1. Volume 27, Issue 6, Pages 1934-1947.e5 (May 2019)
Dissecting the Single-Cell Transcriptome Network Underlying Gastric Premalignant Lesions and Early Gastric Cancer 
Peng Zhang, Mingran Yang, Yiding Zhang, Shuai Xiao, Xinxing Lai, Aidi Tan, Shiyu Du, Shao Li 
Cell Reports 
Volume 27, Issue 6, Pages e5 (May 2019)
DOI: /j.celrep
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2. Cell Reports 2019 27, 1934-1947.e5DOI: (10.1016/j.celrep.2019.04.052)
Copyright © 2019 The Author(s) Terms and Conditions

3. Figure 1
A Single-Cell Atlas of Gastric Antral Premalignant and Early-Malignant Mucosae
(A) Schematic diagram highlighting the experimental workflow for the whole study. Thirteen mucosa biopsies were collected from nine patients with wild superficial gastritis (non-atrophic gastritis [NAG], three biopsies) that were viewed as normal controls, chronic atrophic gastritis (CAG, 3 biopsies), intestinal metaplasia (IM, 6 biopsies) and early gastric cancer (EGC, 1 biopsy). The transcriptome of single cells was sequenced using the 10x Chromium system. See also Figures S1, S2, and S3 and Tables S1 and S2.
(B) The t-SNE plot of 32,332 high-quality cells to visualize cell-type clusters based on the expression of known marker genes. EC, endothelial cell; GMC, antral basal gland mucous cell; MSC, metaplastic stem-like cell; PC, proliferative cell; PMC, pit mucous cell; SM cell, smooth muscle cell. See also Figure S4 and Table S3.
(C) Cell-type markers. The relative expression level of genes across cells is shown, sorted by cell type. Cell-type marker genes were identified in an unbiased fashion (Wilcoxon rank-sum test, FDR <0.01, and fold change >1.5) and only the top three are shown in the figure. See also Figures S5, S6, S7, S8, and S9 and Data S1.
(D) Dot plot of representative genes in the NF-κB signaling pathway and cytokines mapped onto cell types.
(E) The t-SNE plot of both epithelial and stromal cells (color coded, right) from H. pylori-infected IM samples (IMW1, IMS1, and IMS2) and uninfected IM samples (IMW2, IMS3, and IMS4).
(F) The expression distribution for H. pylori infection-associated genes in specific cell types (red) and all other cells (gray). See also Figure S10.
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4. Figure 2
The Single-Cell Transcriptomes of Epithelial Cells in the Cascade from Gastritis to EGC
(A) t-SNE plot of 24,223 epithelial cells from the gastric mucosae by stages (NAG, 3,306 cells; CAG, 10,966 cells, IM, 7,815 cells; EGC, 2,136 cells) and cell types (right).
(B) The proportion of epithelial cell types across multiple lesions. The cell types were annotated based on Figure 1B. See also Figure S11.
(C) Violin plots display the distribution of expression of TFF1, TFF2, and TFF3 across diverse epithelial cell types.
(D) Heatmap showing the expression patterns of multiple epithelial cell types in premalignant gastric mucosae and EGC. Genes involved in the heatmap were significantly upregulated genes (FDR <0.01, fold change >1.5, Wilcoxon rank-sum test) for each cell type in each lesion. Pathways that mostly enriched for the gene signature in each lesion are denoted at right. See also Data S1.
(E) The single-cell transcriptome network underlying gastric premalignant and early-malignant lesions. Top (single-cell network): the nodes represent the main epithelial cell types (>1% in our data) present in the mucosae of each lesion, and the thickness of edges in the network denotes the Pearson correlation coefficient between the centroids of any pair of cell types. Center (molecular network markers): the putative network markers (FDR <0.01 and fold change >1.5) for representative cell types of each lesion are shown, where nodes with blue cycles represent high-risk genes inferred by CIPHER for gastritis or GC. Bottom: the dynamic changes for the overall molecular hallmarks implicated for each lesion.
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5. Figure 3
The scRNA Profiles for Gastric Mucous-Secreting Cell Lineages across Different Lesions
(A–C) The t-SNE plot that showed the distribution of gastric mucous-secreting cell lineages (red, n = 14,481) in the atlas (A), marked by the expression of marker genes MUC5AC (B) and MUC6 (C), respectively (red, high; gray, low).
(D) Heatmap showing the expression of selected functionally relevant genes that differentially expressed between the two subcluster types of gastric mucous-secreting cell lineages, GMC and PMC (fold change >1.5, FDR <0.01). See also Data S1.
(E) The most enriched pathways for upregulated genes for PMCs from samples in CAG (top), IM (center), and EGC lesion (bottom), respectively.
