Novel Integration of Spatial and Single-Cell Omics Data Sets Enables Deeper Insights into IPF Pathogenesis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by repetitive alveolar injuries with excessive deposition of extracellular matrix (ECM) proteins. A crucial need in understanding IPF pathogenesis is identifying cell types associated with histopathological regions, particularly local fibrosis centers known as fibroblast foci. To address this, we integrated published spatial transcriptomics and single-cell RNA sequencing (scRNA-seq) transcriptomics and adopted the Query method and the Overlap method to determine cell type enrichments in histopathological regions. Distinct fibroblast cell types are highly associated with fibroblast foci, and transitional alveolar type 2 and aberrant KRT5-/KRT17+ (KRT: keratin) epithelial cells are associated with morphologically normal alveoli in human IPF lungs. Furthermore, we employed laser capture microdissection-directed mass spectrometry to profile proteins. By comparing with another published similar dataset, common differentially expressed proteins and enriched pathways related to ECM structure organization and collagen processing were identified in fibroblast foci. Importantly, cell type enrichment results from innovative spatial proteomics and scRNA-seq data integration accord with those from spatial transcriptomics and scRNA-seq data integration, supporting the capability and versatility of the entire approach. In summary, we integrated spatial multi-omics with scRNA-seq data to identify disease-associated cell types and potential targets for novel therapies in IPF intervention. The approach can be further applied to other disease areas characterized by spatial heterogeneity.

Keywords: Idiopathic pulmonary fibrosis; cell type; gene signature; laser capture microdissection; mass spectrometry; multi-omics integration; protein signature; scRNA-seq; spatial proteomics; spatial transcriptomics.

Figure 1

Cell type enrichments in distinct region types integrating GeoMx spatial transcriptomics and scRNA-seq transcriptomics in human IPF lungs by the Query method. (A) Workflow of the Query method. In this example, differential analysis extracted up-regulated fibroblast foci-specific genes (n = 50) from GeoMx spatial transcriptomics [17], and the sum expression of the whole gene set was queried as a z-score in PLIN2+ fibroblast cells from scRNA-seq transcriptomics [21]. (B) PCA plotting of gene expression pattern. (C) Venn graph of the up-regulated region-specific gene sets in five histopathological region types: 32 up-regulated region-specific differential genes for control alveoli, 27 for IPF blood vessel, 41 for IPF distant alveoli, 50 for IPF fibroblast foci and 106 for IPF immune infiltrate. (D) Enrichment z-score summary of 30 cell types in five histopathological region types from spatial transcriptomics. These 30 cell types are classified into five large types: Epithelium, Mesenchyme, Myeloid, Endothelium and Lymphoid. Abbreviations. ACTA2: Smooth muscle alpha-actin; ADAM12: Disintegrin and metalloproteinase domain-containing protein 12; BMP5: Bone morphogenetic protein 5; VCAN: Versican; PC: principal component; AT1: Alveoli type 1 epithelial cells; AT2: Alveoli type 2 epithelial cells; KRT5: Keratin 5; KRT17: Keratin 17; MUC5AC: Mucin 5AC; MUC5B: Mucin 5B; SCGB3A2: secretoglobin family 3A member 2; SCGB3A1: secretoglobin family 3A member 1; PLIN2+: perilipin 2; cDCs: Conventional dendritic cells; pDCs: Plasmacytoid dendritic cells; NK cells: Natural killer cells. Cell type annotations from all figures follow the same abbreviations.

Figure 2

Cell type enrichments in distinct region types integrating GeoMx spatial transcriptomics [17] and scRNA-seq transcriptomics [21] in human IPF lungs by the Overlap method. (A,B) Two representative workflow examples of the Overlap method. (A) Enrichment example: Differential analysis extracted up-regulated fibroblast foci-specific genes (n = 50) from GeoMx spatial transcriptomics and PLIN2+ fibroblast-specific genes (n = 576) from scRNA-seq transcriptomics and determined their overlap is larger than expected by random, indicative of enrichment. (B) Depletion example: Differential analysis extracted up-regulated fibroblast foci-specific genes (n = 50) from GeoMx spatial transcriptomics and mast cell-specific genes (n = 581) from scRNA-seq transcriptomics and determined their overlap is smaller than expected by random, indicative of depletion. (C) Enrichment p-value summary of 30 cell types in five histopathological region types from spatial transcriptomics. (D) Spearman’s correlations between cell type rankings in five histopathological defined region types from the Query method and the Overlap method. Abbreviations. ACTA2: Smooth muscle alpha-actin; ADAM12: Disintegrin and metalloproteinase domain-containing protein 12; BMP5: Bone morphogenetic protein 5; VCAN: Versican; ABCA9: ATP binding cassette subfamily A member 9; ABCF2: ATP binding cassette subfamily F member 2; ABL1: ABL proto-oncogene 1; ZNFX1: Zinc finger NFX1-type containing 1; ABCB8: ATP binding cassette subfamily B member 8; ABCC1: ATP binding cassette subfamily C member 1; ABCC4: ATP binding cassette subfamily C member 4; ZNRF1: Zinc and ring finger 1.

Figure 3

Cell type enrichments in distinct region types integrating LCM-directed LC–MS spatial proteomics and scRNA-seq transcriptomics in human IPF lungs by the Query method and the Overlap method. (A) Enrichment z-score summary of 30 cell types in four histopathological region types from spatial proteomics. (B) Enrichment p-value summary of 30 cell types in four histopathological region types from spatial proteomics. (C) Spearman’s correlations between cell type rankings in four histopathological region types from the Query method and the Overlap method.

Figure 4

Cell type enrichment comparisons by combining either GeoMx spatial transcriptomics or LCM-directed LC–MS spatial proteomics with scRNA-seq transcriptomics. (A) z-score comparisons by the Query method from spatial RNA/cellular RNA integration and spatial protein/cellular RNA integration in 30 cell types in three common region types: IPF fibroblast foci, IPF alveoli and control alveoli regions. (B) significance p-value comparisons by the Overlap method from spatial RNA/cellular RNA integration and spatial protein/cellular RNA integration in 30 cell types in three common region types: IPF fibroblast foci, IPF alveoli and control alveoli regions. Spearman’s correlations are calculated in each comparison.