Spatial profiling reveals distinct immune microenvironments in early- vs late-onset colorectal cancer

Incidence of colorectal cancer (CRC) has increased in individuals < 50 years, described as early-onset CRC (EOCRC). Although the precise etiology is unclear, EOCRC is associated with external exposures that may alter the tumor immune microenvironment (TIME). We hypothesized that EOCRC develops a distinct immune landscape compared with late-onset CRC (LOCRC). Using imaging mass cytometry (IMC), we compared the composition and spatial organization of the TIME at single-cell resolution. In total, 142,992 cells from 18 regions of interest (ROIs) from 7 EOCRC and 10 LOCRC patients were analyzed using Steinbock computational pipeline. Overall immune cell abundance was similar between EOCRC and LOCRC patients, with no significant differences observed. Spatial analysis revealed distinct patterns of immune cell organization, including increased Treg avoidance of GZMB + neutrophil-like (CD15+) cells and increased interaction between CD8+ T cells and T-helper cells (CD3+, CD4 +) in EOCRC ROIs. Tregs were significantly enriched within immune-enriched neighborhoods in EOCRC patients (1.6-fold, p = 0.018). Patch-based analysis demonstrated higher Treg enrichment in EOCRC (1.6-fold, p = 0.03). In contrast, EOCRC tumors exhibited lower CD8+ T cells (0.5-fold, p < 0.0001) and T-helper cells (0.5-fold, p = 0.00054) within non-tumor regions, with a trend toward increased intraepithelial lymphocytes (IELs; CD8a + ). Higher Treg abundance (1.6-fold, p <0.0001) in EOCRC was confirmed by immunohistochemistry in four independent EOCRC and four LOCRC tumors. These findings suggest subtle changes in spatial organization of immune cells between EOCRC and LOCRC.