Validation of the DSP protein and RNA profiling approaches using colorectal cancer tissues. (A) ROI selection of normal, tumor, and stromal cells on FFPE tissue slides. Morphology markers (SYTO13 for nuclei staining, blue; Pan-CK for tumor/ epithelial cells, green; CD45 for immune cells, red) were used to guide 12 ROI selections of tumor, stroma, or normal epithelial cells enriched areas. Based on the abundance of tumor-infiltrating lymphocytes (TILs), 2 stromal TILs low, 2 stroma TILs medium, and 2 stroma TILs high ROIs were selected. Two sets of ROIs at the same locations were selected from 2 serial sections of the FFPE tissue for protein and RNA profiling, respectively. (B) Unsupervised hierarchical clustering of 12 ROIs representing different histological regions (normal epithelial cells, tumor, and stroma) based on 38-plex protein profiling. ROIs belonging to the same group are color-coded. (C) PCA clustering of different group assignments in protein profiling. Groups represents ROIs from different histological regions. (D) Unsupervised hierarchical clustering of 12 ROIs representing different histological regions (normal epithelial cells, tumor, and stroma) based on 78-plex RNA profiling. ROIs belonging to the same group are color-coded. (E) PCA clustering of different group assignments in RNA profiling. Groups represent ROIs from different histological regions.