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Spatial organization and prognostic significance of NK and NKT-like cells via multimarker analysis of the colorectal cancer microenvironment

Authors: Väyrynen, Juha P.; Haruki, Koichiro; Lau, Mai Chan; Väyrynen, Sara A.; Ugai, Tomotaka; Akimoto, Naohiko; Zhong, Rong; Zhao, Melissa; Costa, Andressa Dias; Borowsky, Jennifer; Bell, Phoenix; Takashima, Yasutoshi; Fujiyoshi, Kenji; Arima, Kota; Kishikawa, Junko; Shi, Shan-shan; Twombly, Tyler S.; Song, Mingyang; Wu, Kana; Chan, Andrew T.; Zhang, Xuehong; Fuchs, Charles S.; Meyerhardt, Jeffrey A.; Giannakis, Marios; Ogino, Shuji; Nowak, Jonathan A.

Online: https://cancerimmunolres.aacrjournals.org/content/early/2021/12/22/2326-6066.CIR-21-0772

Issue: Cancer Immunol Res. 2022 Feb;10(2):215-227.


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 NK cells (NCAM1+CD3-), NKT-like cells (NCAM1+CD3+), and T cells (NCAM1-CD3+) within the PTPRC+ (CD45+) cell population and to measure their 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 (Ptrend<0.0007). Higher stromal GZMB+ and FCGR3A+ NK cell densities associated with longer cancer-specific survival (Ptrend<0.003). For T and NKT-like cells, greater proximity to tumor cells associated with longer cancer-specific survival (Ptrend<0.0001). These findings indicate that cytotoxic NCAM1+CD3-GZMB+ NK cells and NCAM1+CD3+ 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 in situ, single-cell immune biomarker evaluation and underscore the importance of spatial context for tumor microenvironment characterization.