Spatial transcriptomics identifies enriched gene expression and cell types in human liver fibrosis

Abstract

Liver fibrosis and cirrhosis have limited therapeutic options and represent a serious unmet patient need. Recent use of single-cell RNA sequencing (scRNAseq) has identified enriched cell types infiltrating cirrhotic livers but without defining the microanatomical location of these lineages thoroughly. To assess whether fibrotic liver regions specifically harbor enriched cell types, we explored whether whole-tissue spatial transcriptomics combined with scRNAseq and gene deconvolution analysis could be used to localize cell types in cirrhotic explants of patients with end-stage liver disease (total n = 8; primary sclerosing cholangitis, n = 4; primary biliary cholangitis, n = 2, alcohol-related liver disease, n = 2). Spatial transcriptomics clearly identified tissue areas of distinct gene expression that strongly correlated with the total area (Spearman r = 0.97, p = 0.0004) and precise location (parenchyma, 87.9% mean congruency; range, 73.1%-97.1%; fibrosis, 68.5% mean congruency; range, 41.0%-91.7%) of liver regions classified as parenchymal or fibrotic by conventional histology. Deconvolution and enumeration of parenchymal and fibrotic gene content as measured by spatial transcriptomics into distinct cell states revealed significantly higher frequencies of ACTA2+ FABP4+ and COL3A1+ mesenchymal cells, IL17RA+ S100A8+ and FCER1G+ tissue monocytes, VCAM1+ SDC3+ Kupffer cells, CCL4+ CCL5+ KLRB1+ and GZMA+ IL17RA+ T cells and HLA-DR+, CD37+ CXCR4+ and IGHM+ IGHG+ B cells in fibrotic liver regions compared with parenchymal areas of cirrhotic explants. Conclusion: Our findings indicate that spatial transcriptomes of parenchymal and fibrotic liver regions express unique gene content within cirrhotic liver and demonstrate proof of concept that spatial transcriptomes combined with additional RNA sequencing methodologies can refine the localization of gene content and cell lineages in the search for antifibrotic targets.

FIGURE 1

Classification of parenchymal and fibrotic regions by spatial transcriptomics. (A) Overview of spatial transcriptomics pipeline. Snap‐frozen liver tissue was sectioned, stained, and permeabilized on RNA capture slides. Whole‐tissue transcriptomes were tagged with region barcodes, amplified, sequenced, aligned to the human genome (GRCh38), and analyzed by graph‐based clustering. (B) Classification of parenchymal and fibrotic liver regions (i.e., RNA capture spots) by conventional histology or spatial transcriptomics. Unassigned regions represent areas of low RNA counts as measured by spatial transcriptomics. Hematoxylin and eosin (HE) staining for assessment by conventional histological shown for reference. (C) Spearman correlation (r) of parenchymal and fibrotic classification using spatial transcriptomics and histology (n = 8 liver samples). (D) Percentage of classification congruency between identical RNA capture spots defined as parenchymal or fibrotic by spatial transcriptomics and histology (n = 8 livers). (E) Differentially expressed gene analysis of parenchymal and fibrotic liver regions for each explant sample. Significantly up‐regulated genes are shown (p < 0.05) and grouped into biological processes defined by STRING protein–protein pathway analyses.

FIGURE 2

Spatial transcriptomics reveals enriched cell types in liver fibrosis. (A) Transcriptional profiles of 11 major liver cell types generated by CIBERSORTx analysis of single‐cell RNA sequencing (scRNAseq; n = 10,000 cells). Rows denote cell types; columns denote genes; exemplar lineage markers shown. (B) Fractions of cell types in parenchymal and fibrotic regions as calculated by CIBERSORTx analysis of spatial transcriptomes. (C) Location of cell types detected at 2.5‐fold or greater frequencies by CIBERSORTx gene deconvolution of spatial transcriptomes. Hematoxylin and eosin (HE) staining is shown as comparison reference for parenchymal (blue) and fibrotic (red) liver regions classified by spatial transcriptomics (top row). Indicated cell types detected in parenchyma using shown marker genes appear green, and cell types detected in fibrotic regions appear yellow (bottom row). Absence of indicated cell types in parenchyma appear blue; fibrosis regions without indicated cell types appear red. (D) Percentages of each indicated cell type using shown marker genes in parenchymal (P) and fibrotic (F) regions of cirrhotic human liver samples. (E) Immunofluorescence staining of liver sections from the same tissue blocks used for spatial transcriptomics. Hepatocytes are marked by nicotinamide N‐methyltransferase (NNMT; yellow), mesenchyme by collagen type III alpha 1 chain (COL3A1; magenta), monocytes by complement C1q C chain (C1QC; yellow), T cells by CD3D (green), and B cells by immunoglobulin heavy constant mu (IGHM; red). Nuclei stained by DAPI (blue). Scale bars, 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3

