n=11). After platform-specific cell segmentation, we applied a uniform data processing pipeline to all datasets, including transcript detection, cell annotation, differential gene expression, and neighborhood enrichment. Transcriptomic signatures identified with iSCST were validated using external, publicly available bulk transcriptomic datasets.
RESULTS: A custom 290-plex Xenium gene panel exhibited the highest sensitivity and specificity for transcript detection, enabling precise identification and quantification of diverse cell subsets and differentially expressed genes across cell types and disease states. We mapped transcriptionally distinct fibroblast subsets to discrete spatial locations and identified inflammation-associated fibroblasts (IAFs) and monocytes as a colitis-associated cellular neighborhood. We also identified signatures associated with Vedolizumab (VDZ) responsiveness. VDZ non-responders were characterized by an IAF-monocyte transcriptional signature, while responders exhibited enrichment of epithelial gene sets.
CONCLUSIONS: Our optimized iSCST framework for archived FFPE biopsies provides unique advantages for assessing the role of colitis-associated cellular networks in routinely collected clinical samples. FFPE-based biomarkers could integrate with existing clinical workflows and potentially aid in risk-stratifying patients.
METHODS: We performed a comprehensive benchmarking comparison of three iSCST platforms capable of analyzing FFPE specimens. We analyzed FFPE mucosal biopsies (n=57) up to 5 years old from non-IBD controls (HC; n=9) and patients with ulcerative colitis (UC;n=11). After platform-specific cell segmentation, we applied a uniform data processing pipeline to all datasets, including transcript detection, cell annotation, differential gene expression, and neighborhood enrichment. Transcriptomic signatures identified with iSCST were validated using external, publicly available bulk transcriptomic datasets.
RESULTS: A custom 290-plex Xenium gene panel exhibited the highest sensitivity and specificity for transcript detection, enabling precise identification and quantification of diverse cell subsets and differentially expressed genes across cell types and disease states. We mapped transcriptionally distinct fibroblast subsets to discrete spatial locations and identified inflammation-associated fibroblasts (IAFs) and monocytes as a colitis-associated cellular neighborhood. We also identified signatures associated with Vedolizumab (VDZ) responsiveness. VDZ non-responders were characterized by an IAF-monocyte transcriptional signature, while responders exhibited enrichment of epithelial gene sets.
CONCLUSIONS: Our optimized iSCST framework for archived FFPE biopsies provides unique advantages for assessing the role of colitis-associated cellular networks in routinely collected clinical samples. FFPE-based biomarkers could integrate with existing clinical workflows and potentially aid in risk-stratifying patients.