Fig. 5: Comparative analysis of cell clustering, cell type annotation, and spatial alignment with adjacent CODEX.

a Uniform Manifold Approximation and Projection (UMAP) of scRNA-seq and ST data for COAD samples. Each point represents a single cell (for scRNA-seq, Stereo-seq v1.3, CosMx 6K, and Xenium 5K) or an 8 × 8 μm bin (for Visium HD FFPE). Colors denote clusters identified by unsupervised clustering applied independently to each dataset based solely on transcriptomic profiles. b Average silhouette width (ASW) of unsupervised clustering results across platforms, with higher scores indicating better separation between distinct cell states. c Consistency of automated cell type annotations across five reference-based annotation tools. Bars represent the proportion of cells annotated as the same cell type by one to five tools. d Spatial distribution of annotated cell types in ST and CODEX data. Colors denote major cell types. Each ST platform is compared to its adjacent CODEX section. e, f Spatial correlation between CODEX-inferred and ST-inferred cell counts for different cell types over the spatial grids. Panel e shows the correlations for immune and stromal cells, while panel f shows the correlations for epithelial cells. Pearson correlation coefficients are reported. Hollow circles indicate individual correlation values obtained under different grid sizes (n = 5). Data are presented as mean values +/− SEM. g Representative immune-enriched regions (500 × 500 μm) from COAD sections. H&E staining, ST-derived annotations, CODEX-derived annotations, and multiplexed CODEX staining for CD20, CD8, and CD4 are shown. For ST data, each point represents a single cell (for Stereo-seq v1.3, CosMx 6K, and Xenium 5K) or an 8 × 8 μm bin (for Visium HD FFPE), colored by annotated cell type as shown in the legend. Scale bars, 100 μm. Source data are provided as a Source Data file.