Figure 4.

Difference in CD74⁺ macrophages between DB- and nDB-CRCs. (A) Uniform Manifold Approximation and Projection (UMAP) map of 16,748 macrophages in 30 regions from 13 hypermutated CRCs in the discovery cohort. Cells were grouped in 9 clusters based on the expression of 11 phenotypic markers using Seurat⁹ (Supplementary Table 8) and colored according to the mean intensities of representative markers. The circle indicates the cluster enriched in DB-CRCs. (B) Proportions of cluster 3 (CD68⁺CD74⁺ cells) over the total macrophages in DB- and nDB-CRCs. Distributions were compared using the 2-sided Wilcoxon’s rank sum test. Benjamini-Hochberg false discovery rate (FDR) correction was applied for testing over 9 clusters. (C) IMC-derived images of CD74, CD16, and CD163 and tumor-associated markers (E-cadherin and pan-keratin) in 2 representative samples. Scale bar = 100 μm. (D) Comparisons of normalized CD74⁺ area between DB- and nDB-CRCs in the discovery, (E) validation, and (F) both cohorts using the 2-sided Wilcoxon’s rank sum test. For the discovery cohort, Benjamini-Hochberg FDR correction was applied for testing over 9 combinations of macrophage markers (Supplementary Table 6). (G) CD74⁺ macrophages in the validation and (H) combined cohorts were identified by applying a threshold of 0.1 CD74 expression to all macrophages after IMC image histologic inspection. Mean marker intensities in CD74⁺ and CD74⁻ macrophages are reported and normalized across all markers and cells. (I) Comparison of normalized of CD74⁺ macrophages between DB- and nDB-CRCs in the validation and (J) combined cohorts. Distributions were compared using the 2-sided Wilcoxon’s rank sum test. The number of patients in each tumor group is reported in brackets. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively, and the vertical lines mark minimum and maximum of all the data.