The biological sex plays a crucial role in multiple tumor entities, like melanoma, lung cancer, and bladder cancer,33‐36 and is also known to highly influence PDAC pathology. Natural killer (NK) cells are granular innate lymphocytes that represent an efficient endogenous first-line defense against cancer. Notably, this occurred despite the use of the same cell line in both sexes, indicating that host sex significantly shapes the composition and architecture of the tumor microenvironment. These data suggest that T and/or its metabolites may help maintain the physiological balance of autoimmunity and protective immunity by preserving the number of regulatory T cells and the activation of CD8+ T cells. The percentage of NETosing neutrophils was calculated as an average of the number of NETs divided by total number of cells/per image. Non-NK cells were magnetically labeled and depleted via column separation, resulting in an NK cell-enriched population of 85%–100% purity. Spleens were dissociated and filtered to yield single-cell suspensions containing 2%–7% CD3⁻NK1.1⁺ cells. As sex-dependent differences have been reported for the incidence and mortality of PDAC (Figure 1),39 as well as NK cell function, we aimed to elucidate the role of biological sex in NK cell-mediated immunity against PDAC-derived cell lines in vivo and in vitro. However, uncontrollable influences such as social rank, individual characteristics of the animals, cage position within the rack, or the order of treatments may have affected the results. The representative images shown depict male and female samples processed within the same imaging run. CD11b was more affected by co-culture with Panc02 cells than by IL-2 alone, indicating enhanced NK cell maturation in the presence of pancreatic cancer cells59 (Figure 3B). The expression of Ar and Esr1 was confirmed with RT-PCR in positively isolated NK cells (Supplementary Figure S3). We found that freshly isolated NK cells displayed a naïve, non-activated phenotype, characterized by low expression of the maturity markers CD11b and CD27, and the activation markers NCR1 and NKG2D, as well as a low intracellular content of granzymes A and B (Figure 2D). The cell types are displayed as a t-SNE plot (left), while the population frequencies and their distribution among specimens are shown as bar charts (right). Scatter dot plot showing NK cell counts per area; statistical significance was determined by Mann–Whitney U test (2). Multiple blood samples were collected during tumor development and analyzed via flow cytometry. Additionally, the experiments showed that the initial effect triggered by sex hormones vanished within 24 h, which is why hormones presumably had no influence on the in vitro killing assays. Although we detected initial differences in NK cell activity following hormone stimulation, these effects dissipated within 24 hours in the presence of SU.86.86 and in the absence of hormones (Supplementary Figure S6A) and had no impact on the cytotoxic efficacy (Supplementary Figure S6B). Log2-fold changes in gene expression and statistical significance are depicted as a bubble plot below the heatmap. Researchers are investigating whether they can suppress NK cells in animals or test tubes. Low dose naltrexone (LDN) was also researched for increasing NK cells, but its use is controversial The factors listed below are hypothesized to increase NK cell function but not necessarily NK cell levels. Most of the lifestyle, dietary, and supplement factors listed below rely on animal and cellular data. On the other hand, NK cells may, in turn, destroy and suppress Th1 cells . Collectively, these data suggest that IFN-producing cells and DCs are activated by MCMV through a TLR9- and MyD88-dependent pathway to produce type I IFNs and IL-12. Of note, NK cells from MCMV-infected TLR9- and MyD88-deficient mice produced less IFN-γ 4, 5, had impaired proliferation, and displayed reduced cytolytic activity compared with NK cells from MCMV-infected wild-type mice . They respond directly by recognizing virus-infected cells, and indirectly by interacting with dendritic cells (DCs), which express Toll-like receptors (TLRs) and secrete cytokines in response to encounter with microbes. In this manner, NK cells function as important sentinels of the immune system, working as primary responders and alerting the host to the presence of infectious organisms. Natural killer (NK) cells represent a distinct subset of lymphoid cells that have innate immune functions . GFP expression was used to distinguish target cells from effector cells and to model proliferation curves, while single-cell segmentation was performed with the AI Health Module based on brightfield images to determine living and dead tumor cells. This indicates that female NK cells not only restrict growth but also increase the destruction of target cells by approximately 50% compared to males in our study. The differences in the GFP proliferation curves between males and females were confirmed by analyzing AI-based single-cell segmentation of Panc02 cells (Live Count, Supplementary Figure S5A). (K) MICS-derived images of the indicated marker genes indicate sex-specific differences in myCAF localization and endothelial cell organization. Violin plots indicate signal distributions for the indicated cell types (F, G, I) or sexes (H). The cell types were annotated based on protein marker expression and gating strategies as described in Supplementary Table T3. This approach mirrors our experiments with primary NK cells, which were exposed to hormones until isolation, but were not subjected to hormonal influence during the actual killing assay. The growth curves represent the percentage of dead Panc02 cells (2). (B) Histograms (upper panels) and median fluorescence intensity (MFI, lower panels) values of NKG2D, NCR1, CD11b, CD27, granzyme A, and granzyme B in NK cells following isolation (input), 24 h NK mono-culture (NK only), and 24 h Panc02 co-culture.
