Single-Cell RNA Sequencing Unifies Developmental Programs of Esophageal and Gastric Intestinal Metaplasia

Barrett's esophagus and gastric intestinal metaplasia are molecularly similar precancerous lesions, with epithelial cells being mosaics of gastric and intestinal phenotypes and stromal cells showing features of cancer-associated fibroblasts.


Figure S16: Esophageal IM (E-GM and BE-IM) and Gastric IM (NAG, CAG, GIM, CIM) cell do not share features with squamous esophagus, SMG nor Colonic cell types.
UMAP project of columnar cell types from esophageal IM (E-GM and BE-IM) (A, C, E) or gastric IM (NAG, CAG, GIM, CIM (B, D, F) (same coordinates as figure 3A) with MuSiC derived contribution of squamous esophageal (A, B), SGM (C, D) or Colonic (E, F) phenotypes highlighted.

Figure S17: Gastric cancer cells do not share features with squamous esophagus, SMG nor Colonic cell types.
A) UMAP of all cells from this study with cells obtained from normal gastric samples adjacent to gastric cancer and gastric cancer cells overlaid (Kumar et al. 2022) B) UMAP of normal gastric samples adjacent to gastric cancer and gastric cancer cells overlaid (Kumar et al. 2022). Only cells from Kumar et al 2022 are presented. C) UMAP of all cells from this study with cells obtained from normal gastric samples adjacent to gastric cancer and gastric cancer cells overlaid with global tissue type of cells indicated D) Violin plot of MuSiC-derived esophageal (squamous), SMG, Transitional, Gastric, Intestinal and Colonic phenotype contribution to columnar cells from normal cells adjacent to cancer or columnar cells from gastric cancer samples. Cell from teach tissue type were split into Cancer like cells (brown cells in C) or columnar (non-cancer) like cells (green in C).

Figure S18: Monocle3 pseudotime analysis places esophageal and gastric intestinal metaplasia cells and gastric cancer cells between normal gastric and intestinal cells.
A) UMAP of columnar cell types obtained using monocle3 analysis. Only cells from normal tissue types are visualized and tissue types are indicated. B) UMAP of columnar cell types obtained using monocle3 analysis. Only cells from normal tissue types are visualized and cell types are indicated. C) UMAP of columnar cell types obtained using monocle3 analysis. Only cells from metaplastic and cancer tissue types are visualized and tissue types are indicated. Pseudotime trajectory was anchored at the gastric mucous-neck cells, most likely stem-like cells of normal gastric epithelium G) Violin plot of monocle3 pseudotime trajectory with cells split into individual tissue types and segregated into healthy and esophageal or stomach IM groups. NGC samples were collected from healthy stomach or from endoscopically normal gastric tissue adjacent to BE or Gastric IM (hence it listed 3 time, depending on patient status).

Figure S19: Reclustering of columnar cells identifies a new subtype of Neck-like cells.
A) UMAP of all columnar cell types (coordinates as in Fig. 3A) with clusters identified during the reclustering highlighted. B) Stack bar chart of contribution of neck-like cells that overlap cluster 6 and 16 to individual tissue types. For tissue that patient status could be identified, the tissue was split into healthy and disease states. NGB sample were adjacent to gastric cancer samples.

Figure S20: Genetic changes detected in the esophagus with gastric metaplasia
A) Heatmap of copy number alterations identified in the esophagus with gastric metaplasia (E-GM) or normal gastric cardia (NG). B) Histograms of the distribution of variant allele frequency (VAF) in the esophagus with gastric metaplasia (GM) or normal gastric cardia (NG).
To retain privacy, individual samples are named after their library preparation barcodes.      figure S1) with monocle3 pseudotime overlay. The trajectories were fixed at the extreme of S1 cell population (for S1-S2 trajectory) and at the normal gastric terminus of S3 trajectory. E) Heatmap of normalized expression values for gene modules identified in the S3 trajectory. The cells are ordered by pseudotime value from D. F) Heatmap of normalized expression values for gene modules identified in the S1-S2 trajectory. The cells are ordered by pseudotime value from D. Figure S26: Immunofluorescent staining of S1 (CD34+CD31-), S2 (POSTN+aSMA-) and S3 (aSMA+) fibroblasts.
NE, NGC and NGB samples for immunofluorescence were from disease-free organ donors. NGC was defined as the immediate 3 mm long mucosa area adjacent to the Z-line; NGB was defined as the gastric mucosa that 3 to 5 cm away from the Z-line. NE -normal esophagus, NGC -normal gastric cardia, NGBnormal gastric body, E-GM -esophagus with gastric metaplasia, BE-IM -Barrett's esophagus with intestinal metaplasia, GIM -gastric intestinal metaplasia Figure S27: Proportion of each subtype in total S1/S2/S3 fibroblasts derived from immunofluorescent staining of E-GM, BE-IM and GIM.