Journal: Redox Biology

Article Title: Ketogenic diet impairs NK cell cytotoxic function in colorectal cancer liver metastasis by inducing ferroptosis via suppression of the p62-Keap1-Nrf2 pathway

doi: 10.1016/j.redox.2025.103969

Figure Lengend Snippet: Metabolite PE compromises NK cell mitochondrial function and promotes liver CRC metastatic growth. A. Schematic overview of the single-cell RNA sequencing workflow, depicting tumor tissue collection and sample processing. B. UMAP plot displaying dimensional reduction and clustering of all datasets, with point colors representing cell type identity and their relative proportions. C. UMAP visualization showing three distinct NK cell clusters, overlaid with corresponding RNA velocity vectors. D. Volcano plot displaying differentially abundant genes. Red points represent significantly up-regulated genes, blue points represent significantly down-regulated genes (FDR<0.05, |log2FC|>0.25). E. GO enrichment analysis of significantly differentially expressed genes of NK cells. F. Gene set enrichment analysis (GSEA) of mitochondria-related pathways in NK cell populations between groups. G. Representative TEM images of liver tumor tissues across the two groups. Blue boxes highlighted cell-cell interactions, while red arrows indicated mitochondria. H. Mean fluorescence intensity (MFI) measurements of MitoTracker Green staining in liver tumor-associated NK cells. I. Mean fluorescence intensity (MFI) measurements of MitoTracker Deep Red staining in liver tumor-associated NK cells. J. Representative confocal microscopy images of NK-92MI cells stained with MitoTracker (green) and DAPI (blue). K. Mean fluorescence intensity (MFI) measurements of Calcein AM staining in NK-92MI cells following treatment with varying concentrations of PE. L. Measurement of real-time oxygen consumption rates (OCRs) in NK-92MI cells under basal conditions and after sequential treatment with oligomycin, FCCP, and rotenone/antimycin A, following exposure to varying concentrations of PE (n = 3 per group). Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Four weeks post-inoculation, NK-92MI cells were pretreated with 10 μM tert-butylhydroquinone (TBHQ; MCE, New Jersey, USA) for 24 h and subsequently administered to the mice via tail vein injection three times per week for three weeks.

Techniques: Single Cell, RNA Sequencing, Fluorescence, Staining, Confocal Microscopy

Journal: Redox Biology

Article Title: Ketogenic diet impairs NK cell cytotoxic function in colorectal cancer liver metastasis by inducing ferroptosis via suppression of the p62-Keap1-Nrf2 pathway

