Single cell RNA-seq reveals profound transcriptional similarity between Barrett's oesophagus and oesophageal submucosal glands

Abstract

Barrett's oesophagus is a precursor of oesophageal adenocarcinoma. In this common condition, squamous epithelium in the oesophagus is replaced by columnar epithelium in response to acid reflux. Barrett's oesophagus is highly heterogeneous and its relationships to normal tissues are unclear. Here we investigate the cellular complexity of Barrett's oesophagus and the upper gastrointestinal tract using RNA-sequencing of single cells from multiple biopsies from six patients with Barrett's oesophagus and two patients without oesophageal pathology. We find that cell populations in Barrett's oesophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with oesophageal submucosal gland cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barrett's oesophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice.

Fig. 1

Single cell RNA sequencing identifies cell groups in normal upper gastrointestinal epithelia. a Endoscopic sampling sites (yellow, oesophagus; green, gastric cardia; purple, duodenum; orange, Barrett’s oesophagus) with summary of how tissues from patients were used. Two to four biopsies were taken at each site. Patients without BO were sampled from the lower oesophagus 20 mm proximal to the squamous-columnar junction. b From bulk RNA-seq data derived from samples from 13 patients with BO, heatmap of genes differentially expressed between any tissue type (analysis of variance-like test, false discovery rate (FDR) <1 × 10⁻²²) with tissue hierarchy determined by nearest neighbour. Tissue indicated by colours as in a. One duodenal sample from patient Q failed to produce usable data and was excluded. c From bulk RNA-seq data, heatmap of expression of mucin and trefoil factor genes with tissue hierarchy determined by nearest neighbour, in samples from 13 patients with BO. d Upper panels show the cluster consensus matrices for single cells from normal tissue sites in four BO patients. Blue-to-red colours denote the frequency with which cells are grouped together in 250 repeat clusterings of simulated technical replicates (see Methods). Cell clusters are indicated by coloured bars below the matrices. In lower panels, heatmaps show expression of known functionally relevant genes that were differentially expressed between cell clusters (>4 fold change, FDR <1 x 10^-5). e Haematoxylin and eosin staining of normal oesophagus taken from the proximal part of an oesophagectomy specimen resected for Siewert type III junctional tumour in a patient with no BO, showing OSGs (red arrow), OSG ducts (black arrow), and squamous epithelium (marked with dotted black line). Scale bar, 500 µm. f Immunohistochemical staining of KRT14, TFF3 and KRT7 (left, middle and right images, respectively) in adjacent sections from the same specimen as e, showing OSG ducts (black arrows) and OSGs (red arrows) and squamous epithelium (marked with dotted black line). Scale bar, 500 µm. OSG oesophageal submucosal gland

Fig. 2

LEFTY1 and OLFM4 are mainly expressed in Barrett’s oesophagus cells that do not express differentiated secretory cell markers. a Upper panel, cluster consensus matrix of BO cells from 4 BO patients (n = 371 cells). Blue-to-red colours denote the frequency with which cells are grouped together in 250 repeat clusterings of simulated technical replicates (see Methods). Clusters (B1-B4) are indicated by the coloured bars below. Lower panel, heatmaps showing expression of selected functionally relevant genes that are differentially expressed between cell clusters (>4 fold change, FDR <1e-5). b Immunohistochemical staining of MUC2, LEFTY1 and CHGA in sections derived from the same BO resection specimen. Black arrows indicate goblet cells on all sections (positively stained for MUC2; negative for LEFTY1 and CHGA). Scale bars are 50 µm. c Immunohistochemical staining of LEFTY1 in an OSG from a normal squamous endoscopic biopsy obtained from a patient with BO. Scale bars are 300 µm and 50 µm in enlarged image

