Figure 5
Anti-melanoma activity of novel nNOS inhibitors. Metastatic melanoma A375 cells were injected to nude mice subcutaneously on the flank. The growth of tumor was measured daily and tumor volumes were determined using digital calipers (Fisher Sci) by using the formula tumor volume (mm3) = [Length × (Width2)]/2. Data are represented as mean ± SD. (a) nNOS inhibitor HH044 (10 mg/kg, i.p. daily) markedly inhibited the tumor growth of human melanoma in vivo compared to control (Control, n = 5; HH044, n = 4). (b) HH044 significantly decreased the final mass of xenograft tumors with no significant change in lung and body weight. *p < 0.05 compared to control; ns, p > 0.05, compared to control (Control, n = 5; HH044, n = 4). (c) PD-L1 expression of HH044 treated tumors was significantly decreased as detected by flow cytometry. *p < 0.05 compared to control (Control, n = 5; HH044, n = 4). Single cell suspensions of harvested tumor xenografts were stained with Alexa Fluor 488 conjugated PD-L1 antibody. The relative expression levels of PD-L1 were determined by the average fluorescence density as detected by flow cytometry. (d) nNOS inhibitor MAC-3-190 (5 mg/kg, i.p. daily) diminished the tumor growth stimulated by IFN-γ (1000 units, i.p. daily). *p < 0.05 compared to control; #p < 0.05 compared to IFN-γ treatment (Control, n = 7; IFN-γ, n = 11; IFN-γ + MAC-3-190, n = 5). (e,f) Expression levels of PD-L1 induced by IFN-γ treatment were inhibited by nNOS inhibitor MAC-3-190 in vivo. Metastatic melanoma A375 cells were injected to nude mice subcutaneously on the flank. IFN-γ (1000 units/day) was injected intraperitoneally once daily and nNOS inhibitor MAC-3-190 was administered i.p. daily at a dosing of 5 mg/kg for 21 days. The expression of PD-L1 in xenograft tumor samples were detected by immunohistochemistry staining in T-cell non-infiltrated area. By the end of study, xenograft tumors from different treatment groups were collected and specimens were fixed in a 10% formalin solution and embedded in paraffin wax for automatic processing using the Ventana Benchmark Ultra machine. Images of PD-L1 staining (brown) were captured in CD8-negative areas at 20× and 100× magnification, respectively. PD-L1 positive cells were quantified using ImageJ (https://imagej.nih.gov/ij/index.html) and represented as percentage of PD-L1 positive staining in the graph. Representative sections of each condition are shown. *p < 0.05 compared to control group; #p < 0.05, compared to IFN-γ group (n = 4 of each treatment).
