Figure 3 from Single-Cell Atlas of Lineage States, Tumor Microenvironment, and Subtype-Specific Expression Programs in Gastric Cancer

Abstract

<p>Differential TME analysis between histologic subtypes identifies increased plasma cells in diffuse-type tumors. <b>A,</b> Density plot of UMAP representation comparing diffuse and intestinal gastric cancer samples after random downsampling to approximately 25,000 cells each. Each dot represents a single cell. Dashed lines highlight a higher proportion of plasma cells in diffuse gastric cancer. <b>B,</b> IHC staining of IRF4 expression in diffuse (<i>n</i> = 5) and intestinal (<i>n</i> = 12) gastric cancer samples (scale bar, 400 μm). Bar graph showing significantly higher average IRF4 IHC score in diffuse compared with intestinal gastric cancer. <b>C,</b> Plasma cell proportions deconvoluted by CIBERSORTX in diffuse and intestinal gastric cancer in the TCGA data set (diffuse <i>n</i> = 63; intestinal <i>n</i> = 167). <b>D,</b> Bar graph showing enrichment of plasma cell proportions in PL4 and PL5 plasma subclusters in diffuse versus intestinal gastric cancer single-cell samples. <b>E,</b> Pearson correlation plot showing significant positive correlation of plasma cell proportion to average <i>KLF2</i> expression in the epithelial metacluster. <b>F,</b> Bee swarm plot showing increased <i>KLF2</i> expression in diffuse versus intestinal gastric cancer samples in the bulk RNA-seq TCGA-STAD data set. <b>G,</b> Bar graph showing increased <i>KLF2</i> expression in Pan-CK<sup>+</sup> epithelial morphologic regions of diffuse versus intestinal gastric cancer by DSP (<i>n</i> = 10). <b>H,</b> DSP analysis depicting epithelial ROIs proximal (top) and distal to plasma cells (bottom). Analysis is based on immunofluorescence staining for Pan-CK (epithelial, green), CD138 (plasma, pink), smooth muscle actin (SMA; fibroblast, cyan), and DAPI (blue). Each circular ROI is 300 μm in diameter. <b>I,</b> Bar graph showing expression of <i>KLF2</i>, <i>IRF4</i>, and <i>SLAMF7</i> genes in GSU humanized and nonhumanized mice against SNU1750 humanized and nonhumanized mice. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01. <b>J,</b> Western blot (top) showing stable knockdown of <i>KLF2</i> in gastric cancer cell line GSU (sh<i>KLF2</i>) compared with shNT (nontargeting) control. Loss of <i>KLF2</i> in GSU significantly reduces migration of plasma cells derived from peripheral blood mononuclear cells (PBMC) and multiple myeloma cell line KMS-11 (<i>N</i> = 17; bottom). <b>K,</b> Upregulation of immunoglobulin genes in diffuse versus intestinal epithelial metacluster (top). Tree map shows the overlap of upregulated pathways in epithelial metacluster versus subclusters, with the EpiC cluster showing the greatest overlap (bottom).</p>

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