Mesenchymal cell states localize to fibrotic liver septa. (A) Transcriptional profiles of mesenchymal cell types generated by CIBERSORTx analysis of scRNAseq. Rows denote cell types; columns denote genes; exemplar lineage markers are shown. (B) Fractions of mesenchymal lineages in parenchymal and fibrotic regions as calculated by CIBERSORTx analysis of spatial transcriptomes (n = 8 livers). (C) Hematoxylin and eosin (HE) staining shown as comparison reference for parenchymal (blue) and fibrotic (red) liver regions classified by spatial transcriptomics (left). Detection of hepatic stellate cells (HSC) and scar‐associated mesenchymal cell (SAMes) subtypes in parenchyma using shown marker genes appear green, and in fibrotic regions appear yellow (right). Parenchyma without HSC or SAMes cell types appear blue; fibrotic regions without HSC or SAMes appear red. (D) Immunofluorescence staining of primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), and alcohol‐related liver disease (ALD) liver sections show expression of fatty acid–binding protein 4 (FABP4; magenta) on HSC (actin alpha 2, smooth muscle [ACTA2]; green). Nuclei stained by 4′,6‐diamidino‐2‐phenylindole (DAPI; blue). ****p < 0.0001.

FIGURE 4

Distinct monocyte states localize to regions of liver fibrosis. (A) Transcriptional profiles of monocytes generated by CIBERSORTx analysis of scRNAseq. Rows denote cell types; columns denote genes; exemplar lineage markers are shown. (B) Fractions of monocyte states in parenchymal and fibrotic regions calculated by CIBERSORTx analysis of spatial transcriptomes (n = 8 livers). (C) Hematoxylin and eosin (HE) staining shown as comparison reference for parenchymal (blue) and fibrotic (red) liver regions classified by spatial transcriptomics (left). Detection of TM‐1, TM‐3, and KC‐1 mesenchymal subtypes in parenchyma using shown marker genes appear green and in fibrotic regions appear yellow (right). Parenchyma without TM‐1, TM‐3, and KC‐1 cell types appear blue; fibrotic regions without TM‐1, TM‐3, and KC‐1 appear red. (D) Immunofluorescence staining of PSC, PBC, and ALD liver sections shows co‐expression of vascular cell adhesion molecule 1 (VCAM1; magenta) on CD68+ monocytes (yellow). Nuclei stained by DAPI (blue). *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Subsets of T cells and B cells are enriched in regions of liver fibrosis. Transcriptional profiles of T cells (A) and B cells (D) generated by CIBERSORTx analysis of scRNAseq. Rows denote cell types; columns denote genes; exemplar lineage markers are shown. Fractions of T cell (B) and B cell states (E) in parenchymal and fibrotic regions calculated by CIBERSORTx analysis of spatial transcriptomes (n = 8 livers). T cells‐3, T cells‐4, and T cells‐5 (C) and B cells‐1, B cells‐2, and plasma‐cells‐2 (F) are detected using shown marker genes. Hematoxylin and eosin (HE) staining shown as comparison reference for parenchymal (blue) and fibrotic (red) liver regions classified by spatial transcriptomics (left). Detection of indicated cell subtypes in parenchyma using shown marker genes appear green and in fibrotic regions appear yellow (right). Parenchyma devoid of indicated cell types appear blue; fibrotic regions without indicated cell subtypes appear red. *p < 0.05, **p < 0.01, ****p < 0.0001.