Fig. 5: Butyrate upregulates NKG2D ligand expression and increases CRC targeting by DOT cells.
a, Heat maps represent NKR ligand upregulation, assessed by flow cytometry in CRC upon 24-h exposure to different molecules. The fold change (FC) of expression over control with medium is represented. Data are representative of three independent experiments. b, The 3-h killing assays against CRC cell lines with or without treating cell lines with butyrate for 24 h (n = 5 donors, pool of three experiments). Data were analyzed by a one-way ANOVA with Tukey’s post hoc test. c, NKG2D ligand upregulation, assessed by flow cytometry, in primary tumor specimens from participants with CRC upon butyrate exposure for 24 h, represented as the FC of control. d, The 24-h killing assay against primary CRC specimens with or without treating tumor cells with butyrate for 24 h. Lines connect values from the same participants (n = 6 participants, pool of four independent assays). e,f, Viability of DOT cells (e) and expression of NKG2D, DNAM1 and TNF (f), assessed by flow cytometry, upon exposure to different concentrations of butyrate for 3 h. In e and f, lines connect values from the same DOT donors (n = 3 donors). Data are one representative of two independent experiments, Data in d–f were analyzed by a repeated-measures one-way ANOVA with Tukey’s post hoc test. g, The 3-h killing assay of SW620 with or without butyrate pretreatment and/or anti-NKG2D blocking antibodies (n = 3 replicates of tumor alone ± butyrate, n = 6 DOT donors). Data are one representative of three independent experiments and were analyzed by a one-way ANOVA with Tukey’s post hoc test. h, Schematic representation of the administration of 100 mM sodium butyrate in drinking water of intracecal SW620 tumor-bearing mice and DOT cell i.v. treatment. i, Representative in vivo NKG2D ligand expression in the tumor (gated on alive human Epcam+ tumor cells) of control or butyrate-treated mice on day 28 after tumor inoculation, with percentages shown. j, CD69 expression, assessed by flow cytometry, on tumor-infiltrating DOT cells in butyrate-treated (n = 6 mice) and control mice (n = 3 mice). Data are one representative of two independent experiments and were analyzed by an unpaired t-test. k, Kinetics of intracecal SW620 tumor growth measured by luciferase signal using in vivo imaging of mice treated as depicted in h (n = 6 mice per group). Data are presented as the means ± s.e.m. (pool of two independent experiments) and were analyzed by a repeated-measures two-way ANOVA with Šidák’s multiple-comparisons test.
