Spatial distribution and functional heterogeneity of CD56bright and CD56dim natural killer cells and the effects on the immune microenvironment in ovarian cancer.

5593 Background: Natural killer (NK) cells are key components of antitumor immunity, yet their functional heterogeneity and spatial distribution within the tumor microenvironment (TME) remain incompletely understood in ovarian cancer. CD56bright and CD56dim NK cell subsets exhibit distinct immunologic functions, but their relative infiltration patterns and associations with other immune cell populations have not been systematically characterized. Methods: A total of 157 ovarian cancer specimens were analyzed using multiplex immunofluorescence staining. NK cells were classified as CD56bright and CD56dim subsets, and their densities were quantified separately in tumor parenchyma and stroma. The associations between NK cell infiltration patterns and the densities of multiple immune cell populations. Results: Overall, ovarian cancer exhibited a CD56dim NK cell–dominant infiltration pattern. In tumor parenchyma, the mean density of CD56bright NK cells was 149 cells/mm², whereas CD56dim NK cells reached 406 cells/mm². In tumor stroma, CD56bright and CD56dim NK cell densities were 108 cells/mm² and 277 cells/mm², respectively. Patients with higher overall NK cell infiltration showed significantly increased densities of multiple immune cell populations in both tumor parenchyma and stroma, including CD8⁺ T cells (P=0.0091, 0.0167), M1 macrophages (P=0.0002, 0.0001), M2 macrophages (P=0.0011, 0.0008), PD-L1⁺ macrophages (P=0.0164, 0.0006), CD20⁺ B cells (P=0.0001, 0.0193), CD4⁺ T cells (P=0.0001, 0.0001), and CD4⁺FOXP3⁺ regulatory T cells (P=0.0001, 0.0001). When comparing NK cell subset dominance, patients with lower CD56dim than CD56bright NK cell infiltration in tumor parenchyma exhibited significantly higher M1 macrophage density (342 vs 180 cells/mm², P=0.0344) and lower stromal M2 macrophage infiltration (P=0.0356). In tumor stroma, lower CD56dim-dominant cases showed significantly increased infiltration of M1 macrophages (435 vs 242 cells/mm², P=0.0336), M2 macrophages (171 vs 48 cells/mm², P=0.0053), CD8⁺PD-1⁺ T cells (34 vs 10 cells/mm², P=0.0299), CD4⁺ T cells (140 vs 25 cells/mm², P=0.0119), and CD4⁺FOXP3⁺ T cells (73 vs 4 cells/mm², P=0.0016). Conclusions: Ovarian cancer is characterized by predominant infiltration of CD56dim NK cells; however, the balance between CD56bright and CD56dim NK cell subsets is closely associated with distinct immune microenvironment patterns. These findings highlight the functional heterogeneity of NK cell subsets and provide a potential mechanistic explanation for the variable prognostic and therapeutic relevance of NK cells across tumor types and immunotherapy contexts. Spatially resolved profiling of NK cell subsets may offer valuable insights for immunotherapy stratification in ovarian cancer.