Fig 1: Clinical validation of cytokine signature and argininosuccinate. A Plasma VEGF-A, IL-8, CXCL2, and CXCL3 concentrations of patients on the ADAM study randomized to ADI-PEG20 and BSC compared with baseline levels; and B control patients randomized to BSC alone compared to baseline levels (IL-1a not detected in either group). Bar values represent mean and standard deviation: *p < 0.05, ***p < 0.001, ****p < 0.0001; Wilcoxon signed-rank test. C Plasma cytokine concentrations in patients whose disease had progressive disease (PD) compared with patients who had stable disease/partial response (SD/PR) at the 8 week assessment by CT imaging were analyzed in the ADI-PEG20 plus BSC treatment group. Data represent mean values with standard deviation: *p < 0.05; two-way ANOVA followed by Sidak’s multiple comparison’s test, VEGF-A (F1,317 = 12.25, df = 1; p < 0.0005), IL-8 (F1,318 = 56.46, df = 1; p < 0.0001), CXCL2 (F1,295 = 31.0, df = 1; p < 0.0001), and CXCL3 (F1,281 = 0.9333, df = 1; p = 0.335). D Argininosuccinate was detected in plasma from all patients and was only significantly increased in the ASS1-deficient patients with disease progression by week 8 of ADI-PEG20 plus BSC treatment, whereas levels were unchanged in patients displaying SD/PR as analyzed by CT imaging. **p < 0.01; by two-way ANOVA. E Plasma VEGF-A and CXCL2 concentrations of patients receiving ADI-PEG20 plus chemotherapy compared with baseline levels within the first 2 months on the TRAP study. Bar values represent mean and standard deviation: *p < 0.05; Wilcoxon signed-rank test
Fig 2: MPM cytokine induction by ADI-PEG20 and co-ordinate UC enzyme regulation. A Release of the pro-inflammatory cytokines VEGF-A, IL-8, CXCL2, CXCL3, and IL-1a in the cell supernatant of the ASS1-negative MPM cell lines was detected by ELISA at 24 and 48 h following treatment with ADI-PEG20 (n = 3). ELISAs were run in duplicate, with values representing the mean and standard deviation: *p < 0.05, **p < 0.01, ****p < 0.0001; Two-way ANOVA with post hoc Tukey’s test. B Macrophage ASS1 mRNA and C MSTO tumoral cell line ASL mRNA expression following stimulation by IL-1a (2 ng/ml), IL-8 (2 ng/ml), CXCL2 (0.1 ng/ml), CXCL3 (0.5 ng/ml), and VEGF-A (1 ng/ml) individually or in combination using concentrations detected by ELISA above (n = 6), as assessed using qPCR with GAPDH mRNA for normalization. Values represent the mean and standard deviation of the mean: **p < 0.01, ***p < 0.001; One-way ANOVA with post hoc Holm–Sidak test as indicated. D ASL mRNA expression in the MSTO tumor cell line following co-culture with macrophages ± ADI-PEG20 (750 ng/ml). Values represent the mean and standard deviation: p < 0.05 (n = 9); two-way ANOVA with post hoc Tukey’s test
Fig 3: qPCR assay validation of ADI-PEG20-induced MPM cytokine gene signature. A Three ASS1 negative and three ASS1-positive control mesothelioma cell lines were analyzed for induction of cytokine mRNA following ADI-PEG20 treatment at 0, 8, 24, and 48 h: VEGF-A, IL-8, CXCL2, CXCL3, and IL-1a. Experiments were performed in triplicate for each cell line (n = 3), with bars representing the mean and standard deviation: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Two-way ANOVA with post hoc Tukey’s test. Compared with unstimulated levels, ADI-PEG20 significantly increased cytokine levels in the three ASS1-negative MPM cell lines: VEGF-A (F5,45 = 135.8, df = 15; p < 0.0001); IL-8 (F15,42 = 1.912, df = 15; p = 0.05); CXCL2 (F15,41 = 37.82, df = 15; p < 0.0001); CXCL3 (F15,40 = 31.94, df = 15; p < 0.0001); and IL-1a (F15,40 = 62.19, df = 15; p < 0.0001). B Similar results were obtained across the ASS1-negative and ASS1-positive MPM cell lines using arginine-free media: mixed-effects model, *p < 0.05, **p < 0.01, ***p < 0.001; VEGF-A (F8,72 = 11.69, p < 0.0001); IL-8 (F8,45 = 2.604, df = 1; p = 0.0197); CXCL2 (F8,50 = 2.384, df = 8; p < 0.0293); CXCL3 (F8,48 = 31.17.80, df = 1; p < 0.0001); and IL-1a (F8,48 = 6.831, df = 1; p < 0.0001)
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