Immune infiltration and differentially expressed genes between NIC samples and CMS subtypes. a Quantitative analysis of immune cell infiltration by multispectral fluorescent imaging. The number of cells was counted per square millimeter of tissue (total) and epithelium (intra-epithelial). b Representative tissue sections demonstrating variable infiltration of immune cells in MMR-p (NIC3–7) and MMR-d tumors (NIC13). c Heatmaps showing the relative expression of immune regulatory genes for the CRC TCGA dataset and the Leiden cohort. Color saturation indicates the statistical significance; red and blue indicate the direction of change. Volcano plot shows differentially expressed genes between CMS2/3 (left) and CMS4 (right) samples. Statistically significant expressed genes from the immune gene set are depicted. d Box plot representing the gene expression per CMS subtype in the Leiden cohort of the differentially expressed immune genes determined in c.