FIGURE 6 from Single-cell Profiling Uncovers a <i>Muc4</i>-Expressing Metaplastic Gastric Cell Type Sustained by <i>Helicobacter pylori</i>-driven Inflammation
Authors: O'Brien Valerie P. , Kang Yuqi , Shenoy Meera K. , Finak Greg , Young William C. , Dubrulle Julien , Koch Lisa , Rodriguez Martinez Armando E. , Williams Jeffery , Donato Elizabeth , Batra Surinder K. , Yeung Cecilia C.S. , Grady William M. , Koch Meghan A. , Gottardo Raphael , Salama Nina R. 
Published: September 2023

<p>Metaplastic pit cells are detected in human subjects with preneoplasia and gastric cancer. <b>A–G,</b> The expression of MUC4 and the cell proliferation marker Ki-67 was probed in a TMA comprising samples from 47 gastric cancer (“CA”) patients. <b>A,</b> Marker expression is shown in tissue sections from two representative patients. MUC4 is shown in magenta, Ki-67 in green and nuclei (DAPI) in blue. Scale bars, 100 μm. <b>B–D,</b> QuPath was used to segment individual cells within each tissue core and determine their marker expression by pixel intensity. <b>B</b> and <b>C,</b> The percentage of positive cells was calculated for each marker and sample, and is shown as box-and-whisker plots overlaid with individual values for each sample. MUC4 is shown in B and Ki-67 in C. **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; ****, <i>P</i> < 0.0001, Mann–Whitney <i>U</i> test. <b>D,</b> MUC4 and Ki-67 were assessed within every cell in the TMA. The left image shows every cell on a representative slide. On the right, every cell in the slide was binned in quartiles according to its MUC4 expression level and the mean Ki-67 expression level of the cells in each quartile was calculated. Bars indicate the 95% confidence interval of the estimated true mean. <b>E–G,</b> The proportion of MUC4+ cells in the superficial samples (shown in B) was plotted against clinical parameters of the TMA subjects. <b>E,</b> MUC4 levels are reported according to the anatomic site of the cancer. “Other” denotes other gastric anatomical sites (e.g., gastroesophageal junction) and cases where the specific gastric site could not be determined from the available pathology notes (e.g., “diffuse”). <b>F,</b> Gastric <i>Hp</i> status was determined by ddPCR and subjects were categorized as <i>Hp</i>− or <i>Hp</i>+ to indicate whether their analyzed tissues still harbored active <i>Hp</i> infection. <b>G,</b> MUC4 levels are reported according tumor (“T”) stage. **, <i>P</i> < 0.01, Mann–Whitney <i>U</i> test for T1 versus T4. <b>H,</b><i>MUC4</i> transcript levels, reported as log<sub>2</sub>-transformed [<i>MUC4</i> reads per kilobase per million mapped reads (rpkm) +1], were assessed in TCGA and plotted according to tumor (“T”) stage. <b>I</b> and <b>J,</b> Three published gastric scRNA-seq datasets were mined for cell types of interest. Each dot represents one scRNA-seq sample, which are arranged in order of disease severity. <b>I,</b> Pit cells (<i>MUC5Ac</i>+) are shown as the proportion of all cells in each sample. NAG, non-atrophic gastritis; CAG, chronic atrophic gastritis. <b>J,</b> Metaplastic pit cells (<i>MUC5Ac</i>+<i>MUC4</i>+<i>AREG</i>+) are shown as the proportion of non-hematopoietic cells (<i>PTPRC</i>−) in each sample.</p>