Figure 6 from Metabolic Reprogramming of Tumor-Associated Macrophages Using Glutamine Antagonist JHU083 Drives Tumor Immunity in Myeloid-Rich Prostate and Bladder Cancers

JHU083-induced glutamine antagonism causes extensive reprogramming of TILs. A, UMAP plot showing lymphocytic cell subsets identified from scRNA-seq analysis of CD45⁺ cells isolated from B6CaP tumors (JHU083-treated vs. control). B, UMAP density plots showing all NK and T cells. C, Dot plots of normalized expression of selected marker genes in T and NK cell subsets identified in A. D, Changes in proportions of NK cell subsets, stem cell–like CD8⁺ T cells, and CD4⁻ Tregs from scRNA-seq (n = 3/group). E, Surface and intracellular expression of stem-like CD8⁺ T cells and Foxp3⁺ percentage population in B6CaP and MB49 tumors (n = 7/8 per group). F, Schematic workflow of T cell coculture with DON-pretreated human primary macrophages. G, CD8⁺ T cell proliferation measurement using autologous CD8⁺ T cells isolated from human PBMCs and cocultured with monocyte-derived macrophages pretreated with DON either during differentiation (days 0–9) or during polarization (days 5–9) phase. H, gMFI of TNF and IFNγ in cocultured autologous CD8⁺ T cells with DON pretreated monocyte-derived macrophages, and (I) Tumor volume measurement of MB49 and B6CaP tumors during the therapeutic window. Briefly, following the development of palpable tumors, tumor-bearing C57BL/6J mice were injected every third day with anti-PD1 alone or in combination with daily oral gavage of JHU083. The data are presented as mean values ± SEM. Statistical analyses were performed using either t test or a two-way ANOVA using Bonferroni’s multiple comparisons (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). UMAP, uniform manifold approximation and projection for dimension reduction.