Figure 2.
Multiplexed imaging interrogation of gastric biopsies after anti-PD-1 treatment
(A) Stacked barplot showing the number of patients profiled and regions of interest (ROIs) scanned for both healthy and diseased tissue.
(B) Representative image of the sampling approach for the study. Representative ROIs for the gastric biopsies were selected based on the H&E stain and rasterized with MIBI-TOF. The carbon channel from the MIBI-TOF scans reveal the structural similarity with the adjacent H&E section. The stomach image was created using BioRender.
(C) Overview of the MIBI-TOF workflow and panel. MCC, mast cell chymase; CDH2, N-cadherin; α-SMA, alpha-smooth muscle actin; GrnzB, Granzyme B; HH3, histone H3; HLA-DR, human leukocyte antigen – DR isotype; HLA-class I, human leukocyte antigen class I.
(D) Representative marker overlay from a gastric tissue section selected based on paired H&E, along with the MIBI-TOF workflow for segmentation and downstream analyses. Epi, epithelial cell; Mese, mesenchymal cell; Endo, endothelial cell; SMC, smooth muscle cell/myofibroblast; Macro, macrophage; Mono, monocyte; NK, natural killer (NK) cell; H&E, hematoxylin and eosin (H&E) stain.
(E) Cell lineage assignments based on normalized expression of lineage markers (heatmap columns). Columns are hierarchically clustered (Euclidean distance, average linkage), while rows are ordered by cell lineage (top) and immune cell breakdown (bottom). A bar plot with the absolute abundance of each cell type is displayed (left).
(F) Localization of 25 different markers within a representative MIBI image of a gastric tissue section. A 4-color overlay of lineage-specific and functional markers is shown per image.
(G) Pie charts displaying the proportion of immune, epithelial, and endothelial cells and other lineage-specific markers over the total cells and across all regions (left); and the proportion of different immune cell subsets within the total CD45+ cells (right). HH3, histone H3; Pan-CK, pan-cytokeratin; HLA-I, human leukocyte antigen class I; MCC, mast cell chymase; Vim, vimentin.
(H) Scatterplot depicting the total cell density (cell/mm2) per ROI for healthy and diseased gastric tissues.
(I) Scatterplot depicting the lineage cell density (cell/mm2) per ROI for healthy and diseased gastric tissues.
(J) Frequency of immune cell subsets of total immune cells. Lines represent the median and interquartile range (IQR). All p values calculated with a Wilcoxon rank-sum test, where ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
See also Figure S1.