Figure 3 from Sucralose Consumption Ablates Cancer Immunotherapy Response through Microbiome Disruption

<p>Sucralose alters the tumor microenvironment and supports T-cell dysfunction. C57BL/6 mice from Taconic consumed sucralose in the drinking water (0.09 mg/mL) for 2 weeks prior to tumor injection and for the duration of the experiment. Mice were injected with 2.5 × 10⁵ MC38 cells subcutaneously and treated with 200 µg anti–PD-1 on days 9 and 12. <b>A–D,</b> CD45⁺ cells were isolated from the tumor and tdLN prior to single-cell RNA sequencing on day 14 after tumor injection. <b>A</b> and <b>B,</b> Uniform Manifold Approximation and Projection of clusters identified in tdLN (<b>A</b>) and tumor (<b>B</b>) in mice treated with anti–PD-1 ± sucralose. tSNE, t-distributed stochastic neighbor embedding. <b>C,</b> Volcano plot of gene expression from CD8⁺ T cells in the tumor of sucralose + anti–PD-1 vs. anti–PD-1–treated mice. <b>D,</b> Exhaustion signature heatmap for CD8⁺ T cells in the tumor comparing sucralose + anti–PD-1 (purple) with anti–PD-1 (teal). <b>E,</b> Representative flow cytometry plots and quantification of MitoTracker DeepRed staining in CD8⁺ T cells or CD4⁺ T_conv cells from tdLN. <b>F,</b> Representative flow cytometry plots of TNFα and IFNγ staining in CD8⁺ T cells and CD4⁺ T_conv cells in the tumor tissue of mice consuming sucralose and/or anti–PD-1. Responder mice in anti–PD-1 ± sucralose groups are shown as diamonds. Data are representative (<b>E</b>) or a composite (<b>F</b>) of three or one (<b>A–E</b>) independent experiments, respectively, with five mice per group per experiment. Error bars represent the mean ± SEM. A one-way ANOVA with the Tukey multiple comparisons test (<b>E</b> and <b>F</b>) was used. *, <i>P</i> < 0.05; **, <i>P</i> < 0.005.</p>