doi: 10.1016/j.redox.2025.103969

Figure Lengend Snippet: PE induces ferroptosis in intratumoral NK cells and impairs their cytotoxic function. A. Heatmap showing differential expression of mitochondrial function-related genes in NK cells from the PE group versus controls (scRNA-seq). B. Western blot analysis of Nrf2, p62, and Keap1 expression in NK-92MI cells treated with increasing PE concentrations (0, 2, 5, 10 μg/mL) in HT-29-conditioned medium for 24h. C. Representative flow cytometric plots of Annexin V/PI staining in NK-92MI cells treated with PE (0, 2, 5, and 10 μg/mL) in HT-29-conditioned medium for 18 h. D. Quantitative analysis of Annexin V/PI staining, presented as histograms and statistical results. E. Evaluation of PE-induced cell death mechanisms in NK-92MI cells using specific inhibitors: Ferrostatin-1 (Fer-1, 2 μM) for ferroptosis, Necrostatin-1 (Nec-1, 2 μM) for necroptosis, and Z-VAD-FMK (zVAD, 5 μM) for apoptosis. F. Western blot analysis of GPX4 expression in NK-92MI cells treated with increasing PE concentrations (0, 2, 5, 10 μg/mL) in HT-29-conditioned medium for 24h. G. Lipid peroxidation analysis of NK-92MI cells treated with PE (0, 2, 5, 10 μg/mL) in HT-29-conditioned medium for 18h. Representative histogram data and statistical results were shown. H. Representative fluorescence micrographs of intracellular Fe 2+ in PE-treated (0, 10 μg/mL) NK-92MI cells stained with FerroOrange. I. Quantification of reduced-to-oxidized glutathione (GSH/GSSG) ratio in NK-92MI cells following treatment with indicated PE concentrations (0, 2, 5, 10 μg/mL). J. Representative TEM images of NK-92MI cells treated with or without PE for 24h. K. Representative immunofluorescence images of liver tumor sections showing NCR1 (green) and GPX4 (red) and nuclei (DAPI, blue) expression in Ctrl vs PE-treated groups of mice. L. Flow cytometry analysis of Granzyme B, IFN-γ and TNF-α production by NK cells isolated from mouse treated with or without PE for 24h. M. Migration assay showing HT-29 cells co-cultured with PE-treated (5 μg/mL) or untreated NK-92MI cells. N. Left: Cytotoxicity of MC38 cells mediated by murine NK cells treated with or without PE at various E:T ratios. Right: Growth kinetics of HT-29 cells co-cultured with PE-treated (5 μg/mL) or untreated NK-92MI cells. Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Four weeks post-inoculation, NK-92MI cells were pretreated with 10 μM tert-butylhydroquinone (TBHQ; MCE, New Jersey, USA) for 24 h and subsequently administered to the mice via tail vein injection three times per week for three weeks.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Staining, Fluorescence, Immunofluorescence, Flow Cytometry, Isolation, Migration, Cell Culture

Journal: Redox Biology

Article Title: Ketogenic diet impairs NK cell cytotoxic function in colorectal cancer liver metastasis by inducing ferroptosis via suppression of the p62-Keap1-Nrf2 pathway

doi: 10.1016/j.redox.2025.103969

Figure Lengend Snippet: Activation of the Nrf2 pathway protects against PE-induced ferroptosis and restores the anti-tumor activity of NK cells. A. Analysis of cell death in NK-92MI cells following treatment with TBHQ, PE, or their combination (PE + TBHQ) for 24h. B. Western blot analysis of Nrf2, p62, Keap1 and GPX4 expression in NK-92MI cells treated with TBHQ, PE, or their combination (PE + TBHQ) for 24h. C. Representative images of NK-92MI cells treated with TBHQ, PE, or their combination (PE + TBHQ) for 24h by Imaging streaming. D. Flow cytometric analysis of Nrf2 expression of NK-92MI cells treated with TBHQ, PE, or their combination (PE + TBHQ) for 24h. E. Lipid peroxidation analysis of NK-92MI cells treated with TBHQ, PE, or their combination (PE + TBHQ) for 24h. F. Quantification of reduced-to-oxidized glutathione (GSH/GSSG) ratio in NK-92MI cells following treatment with TBHQ, PE, or their combination (PE + TBHQ) for 24h. G. Representative fluorescence micrographs of intracellular Fe 2+ in NK-92MI cells treated with TBHQ, PE, or their combination (PE + TBHQ) for 24h. H. Representative immunofluorescence images showing p62 (yellow), Nrf2 (green), CD56 (red), and DAPI-stained nuclei (blue) in colon tumor tissues from CRC patients and CRC liver metastasis patients. I. Schematic diagram of the experimental mouse model: HT29 cells were injected intrasplenically to establish liver metastasis, followed by intravenous administration of NK-92MI cells (either TBHQ-treated or untreated) via tail vein. J. Representative macroscopic appearance of hepatic metastases in the liver. K. Measurement of tumor weight and enumeration of nodules (n = 6 per group). Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Four weeks post-inoculation, NK-92MI cells were pretreated with 10 μM tert-butylhydroquinone (TBHQ; MCE, New Jersey, USA) for 24 h and subsequently administered to the mice via tail vein injection three times per week for three weeks.

Techniques: Activation Assay, Activity Assay, Western Blot, Expressing, Imaging, Fluorescence, Immunofluorescence, Staining, Injection