Fig. 4

Tumor entrained neutrophils produce greater amounts of ROS than naïve neutrophils. a Conditioned media was collected after overnight incubation and tested for ROS using L-012 luminescent probe. Since this reagent measures all ROS, the addition of catalase determined the presence of hydrogen peroxide. 67NR TEN produce more ROS, including hydrogen peroxide, than naïve neutrophils (p = 0.008). b ROS Levels do not correlate with tumor cell killing. Intra- and extracellular ROS were measured in the Luminol assay. Single cell suspensions of tumor cells or freshly isolated naïve neutrophils were incubated with Luminol for 15 min at room temperature. CCL2 or tumor cells were then added to naïve neutrophils and luminescence was immediately measured. CCL2 did not significantly increase the ROS signal (p = 0.212); moreover, a decrease in ROS signal was observed when tumor cells and neutrophils were co-cultured (p = 0.003 for 4 T1 vs. Neutrophils + 4 T1 and p < 0.001 for 67NR vs. Neutrophils + 67NR). c Granzyme-B release when neutrophils are co-cultured with tumor cells and CCL2. Granzyme-B levels in conditioned media from cultured cells were determined by ELISA. 4T1 cells co-cultured with naïve neutrophils exhibited higher granzyme-B release than neutrophils alone (adj. p = 0.025). Also 67NR cells co-cultured with naïve neutrophils resulted in a significant increase in granzyme-B release over that of neutrophils alone (adj. p < 0.001). For Fig. 4a and b, Kruskal-Wallis test with Dunn’s test for multiple comparisons. For Fig. 4c, a log transformation was used to meet the normality assumption. ANOVA for an overall comparison and t-test for multiple comparisons with Bonferroni p-value adjustment was used. Values are graphed as mean ± SD