Figure 2 from Metabolic Reprogramming of Tumor-Associated Macrophages Using Glutamine Antagonist JHU083 Drives Tumor Immunity in Myeloid-Rich Prostate and Bladder Cancers

JHU083-mediated glutamine antagonism reprograms TME, differentially induces TAMs, TIMs, inflammatory TNF signaling and proliferation in TAMs/TIMs. A, UMAP and stacked bar plots showing the diversity and differential abundance of TAMs and monocyte (TIM) clusters in B6CaP tumors at an early point (day 7 posttreatment) using scRNA-seq (n = 6) across samples. UMAP, uniform manifold approximation and projection for dimension reduction. B, Density plots of different TAM and TIM clusters in control and JHU083-treated B6CaP tumors (identified in A). C, DEG scores were calculated for each TAM cluster based on the GSEA of significant DEGs from bulk RNA-seq of JHU083-treated vs. control TAMs (day 14 posttreatment, late time point; n = 6/group). D, UMAP plot based on the DEG scores identified using bulk RNA-seq of B6CaP-TAMs from C. UMAP, uniform manifold approximation and projection for dimension reduction. E, GSEA between JHU083-treated vs. control cells from the proliferative TAMs and TAM2. F, GSEA between bulk RNA-sequenced JHU083-treated TAMs and control TAMs sorted from B6CaP tumors (day 18 posttreatment; n = 6/group). G, Volcano plot of top DEGs from the TAM2 cluster between JHU083-treated vs. control. H, Volcano plot of top DEGs between JHU083-treated vs. control samples from bulk RNA-seq data. I, Intracellular quantification of the percentage (%) TNF+ population in TAMs and TIMs in B6CaP tumors. J, Intracellular expression of percentage (%) Ki-67+ TAMs in JHU083-treated TAMs vs. control B6CaP tumors, and (K) clusters identified from A overlaid using RNA velocity analysis. The root cells are the undifferentiated cells, and the developmental endpoints are the differentiated cells connected via arrows pointing to the likely developmental paths. Briefly, DEGs were calculated with DESeq2 in the bulk RNA-seq data, and with the Wilcoxon rank-sum test for the scRNA-seq data. 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 P P P