Fig. 4

Exposure to Activated Immune Cells Altered the Arginine Metabolic Landscape in Cancer Stem Cells (CSCs). The heatmap compares gene expression involved in Arginine metabolism in 4T1 CSCs that were not exposed to splenocytes (N), exposed to in vivo tumor antigen primed splenocytes (P) or exposed to in vivo tumor antigen primed splenocytes plus reinforced activation by anti-CD3/CD28 dynabeads (R). Significant increases in genes were noted in CSCs exposed to the in vivo tumor antigen-primed splenocytes with reinforced activation by anti-CD3/CD28 dynabeads (A). An increase in dansyl-arginine uptake (green fluorescence) was seen in 4T1 CSC spheroids with prior exposure to in vivo tumor antigen primed splenocytes and even more uptake of dansyl-arginine with in vivo tumor antigen primed splenocytes with reinforced by anti-CD3/CD28 dynabeads (B). Additionally, the activity of Arginase (C) was also increased in 4T1 CSCs that were exposed to in vivo tumor antigen primed splenocytes with reinforced activation by anti-CD3/CD28 dynabeads. The activity of Arginase was measured through the production of urea (C). Immune challenge of 4T1 CSCs by the in vivo tumor antigen primed splenocytes or these with reinforced activation by anti-CD3/CD28 dynabeads decreased tumor cell counts (examined via MTS) and increased secretion of ADMA into the conditioned medium (D, left and middle). The ADMA production per 4T1 CSC was plotted (D, right panel). ADMA concentration was expressed as ng/mL (left Y axis) or µM (right Y axis). Data are presented as individual replicates with the mean ± SD. (*p < 0.05, ** p  0.01, ****p < 0.0001), N = 3 for ELISA and N = 4 for ARG enzymatic assay