Figure 3 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 promotes phagocytosis of tumor cells by TAMs and decreases angiogenesis in the TME. A, Tumor volume measurement of MB49 tumors in different treatment conditions. JHU083 treatment decreases tumor growth in MB49-RFP+ tumors relative to nontreated (n = 5 or 6/group) tumors. B, Representative flow cytometry plots of RFP+ MB49 tumor cells in TAMs in JHU083-treated vs. vehicle-treated tumors. C, Percentage of RFP+ cells within overall TAMs, CD206+ TAMs (M2-TAMs), and CD86+ MHCII+ TAMs (M1-TAMs) in MB49 tumors in control vs. JHU083-treated tumors. D, Percentage of RM1-RFP+ cells and corresponding mean fluorescence intensity (gMFI) of TAMs (n = 4/group) in control vs. JHU083-treated tumors. E, Schematic diagram showing the determination of the phagocytic activity of TAMs. Briefly, DON-treated, PBMC-derived macrophages were cocultured with CFSE-labeled PC3 cells, and phagocytic activity was determined using flow cytometry. F, Representative photomicrographs showing increased phagocytosis in DON-treated and PKH26-labeled macrophages (PKH26 MAC) cocultured with CFSE-labeled PC3 (CFSE PC3) by immunofluorescence microscopy and quantification using flow cytometry. G, Violin plots of phagocytosis UCell scores on TAM₁, TAM₂, InflamTAM, and ProlifTAM (Wilcoxon rank-sum test) identified in scRNA-seq analysis of CD45+ cells from B6CaP tumors. H, Violin plot for Myo1e expression in all TAMs in B6CaP-derived TAMs identified in scRNA-seq analysis of CD45+ cells from B6CaP tumors. I, IHC-based quantification of CD31+ area intensity in B6CaP tumors (n = 3/group), and (J) IHC quantification of CD31 intensity in MB49 tumors. Statistical analyses were performed using t test or two-way ANOVA using Bonferroni’s multiple comparisons. Violin plots from scRNA-seq studies are analyzed using the Wilcoxon rank-sum test. (*, P P P P