(F) The t-SNE plot showing GMCs across diverse lesions.
(G) Volcano plot showing fold change and the adjusted p value of genes differentially expressed between two goblet cell subclusters (FDR <0.01). Selected highly significant genes are labeled. See also Figure S8F.
(H and I) Double IF staining images of the pair of MUC6 and LEFTY1 (H) and the pair of MUC6 and OLFM4 (I) in the IM biopsy. Scale bar, 100 μM; nuclei (DAPI) in blue. A representative view of co-staining was shown in the enlarged images at right (scale bar for H, 50 μM; scale bar for I, 20 μM).
(J) Bar chart showing the relative percentage of cells expressing MUC6 or OLFM4 alone or in combinations (MUC6 + OLFM4) across different lesions.
See also Figure S13.
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6. Figure 4
The scRNA Profiles for Enteroendocrine Cell Lineages across Different Lesions
(A) The t-SNE plot that showed the distribution of enteroendocrine cell lineages (orange, n = 1,760 cells) within the atlas.
(B) Enteroendocrine cell populations were re-clustered into eight subclusters (color coding).
(C) The distribution of the mean expression of intestinal and gastric endocrine cell canonical markers in each sample. Gastric endocrine cell markers are displayed in blue, while intestinal endocrine cell markers are shown in red. IM-W, wild IM; IM-S, severe IM.
(D) Fraction of cells expressing canonical markers (upper) and cells at different lesions (lower) in each cluster. The y axis represents the proportion of cells expressing canonical markers, while the x axis represents the identified eight clusters. The cluster C8 represents unique endocrine cells in the EGC lesion.
(E) The t-SNE plot shows the expression of the putative EGC endocrine cell marker, OR51E1, in the whole endocrine cell lineage (red, high; gray, low). The cells within the red circle are the endocrine cells in the EGC lesion.
(F) Double IF staining images of OR51E1 and CHGA in the EGC biopsy from patient P8. The representative view of the co-staining of OR51E1 and CHGA is shown in the enlarged images at right. Scale bars, 100 and 20 μm (enlarged images). Nuclei (DAPI) in blue.
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7. Figure 5 HES6 Marks Early Goblet Cells
(A) The t-SNE plot that showed the distribution of the goblet cell cluster (green, n = 565) in the atlas (top) and the enlarged t-SNE plot (bottom) showed that the five subclusters’ goblet cell populations were re-clustered into (color coding). See also Data S1.
(B and C) Heatmap showing the main signatures for the two goblet subtypes (B) and the corresponding five most enriched pathways (FDR <0.01) (C).
(D) The violin plot indicating the expression profile of HES6 across multiple cell types.
(E) Bar chart showing the percentage of cells in the goblet cell cluster expressing MUC2, HES6 alone, or MUC2 and HES6 in combination.
(F) Violin plots displaying the distribution of expression of MUC2 (top) and HES6 (bottom) in goblet subclusters. Cluster 0 is highlighted with a red rectangle.
(G) IF staining images of HES6 (green) in a resected normal colon specimen (blue stain, DAPI), and the triangles indicate HES6-expression cells in the colon glands. Scale bars, 100 μm.
(H and I) Double IF staining images of HES6 and MUC2 (H) and HES6 and KI67 (I) in the IM biopsy. Scale bar, 100 μM. The representative views of co-staining were shown in the enlarged images at right (scale bar, 20 μM).
See also Figure S14.
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8. Figure 6 The scRNA Profile of EGC Cells
(A) The t-SNE plot that showed the distribution of the cancer cell cluster (pink, n = 798) in the atlas. See also Figures S15A and S15B and Data S1.
(B) Boxplot for the distribution of expression of the gastrointestinal cancer marker CEACAM6, cell-cycle-related gene CCND2, and apoptosis-related gene BAX in diverse epithelial cell types, Mann-Whitney U test, FDR <1e−16.
(C) Boxplot of the differential expression for the putative cancer cell-related top six upregulated genes in the GC datasets in TCGA. ∗FDR <1e−5. See also Data S1.
(D) The similarity network among diverse epithelial cell types in our dataset. The thickness of edges in the network was denoted as the Pearson correlation coefficient between the centroids of any pair of cell types. See also Figures S15C and S15D.
(E) The correlations between the centroids of cancer cells and metaplastic stem-like cells (left) and enterocytes (right).
(F) Violin plots display the distribution of expression of previously reported EGC-related markers (FABP1 [left], CDH17 [center], and GRN [right]) in cancer cells, enterocytes, gastric mucous cells, and stem-like cells.
(G) The signature of EGC cells.
(H) The IF staining for the representative gene in the EGC signature, KLK10, in sections derived from IM, EGC, and advanced gastric cancer (AGC) biopsies. Scale bar, 200 μM.
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