Figure 5.

Functional characterization of CD68⁺CD74⁺ cells. (A) CD68⁺CD74⁺ cells in 10 DB-CRCs from both cohorts were identified by applying a threshold of 0.35 CD74 expression to all CD68⁺ cells after IMC image histologic inspection. (B) Mean marker intensities in CD68⁺CD74⁺ and CD68⁺CD74 cells. Values were normalized across all markers and cells. Marker distributions were compared with the 2-sided Wilcoxon’s rank sum test, and Benjamini-Hochberg false discovery rate (FDR) correction was applied to account for testing over 14 markers. Fold change between the mean expression in CD68⁺CD74⁺ and CD68⁺CD74 cells is reported. DC, dendritic cells. (C) Percentage of CD68⁺CD74⁺ cells expressing selected markers associated with antigen presentation and M1 and M2 phenotypes. (D) Uniform Manifold Approximation and Projection (UMAP) maps of 2726 CD68⁺CD74⁺ cells in 17 regions from 10 DB-CRCs. Cells were grouped in 6 clusters based on the expression of 16 phenotypic markers using Seurat⁹ and colored according to the mean intensities of representative markers. The circle indicates a CPDL1-expressing cluster. (E) Single-cell segmentation (upper panel) and IMC images (lower panels) of selected CD68⁺CD74⁺ cell-associated markers from a representative DB-CRC. The right bottom panel reports the combination of all the selected markers. Scalebar = 100 μm.