Abstract
<div>Abstract<p>Although tumor-infiltrating T cells hold a beneficial prognostic role in colorectal cancer, other lymphocytic populations are less characterized. We developed a multiplexed immunofluorescence assay coupled with digital image analysis and machine learning to identify natural killer (NK) cells (NCAM1<sup>+</sup>CD3<sup>−</sup>), natural killer T-like (NKT-like) cells (NCAM1<sup>+</sup>CD3<sup>+</sup>), and T cells (NCAM1<sup>−</sup>CD3<sup>+</sup>) within the PTPRC<sup>+</sup> (CD45<sup>+</sup>) cell population and to measure their granzyme B (GZMB; cytotoxicity marker) and FCGR3A (CD16a; NK-cell maturity marker) expression. We evaluated immune cell densities and spatial configuration in 907 incident colorectal carcinoma cases within two prospective cohort studies. We found that T cells were approximately 100 times more abundant than NK and NKT-like cells. Overall, NK cells showed high GZMB expression and were located closer to tumor cells than T and NKT-like cells. In T and NKT-like cells, GZMB expression was enriched in cells in closer proximity to tumor cells. Higher densities of both T and NKT-like cells associated with longer cancer-specific survival, independent of potential confounders (<i>P</i><sub>trend</sub> < 0.0007). Higher stromal GZMB<sup>+</sup> and FCGR3A<sup>+</sup> NK-cell densities associated with longer cancer-specific survival (<i>P</i><sub>trend</sub> < 0.003). For T and NKT-like cells, greater proximity to tumor cells associated with longer cancer-specific survival (<i>P</i><sub>trend</sub> < 0.0001). These findings indicate that cytotoxic NCAM1<sup>+</sup>CD3<sup>−</sup>GZMB<sup>+</sup> NK cells and NCAM1<sup>+</sup>CD3<sup>+</sup> NKT-like cells are relatively rare lymphocytic populations within the colorectal cancer microenvironment and show distinct spatial configuration and associations with patient outcome. The results highlight the utility of a quantitative multimarker assay for <i>in situ</i>, single-cell immune biomarker evaluation and underscore the importance of spatial context for tumor microenvironment characterization.</p></div>
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