Fig. 3

The majority of Barrett’s oesophagus cells have a similar transcript profile to oesophageal submucosal gland (OSG) cells. a t-Distributed Stochastic Neighbour Embedding (t-SNE) plots of cells from all samples from four BO patients (n = 1107 including brain control), showing similarity of cells in two dimensions, coloured by tissue type (yellow, oesophagus; green, gastric cardia; purple, duodenum; orange, Barrett’s oesophagus; pink, brain). Brain was used as a control. b t-SNE plot of cells from four BO patient samples (A–D), as in a, coloured by how cells contribute to clusters generated by SC3 analysis with 250 repeat clusterings of simulated technical replicates (see Methods). Names given to the clusters are based on expression of known marker genes (see text and Supplementary Fig. 3). c Sankey diagram showing how each tissue type sampled contributes to the clusters shown in b. Colours and labels on the left indicate sampled tissue (as in a); colours and labels on the right indicate cluster (as in b). d Mean BEARscc score for each grouping of ‘gland-like’ cells (n = 372), which are a sub-set of gastric (G, n = 175), BO (n = 78) and OSG cells (n = 119): excluding gastric and BO cells that expressed CHGA or MUC2 (to exclude enteroendocrine and goblet cells, respectively) and excluding oesophageal cells that did not express TFF3 (to exclude squamous cells). ‘Ensemble’ refers to all cells grouped together. Thresholds were set at the tenth centile of cells in which at least one transcript was detected from each gene of interest

Fig. 4

SPINK4 and ITLN1 mark early goblet cells. a Volcano plot showing fold change and P value of genes differentially expressed in the ‘goblet-type’ cell cluster as compared to all other cell clusters (see Fig. 3). Points coloured red indicate genes significant at 5% permutation test. Selected highly significant genes are labelled. b Bar chart showing the percentage of cells in the ‘goblet-type’ cell cluster (n = 98) expressing MUC2, ITLN1 or SPINK4 alone or in different combinations (thresholds set at the tenth centile to include 90% of cells in which at least one transcript was detected from each gene). c Triple immunofluorescence staining images of MUC2 (red), ITLN1 (white) and SPINK4 (green) in normal colon from a resection specimen (blue stain is DAPI). Scale bar, 100 µm. d Triple immunofluorescence staining images of MUC2 (red), ITLN1 (white) and SPINK4 (green) in normal oesophageal epithelium obtained by endoscopic biopsy (blue stain is DAPI). OSGs encroaching on the surface epithelium are shown in the enlarged images on the right. Scale bars are 200 µm and 50 µm in enlarged images. e Triple immunofluorescence staining images of KRT14 (white), KRT7 (green) and MUC2 (red) in an OSG beneath normal squamous epithelium from an endoscopic biopsy of normal squamous epithelium from a patient with BO biopsy (blue stain is DAPI). Scale bar 50 µm. f Representative immunofluorescence staining of Barrett’s EMR specimen containing intestinal metaplasia but no dysplasia for MUC2 (red), ITLN1 (white) and SPINK4 (green); nuclei (DAPI) in blue. Scale bars are 400 µm and 100 µm in enlarged images

Fig. 5

OLFM4 is upregulated in BO and OSG cells with stem-like transcript profiles. a Bar plot on left shows StemID scores across all RaceID2 clusters (see Methods) applied to all non-squamous oesophageal cells (BO and oesophageal cells with <5 KRT14 counts to exclude squamous cells, n = 533). Scores are calculated from multiplication of the entropy (spread from the cluster mean) and the number of cluster links arising from a given cluster. Differentially expressed genes in the highest scoring cluster (C3, blue asterisk) and second highest scoring cluster (C7, red asterisk) are shown in the volcano plots in the centre and right plots, respectively. Points coloured red indicate the most significant genes with a fold change >2. Selected highly significant genes are labelled. b Immunohistochemical staining of OLFM4 in human colon (close-up of base of crypt inset). Scale bars are 100 µm and 20 µm in inset. c Immunohistochemical staining of OLFM4 in BO mucosal resection containing intestinal metaplasia but no dysplasia, with enlarged image. Scale bars are 1000 µm, 200 µm in enlarged image and 50 µm in inset. d Immunohistochemical staining of OLFM4 in OSG under normal oesophagus taken from the proximal part of an oesophagectomy specimen resected for Siewert type III junctional tumour in a patient with no BO. Red dashed area and arrow indicates OSG, black arrow indicates OSG duct. Scale bars are 300 µm and 20 µm in enlarged image. e Immunohistochemistry in OSGs from endoscopic biopsy of normal squamous oesophagus in patients with BO. Scale bars are 300 µm and 50 µm in enlarged image